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TRANSCRIPT
Investigation on intercropping of Ziziphus mauritiana with Cajanus
cajan for fruit and fodder at marginal land and cultivation of Carissa
carandas for fruits through saline water irrigation
TAYYAB
DEPARTMENT OF BOTANY
UNIVERSITY OF KARACHI
2015
ii
Investigation on intercropping of Ziziphus mauritiana with Cajanus
cajan for fruit and fodder at marginal land and cultivation of Carissa
carandas for fruits through saline water irrigation
PhD Thesis
Submitted to the Board of advance Studies and Research in fulfillment of
the Degree of Doctor of Philosophy in the Department of Botany
University of Karachi
TAYYAB
DEPARTMENT OF BOTANY
UNIVERSITY OF KARACHI
2015
iii
Investigation on intercropping of Ziziphus mauritiana with Cajanus
cajan for fruit and fodder at marginal land and cultivation of Carissa
carandas for fruits through saline water irrigation
Thesis Approved
RESEARCH SUPERVISOR EXTERNAL EXAMINER
PROF DR RAFIQ AHMAD
FPAS FTWAS
Professor (Retd) Botany (Plant Physiology)
PI Biosaline Research Projects
Department of Botany
University of Karachi
iv
CERTIFICATE
It is hereby certified that this thesis is based on the results of the experimental work carried
out by Mr TAYYAB SO MUHAMMAD HANIF under my supervision on the topic
ldquoInvestigation on intercropping of Ziziphus mauritiana with Cajanus cajan for fruit
and fodder at marginal land and cultivation of Carissa carandas for fruits through
saline water irrigationrdquo
Mr TAYYAB had been enrolled under my guidance for the award of PhD in
Department of Botany University of Karachi I have personally checked all the research
work reported in the thesis and certify its accuracyvalidity It is further certified that the
materials included in this thesis have not been used partially or fully in a manuscript
already submitted or in the process of submission in partialcomplete fulfillment for award
of any other degree from any other university Mr TAYYAB has fulfilled requirements of
the University of Karachi for the submission of this dissertation and I endorse its
evaluation for the award of PhD Degree
RESEARCH SUPERVISOR
PROF DR RAFIQ AHMAD
FPAS FTWAS
Professor (Retd) Botany (Plant Physiology)
PI Biosaline Research Projects
Department of Botany
University of Karachi
Karachi-75270 Pakistan
v
DEDICATED TO MY FAMILY
MUHAMMAD HANIF (MY FATHER)
MRS ARIFA (LATE)
(MY BELOVED MOTHER)
SHAHEEN TAYYAB (MY WIFE)
vi
ACKNOWLEDGMENTS
All the praises for almighty Allah and all respects for Prophet Muhammad (Peace be Upon
Him) who has shown me the straight path
I am grateful to my supervisor Prof Dr Rafiq Ahmad for his keen interest
patronage and guidance during this research work which made successful submission of
this thesis
I also obliged to Prof Dr Ehtesham Ul Haque and Prof Dr Javed Zaki (Present
and Former Chairmen Department of Botany respectively) for providing me all the
necessary facilities and administrative support
Being employed as lecturer in Department of Botany Govt Islamia Science
College Karachi I am also thankful to Education and literacy Department Govt of Sindh
(Pakistan) for providing me facilities to perform this study
Thanks are due to Dr D Khan in assessing statistical data analysis and colleague
of Biosaline lab Dr M Azeem Dr Naeem Ahmed and M Wajahat Ali Khan for their
cooperation throughout the course of study
I am also gratefully acknowledged to Mr Noushad Raheem and Mr Noor Uddin
of Fiesta Water Park for providing field plot and facilities to perform this study I am also
thankful to Pakistan Metrological Department for providing environmental data
I am also obliged to Dr M Qasim and Dr M Waseem Abbasi for their suggestions
and support in writing this thesis
Assistance of Abbul Hassan (Lab attendant) Tajwar Khan (Biosaline field
Attendant) and Mr Wahid (Plant Physiology Lab Assistant) is also acknowledged
Thanks are also due to my friends Dr Rafat Saeed Dr Kabir Ahmad Dr Zia Ur
Rehman Farooqi Dr Noor Dr M Yousuf Adnan Asif Bashir Dr A Rauf A Hai Faiz
Ahmed MA Rasheed Jallal Uddin Saadi Ahsan Shaikh Saima Fehmi A Mubeen
Khan Dr Noor Ul Haq Saima Ahmad S Safder Raza SM Akber and my college
colleagues for giving me encouragement during this research work
vii
I can never forget the support and encouragement and good wishes of Mr M
Wilayat Ali Khan Mrs Shahnaz Rukhsana Mr Mansoor Mrs Rabia Mansoor Mrs
Chand Bibi and Mrs Saeeda Anwar
In the last I am highly grateful to my beloved father Muhammad Hanif my loving
mother Arifa (when she alive) my caring wife Shaheen and sweet childrenrsquos Sara and
Sarim my supportive brothers and sisters and all family members for their prayers love
sacrifices and encouragements provided during course of this research work
viii
TABLE OF CONTENTS
No Title Page no
Acknowledgement vi
Summary xix
Urdu translation of summary xxi
General introduction 1
Layout of thesis 11
1 Chapter 1 13
11 Introduction 13
12 Experiment No 1 15
121 Materials and methods 15
1211 Seed collection 15
1212 Experimental Design 15
122 Observations and Results 17
13 Experiment No 2 22
131 Materials and methods 22
1311 Seed germination 22
132 Observations and Results 23
14 Experiment No 3 28
141 Materials and methods 28
1411 Seedling establishment 28
142 Observations and Results 29
1421 Seedling establishment 29
1422 Shoot height 29
15 Experiment No 4 31
151 Materials and methods 31
1511 Drum pot culture 31
1512 Experimental design 31
1513 Vegetative and Reproductive growth 32
1514 Analysis on some biochemical parameters 32
152 Observations and Results 34
1521 Vegetative and Reproductive growth 34
ix
No Title Page no
1522 Study on some biochemical parameters 34
16 Experiment No 5 41
161 Materials and methods 41
1611 Isolation Identification and purification of bacteria 41
1612 Preparation of bacterial cell suspension 41
1613 Study of salt tolerance of Rhizobium isolated from root
nodules of C cajan
41
162 Observations and Results 42
17 Experiment No 6 44
171 Materials and methods 44
1711 Experimental design 44
1712 Vegetative and reproductive growth 45
1713 Analysis on some biochemical parameters 45
172 Observations and Results 46
1721 Vegetative and Reproductive growth 46
1722 Study on some biochemical parameters 46
18 Discussion (Chapter 1) 51
2 Chapter 2 59
21 Introduction 59
22 Experiment No 7 60
221 Materials and Methods 60
2211 Growth and Development 60
2212 Drum pot culture 60
2213 Experimental Design 60
2214 Irrigation Intervals 61
2215 Estimation of Nitrate content 62
2216 Relative Water content (RWC) 62
2217 Electrolyte leakage percentage (EL) 62
2218 Photosynthetic pigments 63
2219 Total soluble sugars 63
22110 Proline content 63
22111 Soluble phenols 64
x
No Title Page no
22112 Total soluble proteins 64
22113 Enzymes Assay 64
222 Observations and Results 67
2221 Vegetative growth 67
2222 Photosynthetic pigments 70
2223 Electrolyte leakage percentage (EL) 70
2224 Phenols 70
2225 Proline 71
2226 Protein and sugars 71
2227 Enzyme essays 71
2228 Vegetative growth 73
2229 Photosynthetic pigments 75
22210 Electrolyte leakage percentage (EL) 76
22211 Phenols 76
22212 Proline 77
22213 Protein and Sugars 77
22214 Enzyme assay 77
23 Experiment No8 90
231 Materials and Methods 90
2311 Selection of plants 90
2312 Experimental field 90
2313 Soil analysis 90
2314 Experimental design 91
2315 Vegetative and reproductive growth 93
2316 Analysis on some biochemical parameters 93
2317 Fruit analysis 94
2318 Nitrogen estimation 94
2319 Land equivalent ratio and Land equivalent coefficient 95
23110 Statistical analysis 95
232 Observations and Results 96
2321 Vegetative parameters 96
2322 Reproductive parameters 96
xi
No Title Page no
2323 Study on some biochemical parameters 97
2324 Nitrogen Contents 98
2325 Land equivalent ratio land equivalent coefficient 98
24 Discussion (Chapter 2) 108
3 Chapter 3 113
31 Introduction 113
32 Experiment No 9 114
321 Materials and methods 114
3211 Drum Pot Culture 114
3212 Plant material 114
3213 Experimental setup 114
3214 Vegetative parameters 115
3215 Analysis on some biochemical parameters 115
3216 Mineral Analysis 116
322 Observations and Result 117
3221 Vegetative parameters 117
3222 Reproductive parameters 117
3223 Study on some biochemical parameters 118
3224 Mineral analysis 118
33 Discussion (Chapter 3) 127
4 Conclusion 129
5 References 130
6 Appendices 168
7 Publications 181
xii
LIST OF FIGURES
Figure Title Page no
11 Effect of irrigation water of different sea salt solutions on seed
germination indices of C cajan
27
12 Effect of irrigating water of different sea salt solutions on
seedling emergence (A) and shoot length (B) of C cajan
30
13 Environmental data of study area during experimental period
(July-November 2009)
36
14 Effect of salinity using irrigation water of different sea salt
concentrations on height of C cajan during 18 weeks treatment
36
15 Effect of salinity using irrigation water of different sea salt
concentrations on initial and final biomass (fresh and dry) of C
cajan
37
16 Percent change in moisture succulence relative growth rate
(RGR) and specific shoot length (SSL) of C cajan under
increasing salinity using irrigating water of different sea salt
concentrations
37
17 Effect of irrigating water of different sea salt solutions on
reproductive growth parameters including number of flowers
pod seeds and seed weight of C cajan
38
18 Effect of irrigating water of different sea salt solutions on leaf
pigments including chlorophyll a chlorophyll b total
chlorophyll and carotenoids of C cajan
39
19 Effect of irrigating water of different sea salt solutions on total
proteins soluble insoluble and total sugars in leaves of C cajan
40
110 Growth of nitrogen fixing bacteria associated with root of C
cajan under different NaCl concentrations
42
111 Photographs showing growth of Rhizobium isolated from the
nodules of C cajan in vitro on YEM agar supplemented with
different concentrations of NaCl
43
xiii
Figure Title Page no
112 Effect of salinity using irrigation water of different sea salt
concentrations on height number of branches fresh weight and
dry weight of shoot of Z mauritiana after 60 and 120 days of
treatment
47
113 Effect of salinity using irrigation water of different sea salt
concentrations on succulence specific shoot length (SSL)
moisture and relative growth rate (RGR) of Z mauritiana
48
114 Effect of salinity using irrigation water of different sea salt
concentrations on number of flowers of Z mauritiana
49
115 Effect of salinity using irrigation water of different sea salt
concentrations on leaf pigments including chlorophyll a
chlorophyll b total chlorophyll and chlorophyll ab ratio of Z
mauritiana
49
116 Effect of salinity using irrigation water of different sea salt
concentrations on total sugars and protein in leaves of Z
mauritiana
50
21 Vegetative parameters of Z mauritiana and C cajan at grand
period of growth under sole and intercropping system at two
irrigation intervals
79
22 Fresh and dry weight of Z mauritiana and C cajan plants under
sole and intercropping system at 4th and 8th day irrigation
intervals
80
23 Leaf weight ratio (LWR) root weight ratio (RWR) shoot weight
ratio (SWR)specific shoot length (SSL) specific root length
(SRL) plant moisture Succulence and relative growth rate
(RGR) of Z mauritiana and C cajan grow plants under sole and
intercropping system at 4th and 8th day irrigation intervals
81
24 Leaf pigments of Z mauritiana and C cajan grow plants under
sole and intercropping system at 4th and 8th day irrigation
intervals
83
xiv
Figure Title Page no
25 Electrolyte leakage phenols and proline of Z mauritiana and C
cajan at grand period of growth plants under sole and
intercropping system at 4th and 8th day irrigation intervals
84
26 Total protein in leaves of Z mauritiana and C cajan plants
under sole and intercropping system at 4th and 8th day irrigation
intervals
86
27 Enzymes activities in leaves of Z mauritiana and C cajan plants
under sole and intercropping system at 4th and 8th day irrigation
intervals
87
28 Nitrate reductase activity and nitrate concentration in leaves of
Z mauritiana and C cajan plants under sole and intercropping
system at 4th and 8th day irrigation intervals
89
29 Soil texture triangle (Source USDA soil classification) 99
210 Vegetative growth of Z mauritiana and C cajan growing under
sole and intercropping system
100
211 Reproductive growth of Z mauritiana and C cajan growing
under sole and intercropping system
101
212 Leaf pigments of Z mauritiana and C cajan growing under sole
and intercropping
102
213 Sugars protein and phenols in leaves of Z mauritiana and C
cajan at grand period of growth under sole and intercropping
system
103
214 Sugars protein and phenols in fruits of Z mauritiana grown
under sole and intercropping system
105
215 Nitrogen in leaves and in soil of Z mauritiana and C cajan
growing under sole and intercrop system
106
31 Chlorophyll a chlorophyll b total chlorophyll chlorophyll a b
ratio carotenoids contents of C carandas growing under
salinities created by irrigation of different dilutions of sea salt
124
xv
Figure Title Page no
32 Total protein sugars and phenolic contents of C carandas
growing under salinities created by irrigation of different
dilutions of sea salt
125
33 Mineral analysis including Na and K ions was done on leaves of
C carandas growing under salinities created by irrigation of
different dilutions of sea salt
126
xvi
LIST OF TABLES
Table Title Page no
11 Electrical conductivities of different sea salt solutions
used in germination of C cajan
18
12 Effect of irrigation water of different sea salt solutions
on germination percentage (GP) per day of C cajan
seeds pre-soaked in non-saline water prior to
germination with duration of time under various salinity
regimes
19
13 Effect of irrigation water of different sea salt solutions
on germination rate (GR) per day of seeds C cajan pre-
soaked in non-saline water prior to germination with
duration of time under various salinity regimes
20
14 Effect of irrigation water of different sea salt solutions
on mean germination rate (GR) coefficient of
germination velocity (GV) mean germination time
(GT) mean germination index (GI) and final
germination (FG) of C cajan seeds pre-soaked in non-
saline water prior to germination under various salinity
regimes
21
15 Electrical conductivities of different sea salt solutions
used in germination of C cajan
24
16 Effect of irrigation water of different sea salt solutions
on germination percentage (GP) per day of C cajan
seeds pre-soaked in respective sea salt concentrations
with duration of time
25
17 Effect of irrigation water of different sea salt solutions
on germination rate (GR) per day of C cajan seeds pre-
soaked in respective sea salt concentrations with
duration of time
26
xvii
Table Title Page no
18 Electrical conductivities of different Sea salt
concentrations and ECe of soil saturated paste at the end
of experiment
30
21 Soil analysis data of Fiesta Water Park experimental
field
99
22 Land equivalent ratio (LER) and Land equivalent
coefficient (LEC) with reference to height chlorophyll
and yield of Z mauritiana and C cajan growing under
sole and intercropping system
107
31 Electrical conductivities of different sea salt
concentration used for determining their effect on
growth of C carandas
119
32 Vegetative growth in terms of height and volume of
canopy of C carandas growing under salinities created
by irrigation of different dilutions of sea salt
120
33 Vegetative growth in terms of height and volume of
canopy of C carandas growing under salinities created
by irrigation of different dilutions of sea salt
121
34 Reproductive growth in terms of flowers and fruits
numbers flower shedding percentage fresh and dry
weight of ten fruit and their totals per plant fruit length
and diameter of C carandas growing under salinities
created by irrigation of different dilutions of sea salt
123
xviii
LIST OF ABBREVIATIONS
APX Ascorbate peroxidase
CAT Catalase
DAP Diammonium Phosphate (fertilizer)
dSm-1 Deci Siemens per meter
ECe Electrical conductivity of the Soil saturated extract
ECiw Electrical conductivity of the irrigation water
GPX Guaiacol Peroxidase
GR Glutathione reductase
GSH Reduced glutathione
LEC Land equivalent coefficient
LER Land equivalent ratio
NPK Nitrogen Phosphate Potash (fertilizer)
NR Nitrate reductase
RGR Relative growth rate
ROS Reactive oxygen species
RWR Root weight ratio
SOD Superoxide dismutase
SRL Specific Root Length
SSL Specific Shoot Length
SWR Shoot weight ratio
xix
Summary
Salinity is a growing threat to crop production which affects sustainability of agriculture
in aridsemiarid areas Growth responses of plant to salinity vary considerably among
species Cajanus cajan Ziziphus mauritiana and Carissa carandas are sub-tropical crops
grown worldwide particularly in Asian subcontinent for edible and fodder purposes but
not much is known about their salinity tolerance and intercropping
Effect of salinity has been initially studied in present work at germination of C cajan
under different sea salt salinities using presoaked seeds with water and respective salt
solutions Seed germination decreased with increasing salinity and it was more sever in
presoaking under water of different salinities The 50 threshold reduction started at
ECiw= 35 dSm-1 sea salt in presoaking treatments However this threshold was decreased
up to ECiw= 168 dSm-1 sea salt at further seedling establishment stage Growth experiment
of C cajan in drum pot culture (Lysimeter) also showed a salt induced growth reduction
in which plant tolerate salinity up to 42 dSm-1 At this salinity leaf pigments (chlorophylls
and carotenoids) proteins and insoluble sugars decreased up to 50 whereas soluble
sugars were increased (~25) Reproductive growth was also affected at this salinity in
which at least 70 reduction in flowers pods and seeds were observed
Salt tolerance of symbiotic nitrogen fixing bacteria associated with root of C cajan
showed salinity tolerance up to ECw= 366 dSm-1 NaCl salinity invitro environment For
intercropping experiments Ziziphus mauritiana (grafted variety) was selected with C
cajan Preliminary investigations showed a growth promotion in Z mauritiana at low
salinity (ECe= 72 dSm-1) and growth was remained unaffected up to ECe= 111 dSm-1
Intercropping of C cajan with Z mauritiana was primarily done in drum pot (Lysimeter)
culture Result showed better growth responses of both species when growing together as
intercrops than sole in which encouraging results were found in 8th day irrigation interval
rather than of 4th day Biochemical parameters eg photosynthetic pigments protein
phenols electrolyte leakage and sugars of these species displayed increase or decrease
according to their growth responses Increased activity of antioxidant enzymes and that of
nitrate reductase and its substrate (NO3) also contributed in enhancement of growth
Field experiment of intercropping of above mentioned plants at marginal land
irrigated with underground water (Eciw= 28 dSm-1) showed better vegetative growth of
xx
both species than sole crop The overall reproductive growth remained unaffected
although the numbers size and weight of fruit were better in intercropping system
Photosynthetic pigments were mostly increased whereas leaf protein and sugars remained
unchanged In addition higher values of LER and LEC (gt 1) indicated the success of
intercropping system
Experiment on salinity tolerance of Carissa carandas (varn karonda) using drum
pots culture showed improvement at low salinity (up to ECiw= 42 dSm-1 sea salt) whereas
higher salinity (ECiw= 129 dSm-1 sea salt) adversely affected vegetative and reproductive
growth Plant managed to tolerate up to ECiw= 99 dSm-1 sea salt Salinity severely affected
biochemical parameters including photosynthetic pigments proteins and sugars whereas
leaf phenolics were increased Leaf accumulated high amount of Na+ whereas affect
absorption of essential minerals like K+ was decreased
In the light of above mentioned investigations it appears that C cajan can be
propagated in saline soils with good presoaking techniques in non-saline water which
would helped to grow at moderately saline conditions It could be a good option used as
intercrop species because of its ability to improve soil fertility even under water deficit
conditions The proposed Cajanus-Ziziphus intercropping system could help poor farmers
to generate income from unproductive soils by obtaining sufficient fodder from C cajan
for their cattle and producing delicious edible fruits from Z mauritiana for commercial
purposes Carissa carandas could also be introduced as new crop for producing fruits from
moderate saline waste lands and used for preparing prickle jam and jelly for industrial
purposes
xxi
لاصہ خ
کا عمل ے ں ب ڑھئ لف پ ودوں می ی ےمخ طرہ ہ
وا خ ا ہ ے ب ڑھی لئ داوار کے ی ں زرعی ب وں می
ر علاق ج
ن ی م ب
ر و ب ج ن کھاری پ ن کھاری پ ن ب
دا کروت ی ر اور ر ب ے ارہ ا ہ وت لف ہ ی ی مخ کاف ں ودگی می اص Subtropical کی موج ا اور خ ی و پ وری دب ں ج ی ں ہ صلی
کی ف طے
خ
وراک و ں ج می
ی ملکوں
ائ ی ش کھاکر ای کی ی ان پ ودوں کم لوگ ہ ہت کن ب ں لی ی ی ہ
وئ عمال ہ
ارے کے طور ب ر است ری پ ن سے خ
ں ی ے ہ ں علم رکھئ ارے می ے عمل کے ت گئ ے گائ
کر ا ھ ملا
ی سات ک ہ رواداری اور ات
وں ج ن ر کےب ے ارہ
ھگوئ ہلے سے ت ں ب کاز والے محلول می لف ارت ی
مک کے مخ
دری ں ں سمی ی مطالعہ می
دائ ی کھاری اب کا
کہ پ ن کے و ی ج وئ ع ہ
کمی واف ں ی ت می ب
کی طن وں ج ن
ھ ب ہ کے سات
اف ں اض کھاری پ ن می ا گی ا کی دہ اہ کا مش رات
iwEC =اب
1-35 dSm می خ ی کہ ت ی ج مک کے ب راب ررہ
دری ں زی سمی کا
ہ ارت ں ی ام می ی ت صدی dSm= iwEC 168-1پ ودوں کے ق
ق
ی ک رہ ں Lysemeterت ے والے پ ودوں می ڑھئ ں ب روان چ می 1-dSm 24 ں جوضلہ مک محلول می
دری ں زی سمی کا
ارت
ں کر می ر خل ب زب ر س ی
ات اور غ روز مادوں لمخی
گ اف الت ف کے رت ی ت
ائ ی ں ض کھاری پ ن می ی اس
گئ کھی
ت ت د زا ب رداش
ت صدی 05اف
ق
ی ش کم وب ں کر می ی کہ خل ب زب ر س ں 50کمی ج وں می ج ن
ھلی اورب ھول ت ں ت ن می ری ج دی ب ڑھوب ولی
ا پ ا رہ مات
ہ ں اف ت صدی اض
05ق
ی گئ کھی
ت ح طور د
کمی واض ت صدی
ق
ی وی شلک سہب ڑ سے می کی چ ر مک (Symbiotic)ارہ
کی ں ا رت ی
کٹ ی ے والے ب
کرئ مد خ
ن من روج ی
اب سے (NaCl)ت
ی ر کے سا dSmwEC 366 =-1رواداری ں ب ری ہ می ج ے عمل کے ت گئ ے
گائ
کر ا ھ ملا
ی سات ک ہ یات
گئ کھی
ت ک د ر ت ھ ارہ
ت
بی ق کے ب
حق ی ت دائ ی ا اب گی ا ی
کھاری پ ن کو ج کم ں ے می ج ں dSme (Ec 72 =-1(ن ی کہ می ری ج ں ب ڑھوب ی ر می e (Ec =ب
)1-111 dSm ہل ہلے ب ے عمل ب گئ ے
گائ
کر ا ھ ملا
ی سات ک ہ کو ات ر ی ر اور ب ی ارہ
ر رہ اب ر می ی
ک غ کی خد ت
Lysemeter ج ب رآم ت ا ی زا ب ی کے جوضلہ اف
اش ی ے سے آب
ف ف ھ دن کے و
سی ت آت
کی ی ار دن ی خ
گئ کی ں ں دمی ن می ے ج
وئ ہ
ے عمل گئ ے گائ
کر ا ھ ملا
ی سات ک ہ سی ت ات
کی ی ے پ ودوں
گائ
ن ہا ا کی پ ودوں ب شام
وں اق
ے دوپ ج گئ
ت ا ی زا ب ادہ جوضلہ اف ں زت می
ی ول ب ات ف روزمادوں لخمی
گ اف الت ف کے رت ی ت
ائ ی ضلاات می درخ ی می
ائ کی می ی
ائ ےجی
وئ Electrolyteب رآمد ہ
Leakage کی کر ں س ی وں می ب ی ان پ ودوںاور ب
ی ش کمی ب ں دار می ی دپ ں مق
ں دکھائ ر می
اظ ی ری کے ب
کے ب ڑھوب
xxii
Antioxidant ی ظرح سے ہ اور اس ہ اف ں اض کی سرگرمی وں می امروں
اور اس کے Nitrate Reeducatesخ
Substrate )3(NO ا ی کا سی ب ب ہ اف ں اض ما می وں
ش ھی ی
ت
ےdSmiw(Ec 28 =-1(معمولی گئ ے ئ کب راب ں سی ی می ائ ہ ت والے ت درج ں می ری ہ می ج
ی ت ئ ن ہا زمب کی ب الا پ ودوں
ے عمل گئ ے گائ
کر ا ھ ملا
ی سات ک ہ سی ت ات
کی ی ے پ ودوں
ادوں ب ر لگائ ی
ب ما ب وں
ش دی ی ولی
ے پ
وئ ج خاضل ہ
ت ا ی ر بہی ادہ ب ں زت می
ےض ر رہ ہی ں ب ام می ط ے ت گئ ے
گائ
کر ا ھ ملا
ی سات ک ہ شامت اور وزن ات عداد ج
کی ت ھلوں ی کہ ت ی ج ر رہ اب ر می ی
الت ف ی غ ی ت
ائ
ی وئ ں ہ ہی
ع ب ی دت لی واف ی ب
کوئ ں دار می کی مق کر
ات اور س ں لمخی ی وں می ب ی کہ ب ہ ج
اف ا اض مات
ں ں روزمادوں می
گ اف د کے رت LER مزت
ے LEC (gt1)اور ی ہ کرئ ارہ کی ظرف اس ی ائ کامی کی ام
ط ے ت گئ ے
گائ
کر ا ھ ملا
ی سات ری ات ک ہ
کی ب ڑھوب
ک دا کروت ں ری ہ می ج کھاری پ ن ) Lysemeterو کھاری پ ن روداری کے ت ا کم گی ا ں اگات iwEC = 142می
1-dSm ( کھاری پ ن ادہ ی کہ زت ی ج وئ ری ہ ہی ں ب مک( می
دری ں زی سمی کا
زی dSm= iwEC 129-1 ارت کا دری ارت سمی
ی وئ ر ہ
اب ری ب ری ظرح می
دی ب ڑھوب ولی
ی اور پ
ائ علی
ں ف مک( می
ی کہ ں ک dSm9= iw(Ec 9-1(ج مک ت
دری ں زی سمی کا
ارت
ت کب رداش ات اور س روز مادوں لخمی گ اف الت ف کے رت ی ت
ائ ی ضلاات می درخ ی می
ائ کی می ی
ائ ےجی اب رہ کامی ں ےمی
ر ب ری ظرح کرئ
ں ی وں می ب وا ب ہ ہ
اف ں اض ی ول می ب
ں ف ی وں می
ب ی کہ ب ں ج ی
وب ر ہ اب می
+Na ہ سے کی وج مع ی ج اف رلز کے K+اض روری می
ی سے ض ج
ی وئ ر ہ
اب کی ضلاجی ت می ے
کرئ زب چ
ا ت ق حق الا ت ہ ت درج ے ظر می
وئ ےہ
ھگوئ ں ت ی می
ائ ہلے سے ت کہ ب ی
ے آئ مئ ں ی ہ ت ات سا ی می
ئ کی روش ر ت ہ سے ارہ کی وج ے
ت ف
ھی مدد دے س ں ت ے می گئ ں ا ن می ن زمی مکی دل ں وکہ معی ے ج ا ہ اسکی ا خ ھی لگات
ں ت ن خالات می مکی کو ں وں ج ن
وزہ کے ب ے مج ا ہ کی
داواری ی ر ب ی ے عمل غ گئ ے
گائ
کر ا ھ ملا
ی سات ک ہ ی ر ات ر اور ب ی ضلاجی ت والی ارہ
اف ے اض لئ وروں کے
اپ کی صور ت خ ر ن ارہ زمی
ھی دا ت کروت ے ا ہ وسکی ت ہ اب کا ذرت عہ ت ے ی ب ڑھائ
کی آمدئ وں
کشاپ ی صورت
ارئ ح کی ت ل
ھ ی ت وردئ دار ج ی ر سے مزت ارہ اور ب ی خ
عئصت
صل کے طور ب ی ف ئے ب لئ ے کے
کرئ دا ی ھل ب ن سے ت کارآمد زمی ر ی
ن اور غ مکی
دل ں ے معی
لئ اضد کے ے رمق ا ہ اسکی ا خ کی ی ش ب
1
General Introduction
Intercropping is a major resource conservation technique for sustainable agriculture under
various climatic conditions (Zhang et al 2010 Li et al 2014) It can reduced operational
cost for the production of multiple crops with maintained or even higher level of
productivity (Vandermeer 2010 Perfecto and Vandermeer 2010) It can enhance the
water use efficiency by saving 20 to 40 irrigation water with improved fertilizer
management (Fahong et al 2004 Jat et al 2005 Jani et al 2008) Intercropping system
is more suitable in marginal areas with lower mechanization and cultivation input by
farmers on small tracts of farmlands (Ngwira et al 2012) It can enhance the cumulative
production per unit area and protect the small farmers against market fluctuations or crop
failure ensure the income improve soil fertility and food demands (Rusinamhodzi et al
2012) In this system dominating more compatible and productive species are selected or
replaced in which complementarity effects and beneficial interactions resulting enhanced
yield as compared to monoculture (Huston 1997 Loreau and Hector 2001) It was
estimated that in species diverse systems biomass production is 17 times higher as
compared to monoculture (Cardinale et al 2007)
It is suggested that intercropping is the best suitable cropping system which can
improve the resource-use efficiency by procurement of limiting resources enhanced
phyto-availability and effective plants interactions (Marschner 2012 White and
Greenwood 2013 Ehrmann and Ritz 2014) It is widespread in many areas of world
particularly in latin America it is estimated about 70-90 by small farmers which mainly
grow maiz potatoes beans and other crops under this system whereas intercropping of
maiz with different crops is estimated about 60 (Francis 1986) Additionally
agroforestry is more than 1 billion ha in this area (Zomer et al 2009) The land used for
intercropping system of various crops is greatly varied from 17 in India to 98 in Africa
(Vandermeer 1989 1992 Dupraz and Liagre 2011)
In intercropping system two or more crops or genotypes coexist and growing
together at a same time on a similar habitat (Li et al 2013) It may be divided into various
types such as in mixed intercropping system two or more crops simultaneously growing
without or with limited distinct arrangements whereas in relay intercropping system
second crop is planted when the first is matured while in strip intercropping both the crops
2
are simultaneously growing in strips which can facilitate the cultivation and crop
interactions (Ram et al 2005 Sayre and Hobbs 2004)
Several less-conventional fruit tress including Manilkara zapota (Chicko)
Ziziphus mauritiana (Jujubar) Carissa carndas (Karanda) Annona squamosa (Sugar
apple) and Grewia asiatica (Falsa) has been reported with high nutritional value with
capability to grow at marginal lands (Mass and hoffman 1997) Qureshi and Barrett-
Lennard (1998) suggested few grafted plants that can widely use to improve the quality
and productivity of fruits Grafting is also used to induce stress tolerance in plants against
various abiotic and biotic stresses including salinity stress (Rivero et al 2003) Both root
stocks and shoot stocks contribute to increase the tolerance level of plants Root stocks
represent the first part of defense to control the uptake and translocation of nutrients and
salts throughout the plant (Munns 2002 Santa-cruz et al 2002 Zrig et al 2011) while
shoot stocks develops physiological and biochemical changes to promote plant growth
under stress conditions (Moya et al 2002 Chen et al 2003)
Ziziphus mauritiana Lamk (varn grafted ber) belongs to the family Rhamnaceae
grows widely in most of the dry tropical and subtropical regions around the world Various
grafting methods are used for their propagation including wedge and whip or tongue
methods (Nerd and Mizrahi 1998) Intercropping of these grafted fruit trees with various
leguminous crops is also being successfully practiced in many countries thought the world
Leguminous crops are considered excellent symbiotic nitrogen fixing crops It can
effectively improve soil fertility and offset the critical problems of sub-tropical areas to
fight against desertification and soil degradation These plants are considered as an
excellent source of proteins for humans and animals They can fix the 90 of atmospheric
nitrogen and contribute 40 nitrogen to the soil thus increase the soil fertility (Peoples et
al 1995) However most of the leguminous plants are not salt tolerant while some
species are better drought tolerant and effectively contribute in marginal lands (Zahran
1999)
Among the leguminous plants Pigeon pea (Cajanus cajan (L) Millspaugh) of the
family Fabaceae is widely grown for food fodder and fuel production particularly in
semiarid areas The salinity tolerance of this specie is not well documented both at
germination and seedling stages This crop is still underexploited due to its edible and
3
economic importance While limited investigations has been made to uncover its
nutritional quality medicinal uses and drought tolerance
The identical physiological traits are important in both the mono and intercropping
systems to maximize the resource acquisition The exploitation of best possible
combination of traits of different plants in intercropping system is very important to
maximize the overall performance in intercropping system It depends on the above ground
beneficial plant interactions for light space and optimal temperatures (Wojtkowski 2006
Zhang et al 2010 Shen et al 2013 Ehrmann and Ritz 2014) as well as the
complementary below ground plant interactions with soil biotic factors (Bennett et al
2013 Li et al 2014)
Water is also a major limiting factor intercropping can enhanced the acquisition
of water by root architecture and distribution in the soil profile for effective utilization of
rainfall (Zegada-Lizarazu et al 2006 De Barros et al 2007) and enhanced the water use
efficiency for effective hydraulic redistribution by deep rooted crops and water stored in
the soil profile (Morris and Garrity 1993 Xu et al 2008) Mycorrhizal networks around
the roots of intercrop plants also enhanced the availability of water and available resources
and reduced the surface runoff (Caldwell et al 1998 Van-Duivenbooden et al 2000
Prieto et al 2012)
Intercropping with leguminous plants can enhanced the agricultural productivity in
less productive soils due to enhanced nitrogen availability and also improved the soil
fertility by effective nitrogen fixation (Seran and Brintha 2010 Altieri et al 2012) Due
to weaker soil nitrogen competition intercropping with legumes enhanced the nitrogen
availability to the non-leguminous intercrop which also absorbs the additional nitrogen
released in the soil or root nodules of the leguminous plant (Li et al 2013 White et al
2013a) The use of legumes in many intercropping systems is pivotal According to the
listing of Hauggaard-Nielsen and Jensen (2005) seven out of ten are the legumes among
the most frequently used intercrops around the world
The ecological range of adaptability of legumes reaches from the inner tropics to
arctic regions with individual species expressing tolerance to drought temperature
nutrient deficiency in soil water logging salinity and other environmental conditions
(Craig et al 1990 Hansen 1996) The woody perennial leguminous plants have a number
4
of purposes they can be used to reclaim degraded wastelands retard erosion and provide
shade fuel wood timber and green manure (Giller and Wilson 1991)
Trees with nitrogen fixing capability play an important role to offset the critical
problems of tropical and sub-tropical regions in their fight against desert encroachment
and soil impoverishment These plants are capable to live in N-poor soils through their
association with Rhizobium that fix atmospheric nitrogen Nitrogen fixing activity in the
field depends both on their N2-fixing potential and on their tolerance to existing
environmental stresses (Galiana et al 2002) Symbiotic N2 fixation in leguminous plants
can mainly be considered an excellent source of protein supply for human and animal
consumption They range from extensive pasture legumes to intensive grain legumes and
are estimated to contribution up to 40 of their nitrogen to the soil (Simpson 1987)
The traits in the monocropping system in the selected crop extensively exploit the
acquisition of limiting resources in the environment and continuously focused on the
availably of similar resources for the successful crop production (White et al 2013 ab)
whereas in intercropping with different crops cycling of resources can be optimized to
the complementarity or facilitation traits (Costanzo and Barberi 2014) to overcome
resource limitations during the growing season (Hill 1996 George et al 2014)
For the long term sustainable agriculture and food production in resource limiting
areas with lower input Intercropping systems have the potential to increase the
productivity With efficient mechanization cultural practices and optimized nutrient
management rapid improvements are also possible through this system In future
perspective intercrops with higher resource use efficiency through plant breeding and
genetics is likely to be the most effective option for sustainable agriculture and
development
Increase of world population and demand of additional food production
The demand and production gap of food fodder fuel wood and livestock products is
increasing day by day due to global population which will increase from about 7 billion
(FAO 2014) to 9 billion by 2050 (Haub 2013) The increasing urbanization further
intensifies the problem which will increase from 54 to 66 expected in 2050 (UN
2014) Majority of this rise in urbanization will occur in developing countries around the
5
globe The major problem is to meet the challenge of increasing food demand for this ever
growing population up to 70 more food crops to feed the additional 23 billion population
worldwide by 2050 (FAO 2010 2011) Hence there is great need to increase the re-
vegetation for fuel wood and fodder production (Thomson 1987) An increase in
production could be envisaged through increasing the yield of already productive land or
through more extensive use of unproductive land The high concentration of salts in soil
or water does not let the conventional crops grow and give feasible economic return
Hence it is necessary to search for unconventional crops for foods fodder and fuel which
could give profitable yield under saline conditions (Ahmad and Ismail 1993) Reclamation
of this land through chemical and engineering treatments is very expensive The most
appropriate use of saline wasteland is the production of high yielding salt tolerance fuel
wood timber and forage species (Qureshi et al 1993) Therefore the most attractive
option is to screen a range of species and identify those which have potential of being
commercially valuable for the degraded environments (Ismail et al 1993)
Pakistan is in semi-arid region and the 6th most populated county of the world
Population drastically increased in Pakistan which was 80 million in 1980 and annual
increase in population is about 4 million (UNDES 2011) This is continuously
overburdened and it is estimated that in 2025 it will reach to 250 million and 335 million
in 2050 which decrease the available water per capita to less than 600 m3 resulting 32
shortfall of water requirements causing an alarming condition particularly for Pakistan
Furthermore this shortfall in 2050 leading to severe food shortage upto 70 million tones
which indicates the further development and serious measures for the new resources
(ADB 2002) Subsequent severe food and fodder crises along with all the resource
limitations with continuous increase in urbanization from the current 35 to 52 in 2025
will further intensity the agriculture production and demand
Shortage of good quality irrigation water
On earth surface the major resources of available fresh water is deposited in the form of
ponds lakes rivers ice sheets and caps streams and glaciers whereas underground water
as underground streams and aquifers With the drastic increase in population the water
consumption rise as the twice of the speed of population growth The scarcity of water is
widespread to many countries of different regions Majority of population in developing
countries suffering from seasonal or year round water shortage which will increase with
6
expected climatic changes Currently almost 50 countries around the globe are facing
moderate to severe shortage of water
Due to the greenhouse effect it is estimated that since the start of 20th century 14
degF temperature is already risen which will likely rise at least another 2degF and over the next
100 years it is estimated about more than 11degF due to the consequences of biogenic gases
(El-Sharkawy 2014) This is mainly due to the product of human activities including
industrial malpractices excess fossil fuel consumption deforestation poor land use and
cultural practices
Rising in atmospheric CO2 concentration which probably reached 700 μmol (CO2)
molminus1 resulting severe climatic changes It will accelerate the melting of ice and glacier
resulting the rising rainfall and storms in tropics and high latitude consequently 06 to 1
meter rise in sea level on the expense of costal lowlands across the continents After this
initial high flows the decrease in inflow was very terrifying Due to these climatic changes
humans suffering from socioeconomic changes including degradation of lands with lower
agricultural output and degradation of natural resources will further enhanced the poverty
and hunger resulting dislocation and human migrations (Randalls 2010)
In the mean while scarcity of good quality water is increasing day by day with the
demands of water for domestic agricultural and industrial utilization which will further
increase up to 10 of the total available resources as estimated by 2025 which needs
serious water managements (Bhutta 1999) It is very challenging for the modern
agriculture to ensure the increasing demand of more arable and overburdened population
with the limiting resources including the unavailability of good quality water and
deterioration of even previously productive land (Du et al 2015)
In Pakistan Indus River basin is the back bone of agriculture and socioeconomic
development which contributes 65 of the total river flows and 90 for the food
production with a share of 25 to the GDP It is estimated that about 30-40 of its surface
storage capacity will reduce by 2025 due to siltation of reservoirs and climatic changes It
will impose serious threat to irrigated agriculture in near future consequently with
decreases in groundwater resources resulting shortage of fresh water and 15-20
reduction in grain yield in Pakistan (World Bank 2006)
7
Spread of saline soil and reduction in agricultural yield
Along with scarcity of water soil salinity is one of the major environmental stresses which
severely threaten the agriculture The damages of salinity is widespread around the world
which is so far effected the more than 800 million hectare (more than 6) of land
worldwide including 397 million ha by salinity associated with 434 million ha by sodicity
(FAO 2010) The out of total 230 million hactares of irrigated land more than 45 million
hactares (20) is so far effected by salinity which is about the 15 of total cultivated land
(Munns and Tester 2008)
In Pakistan out of 2036 million hectares of cultivated land more than 6 million
hectares is affected by salinity and water logging of various degrees (Qureshi et al 2004)
About 16 million hectares of tropical arid plains which have been put under crop
cultivation depend extensively on canal irrigation network This area (about 60) is now
seriously affected by water logging and salinity (Qureshi et al 2004) The rise of subsoil
water levels accompanied by its subsequent decline due to irrigation combined with
insufficient drainage has led to salinization of valuable agricultural land in arid zones all
over the world (Ahmad and Abdullah 1982) The dominated cation in salt-affected soil is
Na+ followed by Ca2+ and Mg2+ while the anions Cl and SO4 are almost equal in
occurrence (Qureshi et al 1993) Salt content varies in different regions of the salt-
affected areas but at certain sites could reach up to an ECe of 90-102 dSm-1 (Ahmad and
Ismail 1993)
Salinity is a chief anxiety to meet the ever growing demands of food crops Salinity
adversely affects the plant growth and productivity Plants differentially respond to salt
stress and categories into four classes Salt sensitive moderately salt sensitive moderately
salt tolerant and highly salt tolerant plants on the basis of their tolerance limits Whereas
mainly plants are divided into halophytes (salt tolerant) and glycophytes (salt sensitive) on
the basis of adaptive evolution (Flowers 2004 Munns and Tester 2008) Unfortunately
majority of cultivated crops are not able to withstand in higher salinity regimes and
eventually die under higher saline conditions which proposed serious attentions to manage
the dissemination of salinity (James et al 2011 Rozema and Flowers 2008)
Excessive accumulation of salts in rhizosphere initially reduced the water
absorption capacity of roots leading to hyperosmotic stress followed by specific ion
8
toxicity (Munns 2008 Rahnama et al 2010) Plants initially manage the overloaded salt
by various excluding and avoidance mechanisms depending on their tolerance levels The
management of salt inside the cytosol is depends on the compartmentalization capacity of
plants followed by osmotic adjustments and efficient antioxidant defense mechanisms
Whereas higher salt beyond the tolerance impose injurious effects on various
physiological mechanisms These are including disruption of membrane integrity
increased membrane injuries nutrient ion imbalances osmotic disturbance
overproduction of reactive oxygen species (ROS) compromised photosynthesis and
respiration due to stomatal closure and damages of enzymatic machinery (Munns and
Tester 2008) In specific ion toxicity Na+ and Cl- are the chief contributors in
physiological disorders Excessive Na+ in rhizosphere antagonize the uptake of K+
resulting lower growth and productivity (James et al 2011) Salt load in the cytosol trigger
the overproduction of ROS including H2O2 OH- super oxides and singlet oxygen They
are involved in sever oxidative damages to various vital cellular components including
DNA RNA lipids and proteins (Apel and Hirt 2004 Ahmad and Umar 2011)
Strategies to cope up the salinity problem
The development and cultivation of highly salt tolerant crop varieties for salt affected areas
is the major necessity to meet the future demands of food production whereas the majority
of available food crops are glycophytes Therefore it is an emergent need of crop
improvement methods which are more efficient cost effective and grow on limiting
resource The use of poor quality water for irrigation is also very important under the
proposed shortage of fresh water in near future For the development of salt tolerant
varieties more understanding of stress mechanisms are required at whole plant molecular
and cellular levels
The variability in stress tolerance of salt sensitive genotypes (glycophytes) and
highly salt tolerant plants (halophytes) showed genetic basis of salt tolerance It indicate
that salt tolerance is a multigenic trait which involves variety of gene expressions and
related mechanisms Salt stress induces both the qualitative and quantitative changes in
gene expression (Manchanda and Garg 2008) These multigenetic expressions play a key
role in upregulation of various proteins and metabolites responsible for the management
of anti-stress mechanisms (Bhatnagar-Mathur et al 2008) Plant breeding and transgenic
strategies are intensively used for decades to improve the crop performance under salinity
9
and aridity conditions Few stress tolerant varieties are so far released for commercial
production whereas in natural condition where plant exposed to variety of climatic
conditions the overall performance of plant have changed as compared to controlled in
invitro conditions (Schubert et al 2009 and Dodd and Perez-Alfocea 2012) The success
stories about transgenic approaches for crop improvement under stressful environments
are still very scanty because of the insufficient understanding about the sophisticated
mechanisms of stress tolerance (Joseph and Jini 2010) It indicates that there is less
correlation between the assessment of stress tolerance in invitro and invivo conditions
Although there have been some achievement in this connection in some model plants
including rice tobacco and Arabidopsis (Grover et al 2003) which proposed the
possibilities of success in other crops in future Variety of technicalities and associated
financial challenges are still associated with this strategy
In conventional cultivation practices continuous irrigation with poor quality water
can enhanced the salinization due to evapotranspiration leading to increased saline andor
sodic soils This problem can be cope up by intercropping system in which high salt
tolerant or salt accumulator plants are intercropped with salt sensitive crops which can
accumulate salt thus can reduce the risk of salt increment in soil Additionally better
cultivation practices including the micro-jet or drip irrigation and partial root zone drying
technique is also very fruitful to optimize the water requirements and avoid the risks
associated with conventional flooding irrigation system
In dry land agriculture plantation of deep rooted perennials during off season or
annuals can reduced the risk of salinization They continuously grown and utilize excess
amount of water create a balance between water utilization and rail fall Thus prevent the
chance of salt accumulation on soil surface due to increased water table and
evapotranspiration (Manchanda and Garg 2008) The efficient irrigation and
intercropping strategy is seemed quite attractive cost effective and very beneficial in less
mechanized poor marginal areas It can ameliorate the injurious effects of salinity and
increased production per unit area thus ensure the sustainable agriculture in semi-arid or
marginal lands (Venkateswarlu and Shanker 2009)
A number of plant species are available that are highly compatible with saline
sodic and marginal lands The cultivation of these species with proposed intercropping
system is economically feasible to grow in marginal soil Some plants including Carissa
10
carandus Ziziphus mauritiana and Cajanus cajan was selected to revealed their potential
for intercropping under saline marginal lands These are important plants which can
established well at tropical and subtropical arid zone under high temperatures Hence their
range of salt tolerance and suitability for cultivation at waste saline land or with saline
water irrigation is being undertaken for commercial exploitation
Objective of present investigation
The plan of present investigation has been worked out to look into possibility of increasing
production of an unconventional salt tolerant fruit tree (Z mauritiana) by intercropping
with a legume ( C cajan) which apart from increasing fertility of soil could be able to
provide fodder for grazing animals from salt effected waste land Possibility of making
use of saline water for irrigation has also been considered for growing leguminous plant
(C cajan) and salt tolerant unconventional fruit tree (Crissa carandas) under saline
condition
11
LAYOUT OF THESIS
Chapter 1 Monoculture of Cajanus cajan (Vern Arhar) and Ziziphus mauritiana
(Varn Ber) under different range of salinities created by irrigation of
various sea salt concentrations
A Experiments on Cajanus cajan
Following experiments were performed under A
Experiment No 1 Effect of Pre-soaked seeds of C cajan in distilled water for
germination in water of different sea salt concentrations
Experiment No 2 Effect of Pre-soaked seeds of C cajan in various dilutions of sea salt
for germination in water of respective sea salt concentrations
Experiment No 3 Seedling establishment experiment of C cajan on soil irrigated with
sea salt of different concentrations
Experiment No 4 Growth and development of C cajan in Lysimeter (Drum pot culture)
being irrigated with water of different sea salt concentrations
Experiment No 5 Range of salt tolerance of nitrogen fixing symbiotic bacteria
associated with root of C cajan
B Experiments on Ziziphus mauritiana
Experiment No 6 Growth and development of Z mauritiana in large size clay pot being
irrigated with water of two different sea salt concentrations
Discussion (Chapter 1)
Chapter 2 Intercropping of Ziziphus mauritiana with Cajanus cajan
Experiment No 7 Physiological investigations on Growth of Ziziphus mauritiana and
Cajanus cajan intercropped in drum pot (Lysimeter) culture being
irrigated with water of sea salt concentration at two irrigation
intervals
Experiment No 8 Investigations of intercropping Ziziphus mauritiana with Cajanus
cajan on marginal land under field conditions
12
Discussion (Chapter 2)
Chapter 3 Investigations on rang of salt tolerance in Carissa carandas (varn
karonda) for determining possibility of growing at waste saline land
Experiment No 9 Investigation on the effect of higher range of salinities on growth of
Carissa carandas (varn karonda) created by irrigation of different
dilutions of sea salt
Discussion (Chapter 3)
13
1 Chapter 1
Monoculture of Cajanus cajan (Vern Arhar) and Ziziphus mauritiana
(Varn Ber) under different range of salinity created by irrigation of
various sea salt concentrations
11 Introduction
Scarcity of good quality water enforced the growers to irrigate the crops with
lowmoderately saline water at marginal lands which ultimately enhance soil salinity due
to high evapo-transpiration (Azeem and Ahmad 2011) To overcome this situation people
are now focusing on less-conventional plants which can grow on resource limited areas
and can produce edible biomass for human and animal consumption
Ziziphus mauritiana (varn grafted ber) is salt and drought tolerant plant which can
grow on marginal and degraded land (Morton 1987) It has wide spread crown and a short
bole fast growing tree with average bearing life of 25 years The ripe fruit (drupe) is juicy
hard or soft sweet-tasting pulp has high sugar content vitamins A amp C carotene
phosphorus and calcium (Nyanga et al 2013 2008 Pareek 2013) The leaves contain 6
digestible crude protein and an excellent source of ascorbic acid and carotenoids The
leaves are used as forage for cattlesheepgoats and also palatable for human consumption
(Sharma et al 1982 Bal and Mann 1978 Agrawal et al 2013) The timber is very hard
can be worked to make boats charcoal and poles for house building Roots bark leaves
wood seeds and fruits are reputed to have medicinal properties The tree also used as a
source of tannins dyes silk (via silkworm fodder) shellac and nectar (Dahiru et al 2006
Chrovatia et al 1993 Gupta 1993)
Some atmospherics nitrogen fixing bacterial associated deep rooted drought
tolerent leguminious plants like Cajanus cajan can fix up to 200 Kg nitrogen ha-1 year-1
due to symbiotic association of Rhizobium with its deep penetrating roots (Bhattacharyya
et al 1995) Total cultivated area of Pigeon pea is about 622 million hectare and global
annual crop production is around 474 million tonnes whereas total seed production of
this crop is about 015 million tonnes (FAOSTAT 2013) Its seeds are an excellent source
of good quality protein (up to 24) and foliage is used as animal fodder with high
nutritional value (Pandey et al 2014) Besides being used as food and fodder this plant
14
also have therapeutic value and it is used against diabetes fever dysentery hepatitis and
measles (Grover et al 2002) It also use traditionally as a laxative and was identified as
an anti-malarial remedy beside other medicinal species (Ajaiyeoba et al 2013 Qasim et
al 2010 2011 2014)
Following experiments were conducted to evaluate the seed germination seedling
establishment and growth of C cajan as well as grafted sapling of Z mauritiana under
various salinity regimes Investigations were also undertaken to find-out of their
intercropping has any beneficial effect on growth at marginal saline land saline
environment
15
12 Experiment No 1
Effect of Pre-soaked seeds of Cajanus cajan in distilled water for
germination in water of different sea salt concentrations
121 Materials and methods
1211 Seed collection
Seeds of C cajan were purchased from local seed market Mirpurkhas Sindh and were
tested to determine the effect of salinity on germination at the biosaline laboratory Botany
department Karachi University Karachi The best lot of healthy seeds having 100
germination was selected for further experiments
1212 Experimental Design
Seeds of C cajan were surface sterilized with 01 sodium hypochlorite solution for 2-3
minutes washed in running tap water then soaked in sterilized distilled water for one hour
(Saeed et al 2014) Sterilized glass petri plates (9cm) lined with filter paper were moist
with 10 ml of distilled water at different saline water of different sea salt concentrations
and their germination percentage was observed Their electrical conductivities on these
sea salt dilutions are mentioned in Table 11 Three replicates were used for each treatment
Ten seed were placed in each petri plate which were kept in temperature controlled
incubator (EYELA LTI-1000 Japan) at 28 plusmn 1ordmC in dark Experiment was continued for 7
days Data were recorded on daily bases Analyses of varience by using repeated measures
and the significant differences between treatment means were examined by least
significant difference (Zar 2010) All statistical analysis was performed using SPSS for
windows version 14 and graphs were plotted using Sigma plot 2000
Germination percentage of C cajan was recorded every 24 hours per seedling
evaluation procedure up to 07 days The final percent germination related with salinity in
accordance with Maas and Hoffman (1977) The percent germination was calculated using
the following formula (Cokkizgin and Cokkizgin 2010)
16
Germination index for C cajan was recorded according to AOSA (1990) by using
following formula
Where Gt is the number of germinated seed on day t and Dt is the total number of
days (1 - 7)
Coefficient of germination velocity of C cajan was calculated described by Maguire
(1962)
Where G represents the number of germinated seeds counted per day till the end of
experiment
Mean germination time of C cajan was calculated by Ellis and Roberts (1981) by
using following formula
Where lsquonrsquo is the number of germinated seeds in day d whereas Σn is the total
germinated seeds during experimental period
Germination rate was of C cajan determined according to following formula
(Shipley and Parent 1991)
Where numbers of germinated seeds were recorded from 1 to 7
17
122 Observations and Results
Cajanus cajan (imbibed in distilled water) grown at different salinity regimes showed 50
reduction at 16 salt concentration corresponding ECiw 168 dSm-1 (Table 1 2 Appendix
I)
Rate of germination was inversely correlated with sea salt concentration It was
significantly (p lt 0001) decreased from first day to final (day 7) of observation Higher
germination rate was recorded in control and at lower concentrations of sea salt in early
days of seed incubation with contrast to higher concentrations of sea salt which was
reduced with increasing day of incubation (Table 13 Appendix I)
A significant decrease (p lt 0001) in coefficient of germination velocity was
observed with increasing salinity (Table 14 Appendix I)
A significantly increase (p lt 0001) in mean germination time of seeds was observed
with increasing sea salt concentrations However the difference was insignificant at lower
salinities (Table 14 Appendix I)
A significant decrease (p lt 0001) in mean germination index was observed with
increasing salt concentrations except lower salinities More reduction was observed
byhond 16 and onward sea salt concentration (Table 14 Appendix I)
18
Table 11 Electrical conductivities of different sea salt solutions used in germination of C cajan
Sea salt () ECiw (dSm-1)
Non saline control 06
01 09
02 16
03 35
04 42
05 58
06 62
07 79
08 88
09 99
10 101
11 112
12 128
13 131
14 145
15 159
16 168
ECiw is the electrical conductivity of irrigation water measured in deci semen per meter
19
Table 12 Effect of irrigation water of different sea salt solutions on germination percentage (GP) per day
of C cajan seeds pre-soaked in non-saline water prior to germination with duration of time under
various salinity regimes
Sea Salt
(ECiw= dSm-1)
GP
1st day
GP
2nd day
GP
3rd day
GP
4th day
GP
5th day
GP
6th day
GP
7th day
Control 8333plusmn667 90plusmn00 9333plusmn333 9333plusmn333 9333plusmn333 9333plusmn333 9333plusmn333
09 8667plusmn333 9333plusmn333 9667plusmn333 9667plusmn333 100plusmn00 100plusmn00 100plusmn00
16 7667plusmn667 80plusmn10 8333plusmn882 8333plusmn882 8333plusmn882 8333plusmn882 8667plusmn667
35 6667plusmn333 9333plusmn333 9333plusmn333 9333plusmn333 9333plusmn333 9333plusmn333 9333plusmn333
42 70plusmn00 8667plusmn333 90 plusmn00 90 plusmn00 90 plusmn00 90 plusmn00 90 plusmn00
58 6333plusmn667 7333plusmn333 8333plusmn333 90 plusmn00 90 plusmn00 90 plusmn00 90 plusmn00
62 5667plusmn667 80plusmn577 90 plusmn00 90plusmn00 90 plusmn00 90 plusmn00 90plusmn00
79 5333plusmn333 70plusmn00 8333plusmn333 8333plusmn333 8333plusmn333 8333plusmn333 8333plusmn333
88 4000plusmn00 6667plusmn667 8333plusmn333 8333plusmn333 8333plusmn333 8333plusmn333 8333plusmn333
99 2667plusmn333 60 plusmn00 90 plusmn00 90plusmn00 90 plusmn00 90 plusmn00 90 plusmn00
101 2333plusmn333 70plusmn577 7333plusmn333 7333plusmn333 7333plusmn333 7333plusmn333 7333plusmn333
112 70plusmn577 7667plusmn333 80plusmn00 8333plusmn333 8333plusmn333 8333plusmn333 8333plusmn333
128 6667plusmn333 6667plusmn333 6667plusmn333 6667plusmn333 6667plusmn333 6667plusmn333 6667plusmn333
131 3333plusmn882 50plusmn00 5333plusmn333 5333plusmn333 5333plusmn333 5333plusmn333 5667plusmn333
145 3333plusmn667 40 plusmn00 50 plusmn577 50plusmn577 50 plusmn577 5333plusmn333 5333plusmn333
156 3667plusmn667 40plusmn577 4667plusmn882 4667plusmn882 50plusmn577 50plusmn577 5333plusmn667
168 1667plusmn882 3333plusmn333 3333plusmn333 3333plusmn333 3667plusmn333 3667plusmn333 4333plusmn333
LSD 005 Salinity 18496
Time (days) 13322
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and LSD among the treatments is recorded at p lt 005
20
Table 13 Effect of irrigation water of different sea salt solutions on germination rate (GR) per day
of seeds C cajan pre-soaked in non-saline water prior to germination with duration of
time under various salinity regimes
Sea Salt
(ECiw= dSm-1)
GR
1st day
GR
2nd day
GR
3rd day
GR
4th day
GR
5th day
GR
6th day
GR
7th day
Control 833plusmn067 450plusmn00 311plusmn011 233plusmn008 187plusmn007 156plusmn006 133plusmn005
09 867plusmn033 467plusmn017 322plusmn011 242plusmn008 200plusmn00 167plusmn00 143plusmn00
16 767plusmn067 400plusmn050 278plusmn029 208plusmn022 167plusmn018 139plusmn015 124plusmn010
35 667plusmn033 467plusmn017 311plusmn011 233plusmn008 187plusmn007 156plusmn006 133plusmn005
42 700plusmn00 433plusmn017 300plusmn00 975plusmn750 180plusmn00 150plusmn00 129plusmn00
58 633plusmn067 367plusmn017 278plusmn011 225plusmn00 180plusmn00 150plusmn00 129plusmn00
62 567plusmn067 400plusmn029 300plusmn00 225plusmn00 180plusmn00 150plusmn00 129plusmn00
79 533plusmn033 350plusmn00 278plusmn011 208plusmn008 167plusmn007 139plusmn006 119plusmn005
88 400plusmn00 333plusmn033 278plusmn011 208plusmn008 167plusmn007 139plusmn006 119plusmn005
99 267plusmn033 300plusmn00 300plusmn00 225plusmn00 180plusmn00 150plusmn00 129plusmn00
101 233plusmn033 350plusmn029 244plusmn011 183plusmn008 147plusmn007 122plusmn006 105plusmn005
112 700plusmn058 383plusmn017 267plusmn00 208plusmn008 167plusmn007 139plusmn006 119plusmn005
128 667plusmn033 333plusmn017 222plusmn011 167plusmn008 133plusmn007 111plusmn006 095plusmn005
131 333plusmn088 250plusmn00 178plusmn011 133plusmn008 107plusmn007 089plusmn006 081plusmn005
145 333plusmn067 200plusmn00 167plusmn019 125plusmn014 100plusmn012 089plusmn006 076plusmn005
156 367plusmn067 200plusmn029 156plusmn029 117plusmn022 100plusmn012 083plusmn010 076plusmn010
168 167plusmn088 167plusmn017 111plusmn011 083plusmn008 073plusmn007 061plusmn006 062plusmn005
LSD 005 Salinity 0481
Time (days) 0378
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and LSD among the treatments is recorded at p lt 005
21
Table 14 Effect of irrigation water of different sea salt solutions on mean germination rate (GR)
coefficient of germination velocity (GV) mean germination time (GT) mean
germination index (GI) and final germination (FG) of C cajan seeds pre-soaked in non-
saline water prior to germination under various salinity regimes
Sea Salt
(ECiw= dSm-1) GR GV GT GI FG
Control 2624plusmn100 369plusmn005 027plusmn00 2624plusmn100 9667plusmn333
09 2743plusmn063 365plusmn009 027plusmn001 2743plusmn063 100plusmn00
16 2398plusmn218 423plusmn036 024plusmn002 2398plusmn218 8333plusmn882
35 2467plusmn086 378plusmn005 026plusmn00 2467plusmn086 9333plusmn333
42 3169plusmn733 311plusmn058 035plusmn008 3169plusmn733 9333plusmn333
58 2264plusmn081 399plusmn015 025plusmn001 2264plusmn081 90plusmn00
62 2253plusmn073 400plusmn013 025plusmn001 2253plusmn073 9333plusmn333
79 2074plusmn081 402plusmn00 025plusmn00 2074plusmn081 8333plusmn333
88 1927plusmn043 449plusmn008 022plusmn00 1927plusmn043 90plusmn577
99 1853plusmn033 486plusmn009 021plusmn00 1853plusmn033 90plusmn00
101 1635plusmn056 470plusmn022 021plusmn001 1635plusmn056 8667plusmn882
112 2263plusmn042 369plusmn020 027plusmn001 2263plusmn042 9667plusmn333
128 1953plusmn098 341plusmn00 029plusmn00 1953plusmn098 9667plusmn333
131 1368plusmn059 440plusmn018 023plusmn001 1368plusmn059 6667plusmn333
145 1276plusmn099 446plusmn019 023plusmn001 1276plusmn099 60plusmn577
156 1289plusmn153 447plusmn030 023plusmn002 1289plusmn153 8000plusmn100
168 876plusmn104 589plusmn078 018plusmn002 876plusmn104 8667plusmn333
LSD005 5344 3312 0064 5344 1313
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and LSD among the treatments is recorded at p lt 005
22
13 Experiment No 2
Effect of Pre-soaked seeds of Cajanus cajan in various dilutions of sea
salt for germination in water of respective sea salt concentrations
131 Materials and methods
1311 Seed germination
Procedure of seed germination has been mentioned in Experiment No 1 earlier The seeds
were pre-soaked in various sea salt concentrations instead of non-saline water and
germinated in respective sea salt concentrations Their electrical conductivities mentioned
in Table 15 Data were calculated and analysed according to formulas given in Experiment
No 1
Since these pre-soaked seeds in different sea salt concentration showed 50
germination at 03 equivalent to ECiw= 42dSm-1 sea salt solution any further work
beyond ECiw= 42dSm-1was not continued
132 Observations and Results
The final percent germination related with salinity in accordance with Maas and
Hoffman (1977) linear relative threshold response model as follows
Relative Final Germination = 100-200 (Ke ndash 005)
Where threshold salt concentration was 005 and Ke is the concentration of salts
at which relative final germination may be predicted This model indicated 50
declined in final germination at 030 salt concentration corresponding to ECiw= 42
dSm-1 (Table 16 Appendix II)
Rate of germination was significantly decreased (p lt 0001) from first day to final
(day 07) of observation and it was inversely correlated with sea salt concentration High
germination rate was recorded in control and low sea salt concentrations in early days of
seed incubation compared to higher sea salt concentrations but the difference in rate was
reduced (Table 17 Appendix II)
23
A progressive decline (p lt 0001) in coefficient of germination velocity was
observed with increasing salinity and fifty percent reduction was observed at 021 sea
salt concentration (ECiw = 319 dSm-1 Figure 11 Appendix II)
Final germination percentage was decreased significantly with increasing sea salt
concentrations However the difference was insignificant at lower (ECiw = 16 dSm-1)
salinity (Figure 11 Appendix II)
Mean germination time of seeds was increased significantly (p lt 0001) with
increasing sea salt concentrations However the difference was insignificant at lowest
(ECiw = 09 dSm-1) salinity (Figure 11 Appendix II)
Mean germination index was also significantly decreased (plt0001) with
increasing salt concentrations except for ECiw = 09 dSm-1 salinity Fifty percent reduction
in mean germination index was observed at 0188 sea salt concentration (ECiw = 289
dSm-1 Figure 11 Appendix II)
24
Table 15 Electrical conductivities of different sea salt solutions used in germination of C cajan
Sea salt () ECiw (dSm-1)
0 04
005 09
01 16
015 24
02 32
025 39
03 42
ECiw is the electrical conductivity of irrigation water measured in deci semen per meter
25
Table 16 Effect of irrigation water of different sea salt solutions on germination percentage (GP) per day of C cajan seeds pre-soaked in respective sea salt concentrations
with duration of time
Sea salt
ECiw (dSm-1)
GP
1st day
GP
2nd day
GP
3rd day
GP
4th day
GP
5th day
GP
6th day
GP
7th day
Control 6667plusmn333 8667plusmn333 10000plusmn000 10000plusmn000 10000plusmn000 10000plusmn000 10000plusmn000
09 7000plusmn000 7667plusmn333 9000plusmn000 10000plusmn000 10000plusmn000 10000plusmn000 10000plusmn000
16 4667plusmn333 6000plusmn000 7333plusmn333 8000plusmn000 8667plusmn333 8667plusmn333 9000plusmn577
24 4333plusmn333 5000plusmn000 6000plusmn577 6667plusmn333 7333plusmn333 7333plusmn333 8000plusmn000
32 3000plusmn000 3333plusmn333 3667plusmn333 4333plusmn333 5000plusmn577 6000plusmn577 7000plusmn577
39 1667plusmn333 2333plusmn333 2333plusmn333 4000plusmn577 4333plusmn333 5000plusmn000 6000plusmn000
42 667plusmn333 1333plusmn333 2333plusmn333 2333plusmn333 3333plusmn333 3667plusmn333 5000plusmn000
LSD 005 Salinity 327 Time 327
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and LSD among the treatments was recorded at p lt 005
25
26
Table 17 Effect of irrigation water of different sea salt solutions on germination rate (GR) per day of Ccajan
seeds pre-soaked in respective sea salt concentrations with duration of time
Sea salt
(ECiw= dSm-1)
GR
1st day
GR
2nd day
GR
3rd day
GR
4th day
GR
5th day
GR
6th day
GR
7th day
Control 667plusmn033 433plusmn017 333plusmn000 250plusmn000 200plusmn000 167plusmn000 143plusmn000
09 700plusmn000 383plusmn017 300plusmn000 250plusmn000 200plusmn000 167plusmn000 143plusmn000
16 467plusmn033 300plusmn000 244plusmn011 200plusmn000 173plusmn007 144plusmn006 129plusmn008
24 433plusmn033 250plusmn000 200plusmn019 167plusmn008 147plusmn007 122plusmn006 114plusmn000
32 300plusmn000 167plusmn017 122plusmn011 108plusmn008 100plusmn012 100plusmn010 100plusmn008
39 167plusmn033 117plusmn017 078plusmn011 100plusmn014 087plusmn007 083plusmn000 086plusmn000
42 067plusmn033 067plusmn017 078plusmn011 058plusmn008 067plusmn007 061plusmn006 071plusmn000
LSD 005 Salinity 014
Time 014 Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and LSD among the treatments is recorded at p lt 005)
27
Sea salt (ECiw = dSm-1
)
Contr
ol
09
16
24
32
39
42
Germ
ination Index(s
eedd
ays
-1)
0
2
4
6
8
Fin
al germ
ination (
)
0
20
40
60
80
100
Coeff
icie
nt of
germ
ination v
elo
city
(seedd
ays
-1)
00
01
02
03
04
05
06
07
Sea salt (ECiw = dSm-1
)
Contr
ol
09
16
24
32
39
42G
erm
ination tim
e (
Days
)
0
1
2
3
4
LSD005 = 0086
a = 0664 b = 1572
R2 = 0905 n =21
LSD005 = 062
a = 1239
b = 9836
R2 = 0894 n=21
LSD005 = 053
a = 8560b = -2272
R2 = 0969 n=21
RGF = 100-200 (Ke -005) Ke = 030
Figure 11 Effect of irrigation water of different sea salt solutions on seed germination indices of C cajan
(Bars represent means plusmn standard error of each treatment and significance among the treatments
was recorded at p lt 005)
28
14 Experiment No 3
Seedling establishment experiment of Cajanus cajan on soil irrigated with
sea salt of different concentrations
141 Materials and methods
1411 Seedling establishment
Seedling establishment experiment was carried out in Biosaline research field Department
of Botany University of Karachi Surface sterilized seeds pre-soaked were sown in small
plastic pots filled with 15 Kg sandy loam soil provided with farm manure at 91 ratio (30
water holding capacity) Sea salt solutions of different concentrations mentioned above
were used for irrigation The electrical conductivity of soil saturated paste (ECe) was also
determined at the end of the experiment (Table 18) Data on seedlings emergence was
recorded and their height were measured after 14 days of salinity treatment EC of the soil
(ECe) was initially 054 dSm-1 Statistical analyses were done according to the procedures
given in Experiment No 1
Since germination percentage of seeds pre-soaked in non-saline water was found
better under different concentrations of sea salt the seeds sown in soil for taking for
seedling establishment were pre-soaked in distilled water
29
142 Observations and Results
1421 Seedling establishment
Seedling emergence from soil was reduced significantly (p lt 0001) with increasing salt
concentration of irrigation water Not a single seedling emerged from soil in ge ECiw= 39
dSm-1 saline water irrigation However lower salinities (ECiw= 09 16 dSm-1) showed
slight decrease in seedling emergence with respect to controls Seedling emergence related
with salinity in accordance with a quadratic model as follows
Equation for seedling emergence () = 977751+ 44344 salt ndash 22215238 (salt)2 plusmn
6578 r = 09810 F = 15358 (p lt 00001)
Fifty percent reduction in seedling emergence was noticed at 016 sea salt
concentration (ECiw = 241 dSm-1 Figure 12 Appendix III)
1422 Shoot height
Shoot height was measured after fourteen days of irrigation Shoot length was
significantly decreased (p lt 0001) with increasing salinity A lower decrease was
observed in low sea salt salinity (ECiw= 09 and 16 dSm-1) compared to controls while
higher decrease in shoot height was noticed from ECiw= 2 dSm-1sea salt concentration
Shoot height related with salinity as follows
Equation for shoot height (cm) = 9116714 ndash 3420286 salt plusmn 09221 r = 0968 F =
128893 (p lt 0001)
Fifty percent reduction in shoot height was estimated at 013 sea salt concentration
(ECiw = 210 dSm-1) (Figure 12 Appendix III)
30
Table 18 Electrical conductivities of different Sea salt concentrations and ECe of soil saturated paste at the
end of experiment (ECe = 0447 + 1204 (salt ) plusmn 02797 R = 0987 F = 72301 (p lt
000001)
Sea salt () ECiw (dSm-1) ECe (dSm-1)
0 04 05
005 09 161
01 16 278
015 24 354
02 32 433
025 39 483
03 42 552
Electrical conductivity of soil saturated paste determined after 14 days of saline water irrigation in pots
Figure 12 Effect of irrigating water of different sea salt solutions on seedling emergence (A) and shoot
length (B) of C cajan (Bars represent means plusmn standard error of each treatment where similar
letters are not significantly different at p lt 005)
e f
Sea salt (ECiw = dSm-1
)
Contr
ol
16
27
8
35
4
43
3
48
3
Shoot le
ngth
(cm
)
0
2
4
6
8
10ab
c
de
Contr
ol
16
27
8
35
4
43
3
48
3Seedlin
g e
merg
ence (
)
0
20
40
60
80
100a
bb
c
d
A B
31
15 Experiment No 4
Growth and development of Cajanus cajan in Lysimeter (Drum pot
culture) being irrigated with water of different sea salt concentrations
151 Materials and methods
1511 Drum pot culture
A modified drum pot culture (lysimeter) installed by Ahmad amp Abdullah (1982) at
Biosaline research field (Department of Botany University of Karachi) was used in
present experiment Each drum pot (60 cm diameter 90 cm depth) was filled with 200 kg
of sandy loam mixed with cow-dung manure (91) having 28 water holding capacity
They are fixed at cemented platform at slanting position with basal hole to ensure rapid
drain Over irrigation was practiced to avoid the accumulation of salt in the root zone
1511 Experimental design
Growth and development of C cajan in drum pots was carried out in six different drum
pot sets (each in triplicate) and irrigated with sea salt of following concentrations
Drum pot Sets Sea salt
()
ECiw ( dSm-1) of
irrigation water
Resultant ECe (dSm-1) after
end of experiment
Set I Non saline (C) 04 05
Set II 005 sea salt 09 16
Set III 001 sea salt 16 28
Set IV 015 sea salt 24 35
Set V 02 sea salt 28 38
Set VI 025 sea salt 34 43
Note ECiw is the electrical conductivity of irrigation water and ECe is the electrical conductivity of the saturated soil extract taken after
eighteen weeks at the end of experiment
Ten surface sterilized seeds with 01 sodium hypochlorite were sowed in each
drum pot and were thinned to three healthy and equal size seedlings after two weeks of
establishment in their respective sea salt concentration Each drum pot was irrigated with
15 liters non-saline or respective sea salt solution at weekly intervals Electrical
conductivity of soil was measured by EC meter (Jenway 4510) using saturated soil paste
32
at the end of experiment Experiment was conducted for a period of 18 weeks (July to
November 2009) during which environmental data which includes average humidity
(midnight 76 and noon 54) temperature (low 23oC and high 33oC) wind velocity (14
kmph) and rainfall (~4 cm) was recorded (Pakistan Metrological Department Karachi) is
given in Figure 13Statistics were analysed according to the procedures given in
Experiment No 1
1512 Vegetative and Reproductive growth
Shoot height was measured at every two week interval after seedling establishment Fresh
and dry weight of shoot was recorded at final harvest (18th week when pods were fully
matured) Leaf succulence (dry weight basis Abideen et al 2014) Specific shoot length
(SSL Panuccio et al 2014) and relative growth rate (RGR Moinuddin et al 2014) were
measured using following equations
Succulence (g H2O gminus1 DW) = (FW minus DW) DW
SSL = shoot length shoot dry weight
RGR (g gminus1 dayminus1) = (lnW2 - lnW1) (t2 - t1)
Whereas FW fresh weight DW dry weight W1 and W2 initial and final dry weights and
t1 and t2 initial and final time of harvest in days
Reproductive data in terms of number of flowers number of pods number of seeds
and seed weight per plants was recorded during reproductive period
1513 Analysis on some biochemical parameters
Biochemical analysis of leaves was carried out at grand period of growth Following
investigations was undertaken at different biochemical parameters
i Photosynthetic pigments
Fresh and fully expended leaves (at 2nd3rd nodal part) samples (01g) were crushed in 80
chilled acetone and were centrifuged at 3000rpm for 10 minutes Supernatant were
separated and adjusted to 5ml final volume The absorbance was recorded at 663nm and
645 nm on spectrophotometer (Janway 6305 UVVis) for chlorophyll content while 480
33
and 510 nm for carotenoids Chlorophyll ab ratio was calculated after the amount
estimated The chlorophyll and carotenoid contents were determined according to Strain
et al (1971) and Duxbury and Yentsch (1956) respectively
Chlorophyll a (microgml) = 1163 (A665) ndash 239 (A649)
Chlorophyll b (microgml) = 2011 (A649) ndash 518 (A665)
Total Chlorophylls (microgml) = 645 (A665) + 1772 (A649)
Carotenoids (microgml) = 76 (A480) ndash 263 (A510)
ii Total soluble sugars
Dry leaf samples (01g) were homogenized in 5mL of 80 ethanol and were centrifuged
at 4000 g for 10 minutes 10 mL diluted supernatant in 5mL Anthronrsquos reagent was kept
to boil in 100oC water bath for 30 minutes and were cooled in running tap water Optical
density was taken at 620nm for the determination of soluble carbohydrates according to
Fales (1951)Total soluble carbohydrates was estimated against glucose as standard and
was calculated from the equation mentioned and expressed in mgg-1 dry weight
Total carbohydrates (microgmL-1) = 228462 OD 097275 plusmn004455
iii Protein content
Fresh and fully expended leaves at 2nd3rd nodal part were taken for protein estimation
The protein contents were measured according to Bradford Assay reagent method against
Bovine Serum Albumin as standards (Bradford 1976) Dye stock was made to dissolved
50mg comassie blue in 25 ml methanol The solution is added to 50ml of 85 phosphoric
acid and diluted to 100 ml with distilled water 02g fresh leaf samples were mills in 5 ml
phosphate buffer pH7 5ml of assay reagent (diluting 1 volume of dye stock with 4 volume
distilled water) were added in 01 ml leaf extract used for enzyme assay Absorbance was
recorded at 590nm and was expressed in mgg-1 fresh weight Proteins were calculated
from the following best fit standard curve equation
Protein (microgml-1) = -329196 + 1142755 plusmn 53436
34
152 Observations and Results
1521 Vegetative and Reproductive growth
Effect of sea salt on vegetative growth including height fresh and dry weight of Cajanus
cajan is presented in (Figure 14 and 15 Appendix-VI) Comparative analysis showed
that plant growth (all three parameters) was significantly increased with time (plt 0001)
however it was linearly decreased (plt 0001) with increasing salinity (Figure 16
Appendix-VI) shows the water content succulence relative growth rate (RGR) and
specific shoot length (SSL) of Cajanus cajan Under saline conditions all parameters were
significantly reduced in comparison to control however SSL showed decline after ECe38
dSm-1 Salt induced growth reduction was more pronounced at ECe 38 and 43 dSm-1 in
which plants died before reaching the reproductive maturity after 12 and 14 weeks at sea
salt treatments respectively Therefore further analysis was carried out in plant grown up
to ECe= 35 dSm-1 sea salt concentrations
Salinity significantly reduced (plt 0001) reproductive parameters including
number of flowers pods seeds and seed weight (Figure 17 Appendix-VII) Among all
treatments highest reduction was observed in 315 dSm-1 in which number of flowers and
pods reduced up to 7187 and 70 respectively Similar trend was observed in total
number and weight of seeds which showed 80 and 8793 reduction respectively
1522 Study on some biochemical parameters
i Photosynthetic pigments
Figure 18 Appendix-VII shows the effect of salinity on pigments (chlorophyll a b ab
ratio and carotenoids) of C cajan leaves A slight increase in total chlorophyll contents
(1828) and chlorophyll ab ratio (1215) was observed at low salinity (ECe= 16 dSm-
1) however they were significantly reduced (4125 and 3630 respectively) in high salt
treatment (plt 0001) Chlorophyll a was higher than chlorophyll b in all treatments
however chlorophyll b was un-affected by salinity whereas total chlorophyll content and
ab ratio was disturbed due to change in chlorophyll a This reduction was more
pronounced at high salinity (ECe= 35 dSm-1) in which chlorophyll a total chlorophylls
and ab ratio was decreased by 505 412 and 3630 respectively Carotenoid content
was maintained at ECe= 16 dSm-1 and decreased with further increase in salinity
35
ii Total soluble sugars
Total soluble sugars in leaves of C cajan is presented in Figure 19 Appendix-VII Total
leaf sugars in C cajan were remained un-affected at 16 dSm-1 and subsequently decreased
with further increase in medium salinity Although total sugars were decreased at ECe 28
and 35 dSm-1 a significant increase (~25) of soluble sugars was observed at higher
salinities However this increment was accounted for decrease (504 ) in insoluble sugar
content at that salinity levels
iii Protein
Total protein in leaves of C cajan is presented in Figure 19 Appendix-VII An increase
in leaf protein content in C cajan was found at lower salinity regime (ECe= 16 dSm-1)
which was followed by significant reduction with further increase in salinity This decline
was 2040 at 28 which was more pronounced (5646 ) at high salinity level (ECe=
35dSm-1)
36
Months (2009)
Jun Jul Aug Sep Oct Nov Dec
Valu
es
0
10
20
30
40
50
60
70
80
90
Rainfall (cm)Low Temp (
oC)
High Temp (oC)
Humidity at noon () Wind (kmph)
Humidity at midnight ()
Figure 13 Environmental data of study area during experimental period (July-November 2009)
Time (Weeks)
2 4 6 8 10 12 14 16 18
Pla
nt heig
ht (c
m)
0
30
60
90
120
150
180
210
43 38 35 28 16 Control
Figure 14 Effect of salinity using irrigation water of different sea salt concentrations on height of C cajan
during 18 weeks treatment (Lines represent means plusmn standard error of each treatment represents
significant differences at p lt 005)
37
Sea salt (ECe= dSm
-1)
Cont 16 28 35 38 43
Sea salt (ECe= dSm
-1)
Cont 16 28 35 38 43
Fre
sh w
eig
ht (g
)
0
5
10
15
20
25
30
35Initial Final
a
b b
c c cab b
c c cC 16 28 35 38 43
Fre
sh w
eig
ht
(g)
012345 a
bb
bc ca a ab b c c
Dry weightMoisture
Figure 15 Effect of salinity using irrigation water of different sea salt concentrations on initial and final
biomass (fresh and dry) of C cajan (Bars represent means plusmn standard error of each treatment Different
letters represent significant differences at p lt 005)
Mo
istu
re (
)
0
20
40
60
80
100
Succu
lance
(
)
0
20
40
60
80
100
Sea salt (ECe= dSm
-1)
Co
nt
16
28
35
38
43
RG
R (
)
0
20
40
60
80
100
Co
nt
16
28
35
38
43
SS
L (
)
0
20
40
60
80
100
Sea salt (ECe= dSm
-1)
ab
b b
c c
a
b bc c c
a
b b
c c c
a a a ab
c
Figure 16 Percent change (to control) in moisture succulence relative growth rate (RGR) and specific
shoot length (SSL) of C cajan under increasing salinity using irrigating water of different sea
salt concentrations (Bars represent means plusmn standard error of each treatment Different letters
represent significant differences at p lt 005)
38
Sea salt (ECe= dSm-1)
Control 16 28 35
Tota
l seeds (
Pla
nt-1
)
0
20
40
60
80
100
120
140 Seed w
eig
ht (g
pla
nt -1
)
0
5
10
15
20
25
Num
ber
10
20
30
40
50
60
70 a
b
cc
a
a
b
b
b c
c
a
b
a
c c
Flowers
Pods
Seed weightTotal seeds
Figure 17 Effect of irrigating water of different sea salt solutions on reproductive growth parameters
including number of flowers pod seeds and seed weight of C cajan (Values represent means
plusmn standard error of each treatment Different letters represent significant differences at p lt
005)
39
Sea salt (ECe=dSm-1
)
Control 16 28 35
Caro
tinoid
s (
mg g
-1 F
W)
000
005
010
015
020
025
030
Chlo
rophyll
(mg g
-1 F
W)
00
02
04
06
08
ab
ratio
00
05
10
15
20
25
ab
ab
b
a
cd
b
a
c
d
a
b
c
d
a
a
ab
b
Figure 18 Effect of irrigating water of different sea salt solutions on leaf pigments including chlorophyll a
chlorophyll b total chlorophyll and carotenoids of C cajan (Bars represent means plusmn standard
error of each treatment Different letters represent significant differences at p lt 005)
40
Figure 19 Effect of irrigating water of different sea salt solutions on total proteins soluble insoluble and
total sugars in leaves of C cajan (Bars represent means plusmn standard error of each treatment
Different letters represent significant differences at p lt 005)
Sea salt (ECe= dSm
-1)
C 16 28 35
Pro
tein
(m
g g
-1 F
W)
00
01
02
03
04
05
06
Su
gar
s (m
g g
-1 F
W)
00
02
04
06
08
a ab b
a a
b b
a ab b
a
b
ab
c
SoluableInsoluable
41
16 Experiment No 5
Range of salt tolerance of nitrogen fixing symbiotic bacteria associated
with root of Cajanus cajan
161 Materials and methods
1611 Isolation Identification and purification of bacteria
Nodules of C cajan grow in large clay pots and irrigated with running tap water at
biosaline agriculture research field were collected from the lateral roots (about 15 cm soil
depth) Nodules were surface sterilized with sodium hypochloride (2) for 5 min and
vigorously washed with sterilized distilled water Each nodule was crushed with sterilized
rod in 5 ml distilled water The bacterial suspension was streaked on yeast extract mannitol
agar (YEM) (K2HPO4 05 g MgSO 4 025g Na Cl 01 g Manitol 10g Yeast Extract 1g
Agar 20 g in 1000 ml of Distilled water) with the help of sterilized wire lope Colonies
were identified by studying different phenotypic characters as Rhizobium fredii
(Cappuccino and Sherman 1992 Sawada et al 2003) Pure culture of Rhizobium species
was stored at -20oC temperature
1612 Preparation of bacterial cell suspension
Bacteria were multiplied by growing in YEM broth for 48 hrs on shaking incubator (140
rpm) at 37oC in dark The culture in broth was centrifuged at 4000 rpm for 10 min to
obtained bacterial cell pellet Pellet was washed and centrifuged twice with sterilized
distilled water Pellet then re-suspended in sterilized distilled water before use
1613 Study of salt tolerance of Rhizobium isolated from root nodules of
C cajan
Assessment for salinity tolerance of Rhizobium species was assessed on YEM agar
Salinity levels of 0 05 10 15 20 25 and 30 having electrical conductivity 06 90
188 242 306 366 and 423 dSm-1 respectively were maintained with NaCl Bacterial
cell suspension of 01 ml (5times 103 colony forming unitsml) was poured in each sterilized
Petri dish 10 ml of molten YEM agar was poured immediately and shake well before
solidification of agar Petri plates were incubated at 37deg C in dark Colonies were observed
and counted in colony counter after 48 h and photographed (Dubey et al 2012 Singh and
42
Lal 2015) There were three replicates of each treatment and data were transformed to
log10 before analysis
162 Observations and Results
Colonies of Rhizobium on YEM agar at different salinity levels is presented in Figure 110
and 111 Appendix-VIII A significant decrease (plt0001) in rhizobial colonies was
observed with increasing salinity However the difference between non saline control and
90 dSm-1 and as that of 242 dSm-1 and 302 dSm-1 salt (NaCl) concentration showed
nonsignificant difference in rizobial colonies Whereas drastic decreased was observed on
further salinity levels Rhizobial colonies were not found at 423 dSm-1salt concentration
NaCl (ECw= dSm
-1)
06 9 188 242 306 366 423
Rh
izo
bia
l co
lonie
s (l
og
10)
0
1
2
3
4 a a
b
c c
d
e
Figure 110 Growth of nitrogen fixing bacteria associated with root of C cajan under different NaCl
concentrations (Bars represent means plusmn standard error of each treatment among the treatments
is recorded at p lt 005)
43
Figure 111 Photographs showing growth of Rhizobium isolated from the nodules of C cajan invitro on
YEM agar supplemented with different concentrations of NaCl (ECw)
188
423 90
Control
366
306 242
44
17 Experiment No 6
Growth and development of Ziziphus mauritiana in large size clay pot
being irrigated with water of two different sea salt concentrations
171 Materials and methods
1711 Experimental design
The grafted plants obtained from the local nursery of Mirpurkhas Sindh were transported
to the Biosaline Agriculture Research field Department of Botany University of Karachi
and were transplanted carefully in large earthen pots containing 20 Kg sandy loam soil
mixed with cow dung manure at 91 ratio having about 5 liters of water holding capacity
with a basal hole for drainage of excess salts to avoid accumulation in the rhizosphere
Over irrigation with about 15 liters of non-saline saline water was kept weekly in summer
and biweekly in winter to avoid accumulation of salts in rhizosphere Plants were irrigated
to start with non-saline tap water for about two weeks for establishment All the older
leaves were fallen and new leaves were developed during establishment period Following
irrigation schedule of non-saline (control) and saline water was selected in view of Z
mauritiana being moderately salt tolerant plant which includes both low and as well as
higher concentrations of the salt in irrigation
Sea salt () ECiw (dSm-1)
of irrigation water
Average resultant ECe (dSm-1) of soil
with some fluctuation often over
irrigation
Non saline (Control) 06 12
04 63 72
06 101 111
ECiw = Electrical conductivity of irrigation water ECe = Electrical conductivity of saturated soil
Healthy and well established plants were selected of nearly equal height and
divided into three sets each contain three replicates (total nine pots) Salinity was provided
through irrigation water of different sea salt concentrations All pots except non-saline
control were initially irrigated with 01 sea salt solution and then sea salt concentration
45
in irrigation medium was increased gradually upto the required salinity level The salinity
level of soil was monitored by taken the electrical conductivity of saturated soil paste the
end of experiment The electrical conductivity of soil (ECe) maintained at the level of 12
72 and 111 dSm-1 respectively as described by Mass and Hoffman (1977)
1712 Vegetative and reproductive growth
Vegetative growth in terms of shoot height fresh and dry weight of shoot and number of
branches were noted at destructive harvesting at initial (establishment) 60 and 120 days
of growth For dry weight shoots were dried in oven at 70˚C for three days Shoot
succulence specific shoot length (SSL) moisture percentage and relative growth rate
(RGR) was calculated at final harvest by using formulas given in Experiment No 4
Whereas number of flowers in reproductive data were recorded at onset of reproductive
period
As regard of fruit formation the duration of experiment was not sufficient for fruit
setting and furthermore the amount of sol in pots was not sufficient for healthy growth of
this plant Secondly flowering and fruiting is reported to be poor at the time of 1st initiation
of reproductive period (Azam-Ali 2006) Furthermore statistical significance of flower
and fruit count also become far less due to their excess dropping at early stage Hence it
was decided to proceed with study of fruit formation in forthcoming field trials of their
intercropping culture
1713 Analysis on some biochemical parameters
Biochemical analyses were performed at the grand period (at the time of flower initiation)
in fully expended fresh leaves Chlorophyll contents soluble sugar contents and soluble
proteins were analyzed Leaves samples taken from 3rd 4th node below the apex according
to the procedures given in Experiment No 4
46
172 Observations and Results
1721 Vegetative and Reproductive growth
Effect of sea salt on vegetative growth of Z mauritiana including height fresh and dry
weight is presented in (Figure 112 Appendix-IX) Comparative analysis showed that
plant growth (all three parameters) was significantly increased with time (plt 0001)
however number of branches was decreased (plt 0001) with increasing salinity
Figure 113 shows the moisture content succulence relative growth rate (RGR)
and specific shoot length (SSL) of Z mauritiana A non-significant difference in shoot
succulence SSL and moisture content was observed with time salinity and interaction of
both factors However RGR showed decline Salt induced growth reduction was more
pronounced at higher salinities
In Z mauritiana plants number of flowers showed significant decrease (plt0001)
with increasing salinity treatment Flower initiation seems non-significant at early growth
(60 days) period in controls and salinity treatments However drastic decrease was
observed with increasing salinity in 120 days of observation (Figure 114 Appendix-IX)
1722 Study on some biochemical parameters
i Photosynthetic pigments
The effect of Z mauritiana leaves pigments (chlorophyll a b ab ratio) on salinity shower
a slight difference in chlorophyll lsquoarsquo over control However chlorophyll lsquobrsquo contents
showed increase over control in both salinity treatments due to which the total chlorophylls
were also enhanced compared to controls Chlorophyll ab ratio was significantly
(plt0001) decreased in both salinities as compared to control (Figure 115 Appendix-IX)
ii Sugars and protein
In Z mauritiana plant soluble sugars were significantly decreased (plt0001) over controls
whereas proteins showed little decrease under salinity treatments compared to controls
(Figure 116 Appendix-IX)
47
Control 72 111
Fre
sh w
eig
ht (g
)
0
150
300
450
600
750
900
Sea salt (ECe= dSm
-1)
Control 72 111
Dry
weig
ht (g
)
0
150
300
450
600
750
900
Num
ber
of bra
nches
3
6
9
12
15
18
Heig
ht (c
m)
20
40
60
80
100
120
140
160
Initial 60 days 120 days
AcBb
Ba
AcBb Ba
AcBb Ba
Ac
BbBa
Figure 112 Effect of salinity using irrigation water of different sea salt concentrations on height number of
branches fresh weight and dry weight of shoot of Zmauritiana after 60 and 120 days of
treatment (Bars represent means plusmn standard error of each treatment Different letters represent
significant differences at p lt 005)
48
120 days 60 days InitialS
uccula
nce (
g g
-1 D
W)
00
03
06
09
12
Sea salt (ECe= dSm
-1)
SS
L (
cm
g-1
)
00
01
02
03
04
05
Control 72 111
Mois
ture
(
)
0
10
20
30
40
50
60
Control 72 111
RG
R (
mg g
-1 d
ay
-1)
0
5
10
15
20
a a aa a a a a a a
a aa a a a a a
a a aa a a a a a a a
b
b b
c
Figure 113 Effect of salinity using irrigation water of different sea salt concentrations on succulence
specific shoot length (SSL) moisture and relative growth rate (RGR) of Z maritiana (Bars
represent means plusmn standard error of each treatment Different letters represent significant
differences at p lt 005)
49
Sea salt (ECe= dSm
-1)
Control 72 111
Num
ber
of flow
ers
0
20
40
60
80
100
120
140 60 days120 days
Ac
BbBa
Figure 114 Effect of salinity using irrigation water of different sea salt concentrations on number of flowers
of Z mauritiana (Bars represent means plusmn standard error of each treatment Different letters
represent significant differences at p lt 005)
Sea salt (ECe= dSm
-1)
Control 72 111
Ch
loro
ph
yll
(mg g
-1)
00
03
06
09
12
15
18
bba
bba
bb
a
chl b chl a ab
ab
ra
tio
00
05
10
15
20
Figure 115 Effect of salinity using irrigation water of different sea salt concentrations on leaf pigments
including chlorophyll a chlorophyll b total chlorophyll and chlorophyll ab ratio of Z mauritiana (Values
represent means plusmn standard error of each treatment Different letters represent significant differences at p lt
005)
50
Figure 116 Effect of salinity using irrigation water of different sea salt concentrations on total sugars and
protein in leaves of Z mauritiana (Bars represent means plusmn standard error of each treatment
Different letters represent significant differences at p lt 005)
Sea salt (ECe= dSm
-1)
C 04 06
Pro
tein
s (m
g g
-1)
0
10
20
30
40
50
60
70
80
Solu
ble
sugar
s (m
g g
-1)
0
3
6
9
12
15
18a
a
bb
b b
Control 72 111
51
18 Discussion
Seed germination is the protrusion of radicle from the seed which is adversely affected by
salinity stress (Kaymakanova 2009) Salinity imposes the osmotic stress by accumulation
of Na+ and Cl- which decrease soil water potential that ultimately inhibits the imbibition
process (Othman 2005) Effect of seed germination against salinity is reported in linear
threshold response model of Maas and Hoffman (1977) The germination of a salt tolerant
desert legume Indigofera oblongifolia and a desert graminoid Pennisetum divisum are
also reported to behave to salinity in similar manner (Khan and Ahmad 1998 2007) Many
workers used chemical (organic inorganic) salt temperature biological and soil matrix
priming techniques to enhance seed germination percentage and especially germination
rate in saline medium (Ashraf et al 2008 Ashraf and Foolad 2005)Encouraging results
in most of the species of glycophytes and hydrophytes were found by presoaking in pure
water prior to germinating under saline condition Our study supports this finding and
seeds soaked in distilled water prior to germination performed better than those which
were presoaked in sea salt solutions Salinity adversely affects at all germination
parameters (germination percentage germination rate coefficient of germination velocity
and germination index) directly proportional with increasing salinity (Tayyab et al 2015)
With increase in time a delayed germination at higher salinity was found Higher sea salt
(168 dSm-1 for pure water presoaking and 35 dSm-1 for presoaking in respective
salinities) showed 50 or more reduction in all germination indices as compared to control
(Table 13-16 Figure 11)Our results are parallel with the finding of other workers such
as Kafi and Goldani (2001) who found the same trend in chickpea at higher salinities Pujol
et al (2000) reported that increased salinity inhibit the seed germination as well as delays
germination initiation in various halophyte species as well Similar response was also
found in some other crops such as pepper (Khan et al 2009) sunflower (Vashisth and
Nagarjan 2010) and eggplant (Saeed et al 2014) Salt tolerance within species may vary
at germination and other growth phases (Khan and Ahmad 1998)
According to our results C cajan appeared to be a salt sensitive in initial growth
phase specially when presoaked in saline medium (Figure 12) however at later growth
stages it proved relatively salt tolerant Salt stress delays or either seize the metabolic
activities during seed germination in salt sensitive and even in salt tolerant plants (Khan
and Ahmad 1998 Ali et al 2013b) Salinity also imposes the oxidative stress due to
52
overproduction of reactive oxygen species which may alter metabolic activities during
germination growth and developmental stages (Zhu 2001 Munns 2005
Lauchli and Grattan 2007)
In our study seeds of pigeon pea were unable to emerge beyond ECe39 dSm-1 sea
salt concentration Height of seedling was significantly affected by increasing salinity
(Figure 12) Similar results are also reported in Indian mustered (B juncea Almansouri
et al 2001) some Brassica species (Sharma et al 2013) and tomato cultivars (Jamil et
al 2005) Growth retardation with increasing salinity may be due to reduced
photosynthetic efficiency and inhibition of enzymatic and non-enzymatic proteins
(Tavakkoli et al 2011) Furthermore salt stress also limit the DNA and RNA synthesis
leads to reduced cell division and elongation during germination growth and
developmental stage
Khan and Sahito (2014) found variation in salt tolerance within species subspecies
and provenance level Furthermore the salt tolerance of a species may also vary at
germination and growth phases (Khan and Ahmad 1998 Ali et al 2013a) Srivastava et
al (2006) suggested that the genetic variability influences salinity tolerance eg wild
species like Cajanus platycarpus C scaraboides and C sericea showed better salt
tolerance than C cajan In this connection Wardill et al (2006) has also reported genetic
diversity in Acacia nilotica C cajan in this study appeared to be a salt sensitive at
germination in compression with later stages of growth Seedling establishment at saline
solution faces adverse effects when emerging radicle and plumule come in contact with
salt effected soil particle or saline water hence percent seedling establishment remains
less than germination percentage observed at petri plate Ashraf (1994) found that salinity
tolerance of different varieties of C cajan do not much differ at germination and early
growth stages whereas at adult growth stage show improvement in salt tolerance
Soil salinity is a major limiting factor for plant growth and yield production
particularly in leguminous plants (Guasch-Vidal et al 2013 Tayyab et al 2016) In
present study Plant height RGR fresh and dry biomass were severely reduced with
increasing salinity and plant was unable to grow after ECe= 43 dSm-1(Figure 14-16)
This growth inhibition of C cajan may be accounted for individual and synergistic effect
of water stress nutrient imbalances and specific ions toxicities (Hasegawa et al 2000
Silvera et al 2001) Salt induced ion imbalance results in lower osmotic potential which
53
alter physiological biochemical and other metabolic processes leading to overall growth
reduction (Del-Amor et al 2001) Excessive amount of salt in cytoplasm challenge the
compartmentalization capacity of vacuole and disrupts cell division cell elongation and
other cellular processes (Munns 2005 Munns et al 2006) Our results are parallel with
some other studies in which significant growth inhibition of peas chickpea and faba beans
have been reported against salt stress (El-Sheikh and Wood 1990 Delgado et al 1994)
Singla and Garg (2005) also observed a similar salt sensitive growth response in Cicer
arietinum In our study the fresh and dry biomass of C cajan also showed inhibitory
behavior to salt stress (Figure 15) Hernandez et al (1999) also found significant reduction
in dry biomass of pea plant and common bean (40 and 84 respectively) when grown
in saline medium Mehmood et al (2008) also found similar results in Susbania sasban
Salinity also has imposed deleterious effects on reproductive growth of C cajan
Production of flowers and pods are significantly decreased in response to salinity (Figure
19) Increase in flower shedding leads to decreased number of pods indicating salt
sensitivity of plant at reproductive phase which was more pronounced at high salinity
(Vadez et al 2007) Furthermore seed production and weight of seed per plant was also
linearly decreased Salt induced reduction of reproductive growth has also been found in
mung bean in which 60 and 12 less pods and seeds were produced respectively at 06
saline solution (Qados 2010) Similar results are reported in faba bean (De-Pascale and
Barbieri 1997) tomato (Scholberg and Locascio 1999) maiz sunflower (Katerji et al
1996) and watermelon (Colla et al 2006) Salinity reduces reproductive growth by
inhibiting growth of flowers pollen grains and embryo which leads to inappropriate ovule
fertilization and less number of seeds and fruits (Torabi et al 2013)
On biochemical parameters total chlorophyll and chlorophyll ab ratio has
increased in low salinity in contrast the adverse effect at higher salinity could be due to
high Na+ dependent breakdown of these pigments (Li et al 2010 Yang et al 2011)
Chlorophyll a is usually more prone to Na+ concentration and decrease in total chlorophyll
is mainly attributed to the destruction of chlorophyll a (Fang et al 1998 Eckardt 2009)
This diminution could be due to the destruction of enzymes responsible for green pigments
synthesis (Strogonov et al 1973) and increased chlorophyllase activity (Sudhakar et al
1997) Thus insipid of leaf was a visible indicator of salt induced chlorophyll damage
which was well correlated with quantified values as reported in other legume species
54
(Soussi et al 1998 Al-Khanjari et al 2002) In this study chlorophyll a was found to be
more sensitive than chlorophyll b (Figure 18) Garg (2004) also found similar reduction
in chlorophyll pigments (a b and total chlorophyll) in chickpea cultivars under salinity
stress
At low salinity (16 dSm-1) total carotenoids remained unaffected along with
increased total chlorophyll (Figure 18) which may suggest a role of carotenoids in
protection of photosynthetic machinery (Sharma et al 2012) Similar response was found
in Cajanus indicus and Sesamum indicum (Rao and Rao 1981) however
Sivasankaramoorthy (2013) and Ramanjulu et al (1993) reported slight increase of leaf
carotenoids in Zea maiz and mulberry when exposed to NaCl High salinity was destructive
for both leaf pigments (chlorophyll and carotenoids) of C cajan which was in accordance
with Reddy and Vora (1985) who found similar decrease in some other salt sensitive crops
Salinity led to the conversion of beta-carotene to Zeaxanthin which protect plants against
photo-inhibition (Sharma and Hall 1991)
In present study with increasing salinity water content and succulence of C cajan
were significantly reduced which indicated loss of turgor (Figure 16) Our data suggest
that decreased succulence by lowering water content may help in lowering leaf osmotic
potential when exposed to increasing salinity which is in agreement with findings of Parida
and Das (2005) and Abideen et al (2014) In addition increased production and
accumulation of organic substances is also necessary to sustain osmotic pressure which
provide osmotic gradient to absorb water from saline medium (Hasegawa et al 2000
Cha-um et al 2004) Compatible solutes including carbohydrates amino acids proteins
and ammonium compounds play important roles in water relations and cell stabilization
(Ashraf and Harris 2004) In this study C cajan produce more soluble sugars (Figure 18)
which is considered as a typical plant response under saline conditions (Murakeozy et al
2003) Sugars serve as organic osmotica and their available concentration is related to the
degree of salt stress and plantrsquos tolerance (Ashraf 1994 Murakeozy et al 2003) Sugars
are involved in osmoprotection osmoregulations carbon storage and radical scavenging
activities (Pervaiz and Satyawati 2008) On the other hand insoluble and total sugars were
reduced in higher salinity which is also supported by Parida et al (2002) and Gadallah
(1999) who found similar results in Bruguiera parviflora and Vicia faba
55
Total soluble proteins of C cajan were reduced due to deleterious effects of salinity
(Figure 18) The accumulation of Na+ in cytosol disrupts the protein and nucleic acid
synthesis (Bewley and Black 1985) Gill and Sharma (1993) and Muthukumarasamy and
Panneerselvam (1997) also reported decreased protein content with increasing salinity in
Cajanus cajan seedlings Similar results were found when tomato (Azeem and Ahmad
2011) Zingiber officinale (Ahmad et al 2009) and Sorghum bicolor (Ali et al 2013a)
were grown under variable salt concentrations (Figure 19)
Nodule formation of Rhizobium in Legume depends upon interaction between soil
chemistry of salt composition and osmotic regimes of salt and water (Velagaleti et al
1990 Zahran 1991 Zahran and Sprent 1986) Salinity reduces plant growth directly
through ion and osmotic effects and indirectly by inhibiting Legume-Rhizobium
association (El-Shinnawi et al 1989) Studies demonstrated a more sensitive response of
rhizobial N-fixing mechanism than growth of plant to abiotic stresses including salinity
(Mhadhbi et al 2004) In nodules metabolic disturbance initiated with the production of
ROS leading to tissues injury and loss of nodule function (Becana et al 2000) In general
it slow down the nitrogenase activity and decrease nodule protein and leghemoglobin
content which decreased becteroid development (Mhadhbi et al 2008) In consequence
plant suffer directly by salt induced ion toxicity low water uptake and photosynthetic
damage and indirectly through weak association of symbionts due to high energy demand
for nodule function (Pimratch et al 2008) In our study the isolated rhizobial strain from
nodules of C cajan was found to be tolerant to salinity even up to 2 (ECw= 306 dSm-1)
NaCl (Figure 110 and 111) Some of the other species of Rhizobium such as Brady
Rhizobium have been shown salt tolerant even at higher concentration than their
leguminous hosts (Zahran 1999) For instance a number of rhizobial species can tolerate
up to 06 NaCl (Yelton et al 1983) while Rhizobium meliloti can tolerate 175 to
40 NaCl and R leguminosarum can tolerate can tolerate upto 2 NaCl (Abdel-Wahab
and Zahran 1979 Sauvage et al 1983 Breedveld et al 1991 Helemish 1991
Mohammad et al 1991 Embalomatis et al 1994 Mhadhbi et al 2011) Rhizobia
isolated from soybean and chickpea can tolerate up to 2 NaCl with a difference of fast-
growing and slow growing strains (El-Sheikh and Wood 1990 Ghittoni and Bueno 1996)
Similarly Rhizobium from Vigna unguiculata can survive up to up to 55 NaCl
(Mpepereki et al 1997)
56
Present study shows an increase in vegetative growth in terms of plant height and
fresh and dry weight of shoot with increasing time under non-saline and saline conditions
but the increase was rapid at early period of growth (Figure 112) All the vegetative
growth parameters determined were reduced under salinity stress compared to non-saline
control Measurements of shoot moisture succulence specific shoot length and RGR
(Figure 113) indicate that Z mauritiana adjusted in its water relation over coming
negative water and osmotic potential with increase in salinity levels increased There is
evidence that water and osmotic potentials of salt tolerant plants become more negative in
higher salinities (Khan et al 2000) These altered water relations and other physiological
mechanisms help plants to get by adverse abiotic stress like that of drought and salinity
(Harb et al 2010) However the results clearly showed that salinity had an inhibitory
effect on growth but the decline was less at early sixty days and more during later 60-120
days in compression to controls Growth inhibition in shoot has been observed in number
of plants including different species of halophytes (Keiffer and Ungar 1997) chickpea
(Cicer arietinum Kaya et al 2008) and different wheat cultivars (Triticum aestivum
Moud and Maghsoudo 2008)
Salinity also caused reduction in the number of branches and the number of flowers
in Z mauritiana however reduction in the number of flowers is non-significant in ECe=
72 dSm-1 salinity treatment in comparison with non-saline control (Figure 114) The main
reason for this reduction could be attributed to suppression of growth under salinity stress
during the early developmental stages (shooting stage) of the plants These results are
similar to those reported by Ahmad et al (1991) and Khan et al (1998) As affirmed by
Munns and Tester (2008) suppression of plant growth under saline conditions may either
be due to osmotic effect of saline solution which decreases the availability of water for
plants or the ionic effect due to the toxicity of sodium chloride High salt concentration in
rooting medium also reduced the uptake of soil nutrients a phenomenon which affected
the plant growth thus resulting in less number of branches per plant Various abiotic
stresses such as temperature drought salinity light and heavy metals altered plant
metabolism which ultimately affects plant growth and productivity Amongst these
salinity stress is a major problem in arid and semiarid regions of the world (Kumar et al
2010) Salinity has an adverse effect on several plant processes including seed
germination seedling establishment flowering and fruit formation and ripening (Sairam
and Tyagi 2004) Salinity stress also imposes additional energy requirements on plant
57
cells and less carbon is available for growth and flower primordial initiation (Cheesman
1988) The lesser decrease in number of flowers at lower salinity (ECe= 72 dSm-1) has
been attributed to the fact that the cells of apex are un-vacuolated and the incoming salts
accumulated in the cytoplasm Munns (2002) further suggested a well-controlled phloem
transport of toxic ions from these cells prevented any change in reproductive development
Our findings showed an increase in total chlorophyll contents particularly
chlorophyll b contents were enhanced more than chlorophyll a contents under salinity
stress (Figure 115) In general the total chlorophyll contents decreased under high salinity
stress and this may be due to accumulation of toxic ions in photosynthetic tissues and
functional disorder of stomatal opening and closing (Khan et al 2009) The increase in
total chlorophylls appearing at salinity levels is considered as an important indicator of
salinity tolerance in plants (Katsuhara et al 1990 Demiroglu et al 2001) In another
study on Z mauritiana (cv Banara sikarka) the chlorophyll contents has shown decrease
with increasing salinity and sodicity but the seedlings treated with low salinity (ECe of 5
mmhoscm-1) shows slightly higher values than controls (Pandey et al 1991) Our study
also suggests that increase in total chlorophylls adapted this plant increased its tolerance
to salt stress
Slight decrease in protein has been shown under salinity treatments compared to
controls (Figure 16) Proteins play diverse roles in plants including involvement in
metabolic pathways as enzyme catalyst source of reserve energy and regulation of osmotic
potential under salt stress (Pessarakli and Huber 1991 Mansour 2000) Salts may
accumulate in cell cytoplasm and alter their viscosity depending on the response of plant
to salinity stress (Hasegawa et al 2000 Paravaiz and Satyawati 2008) The decrease in
protein contents under increasing salinity has also been documented in several plants
including Lentil lines (Ashraf and Waheed 1993) sorghum (Ali et al 2013a) and sugar
beet (Jamil et al 2014)
Soluble sugars were also decreased with increasing salinity treatments in our study
(Figure 16) Decrease in soluble sugars due to salinity has also been reported in Viciafaba
(Gadallah 1999) some rice genotypes (Alamgir and Ali 1999) Bruguiera parviflora
(Parida et al 2002) and Lentil (Sidari et al 2008) However the accumulation of soluble
sugars under salinity stress is considered as strategy to tolerate stress condition due to their
58
involvement in osmoprotection osmotic adjustment and carbon storage (Parida et al
2002 Parvaiz and Satyawati 2008)
From these experiments it is evident that C cajan is a salt sensitive plant at every
level of its life cycle starting from germination to growth phases Germination capacity
and salt tolerance ability of this species can be enhanced by water presoaking treatment
Growth reduction with increasing salinity could be attributed to physiological and
biochemical disturbances which ultimately affect vegetative and plant reproductive
growth Its roots are well associated with nitrogen fixing rhizobia and these
microorganisms were salt tolerant in in-vitro cultures Another fruit baring species of
marginal lands Z mauritiana showed growth improvement in lower salinity and its growth
was not much affected in high saline mediums owing to its controlled biochemical
responses
59
2 Chapter 2
Intercropping of Z mauritiana with C cajan
21 Introduction
Increasing soil salinity fresh water scarcity and agricultural malpractice creating shortage
of food crops for human and animal consumption (Bhandari et al 2014) and making
prices high Traditional agriculture which has been practiced since centuries using multi
species at a time in a given space could be a potential solution to narrow down the growing
edges of this supply demand scenario Plant species with innate resilience to abiotic
stresses like salinity and drought could be considered suitable to serve this purpose
especially for arid regions where marginal lands can be utilized to generate economy
Presence of such type of local systems in the region highlight their potential advantage in
crop production income generation as well as sustainability (Somashekar et al 2015)
For instance reports are available on successful intercropping of multipurpose trees
shrubs and grasses like millets pulses and some oil seed and fodder crops Green part of
these species usually mixed and used for cattle feed especially during the lean period The
utilization of the inter-row spaces of fruit trees like Ziziphus mauritiana for growing edible
legumes can generate further income by similar input (Dayal et al 2015) As an option
to this Cajanus cajan could serve as better intercropped as it provides protein rich food
nutritious fodder and wood for fuel which helped to uplift the socio-economic condition
of poor farmers Integrated agricultural practices improve the productivity of each crop by
keeping cost of production under sustainable limits (Arabhanvi and Pujar 2015)
Keeping in mind the above mentioned scenario in present study the possibility to
increase production of a non-conventional salt tolerant fruit tree (Z mauritiana) by
intercropping with a leguminous plant (C cajan) was investigated to produce edible fruits
and fodder simultaneously from salt effected waste lands
60
22 Experiment No 7
Physiological investigations on Growth of Ziziphus mauritiana and
Cajanus cajan intercropped in drum pot (Lysimeter) culture being
irrigated with water of sea salt concentration at two irrigation intervals
221 Materials and Methods
2211 Growth and Development
Experiment was designed to investigate the effect of intercropping on growth and
development of Z mauritiana (a fruit tree) and C cajan (a leguminous fodder) in drum
pot culture irrigated with water of 03 sea salt concentrations at two irrigation intervals
2212 Drum pot culture
Drum pot culture as recommended by Boyko (1966) and modified by Ahmed and
Abdullah (1982) was used for the present investigation as described in chapter 1
2213 Experimental Design
Three sets of 18 plastic drums (lysimeter) were used in this experiment One plant of Z
mauritiana were grown in each lysimeter Three replicates were kept for each treatment
comprising of 06 drums in each set which was further divided in two sub-sets First sub-
set was irrigated at every 4th and second subset at every 8th day
Set ldquoArdquo =Ziziphus mauritiana (Sole crop)
Set ldquoBrdquo = Cajanus cajan (Sole crop)
Set ldquoCrdquo = Ziziphus mauritiana + Cajanus cajan (intercropped)
The effect of salinity on sole crops of C cajan and Z mauritiana on salinity created
by various dilutions of sea salt has been investigated in chapter 1 Concentration of 03
sea salt considered equal level to its 50 reduction has been selected in present
experiment In addition irrigation was given in sub-sets in two intervals to investigate to
have some idea of its water conservation
61
2214 Irrigation Intervals
Sub-set 1 Irrigation was given every 4th day
Sub-set 2 Irrigation was given every 8th day
In set lsquoArsquo and lsquoCrsquo six month old saplings of Ziziphus mauritiana (vern grafted
ber) plants of nearly equal height and good health were transplanted in drum pots Plants
were irrigated to start with non-saline tape water for about two weeks for purpose of
establishment All the older leaves fell down and new leaves immerged during
establishment period
In set lsquoBrsquo and lsquoCrsquo Ten healthy sterilized seeds of Cajanus cajan imbibed in distill
water were sown in each drum pot and irrigated to start with tap water and after
establishment of seedlings only six seedlings of equal size with eqal distance (about one
feet) between C cajan and that of Z mauritiana were kept for further study The sowing
time of cajanus cajan seeds in both sets (B and C) was the same In drum pot lsquoCrsquo it was
sown when sapling of Z mauritiana have undergone two weeks of their establishment
period in tap water
When seedlings of C cajan reached at two leaves stage irrigation in all the sets
(ABC ) was started with gradual increase sea salt concentration till it reached to the
salinity level of treatment (03) in which they were kept up to end of experiment Each
drum was irrigated with enough water sea salt solution which retains 15 liters in soil at
field capacity Rest of water drain down with leaching of accumulated salt in root
rhizosphere
Vegetative growth of Z mauritiana plant was noted monthly in terms of height
volume of canopy while in C cajan height and number of branches was noted Shoot
length root length number of leaves fresh and dry weight of leaf stem and root leaf
weight ratio root weight ratio stem weight ratio specific shoot and root length plant
moisture leaves succulence and relative growth rate was observed and calculated at final
harvest in both the plant species growing individually (sole) or as intercropping at two
irrigation intervals
Investigations were undertaken on nitrate content relative water content and
electrolyte leakage at grand period of growth Amount of photosynthetic pigments soluble
62
carbohydrates proline content soluble phenols and Protein contents were also investigated
in fully expended leaves
Activity of catalase (CAT) ascorbate peroxidase (APX) guaiacol peroxidase
(GPX) superoxide dismutase (SOD) (Anti-oxidant enzymes) and nitrate reductase (NR)
activity was also observed in on both the Z mauritiana and C cajan leaves growing as
sole and as intercropped at two different irrigation intervals
The procedures of above mentioned analysis as follows
Leaf succulence (dry weight basis) Specific shoot length (SSL) and relative
growth rate (RGR) were measured according to the equations given in chapter 1
2215 Estimation of Nitrate content
NO3 was estimated through Cataldo et al (1975) 01g fresh leaf samples were boiled in
50 mL distilled water for 10 min 01mL of sample were added to mixed in 04 mL 50
salicylic acid (wv dissolved in 96 H2SO4 ) and allowed to stand for 20 min at room
temperature 95 mL of 2N NaOH was slowly mixed at last The samples were permissible
to cool NO3 concentration was observed at 410 nm and was calculated according to the
standard curve expressed in mg g-1 fresh weight
2216 Relative Water content (RWC)
Young and fully expended leaf was excise from each plant removing dust particles
preceding to Relative water content (RWC) Fresh weights (FW) were taken to all leaf
samples and were immersed in distilled water at 4 degC for 10 hours The soaked leaf samples
were taken out and surfeit water was removed by tissue paper Weighted again these leaf
samples for turgid weight (TW) and were oven dried at 70 degC Dry weight (DW) was
recorded after 24 hrs The RWC of leaf was calculated by the following formula
RWC () = [FW ndash DW] [TW ndash DW] x 100
2217 Electrolyte leakage percentage (EL)
EL was measured according to Sullivon and Ross (1979) Young and fully expended
leaves removing dust particles were taken 20 disc of 6mm diameter were made through
63
porer and were placed in the test tube containing 10ml de-ionized water First electrical
conductivity (EC lsquoarsquo) was record after shaken the tubes These test tubes now were placed
at 45-50oC warmed water bath for 30 min and observed second Electrical conductivity (EC
lsquobrsquo) Finally tubes were placed at 100oC water bath for ten min and obtained third and final
Electrical conductivity (EC lsquocrsquo) The electrolyte leakage was calculated in percentage by
using following formula
EL () = (EC b ndash EC a) EC b x 100
2218 Photosynthetic pigments
Photosynthetic pigments including chlorophyll a chlorophyll b total chlorophyll
chlorophyll ab ratio and carotinoids were estimated according to the procedure given in
chapter 1
2219 Total soluble sugars
Dry leaf samples (01g) were milled in 5mL of 80 ethanol and were centrifuged at 4000
g for 10 minutes and were estimated according to the procedure described in chapter 1
22110 Proline content
The proline contents were determined through Bates et al (1973) Each dried leaf powder
sample (01 g) was grinded and homogenized in 5 ml of 3 (wv) sulphosalicylic acid and
were centrifuged at 5000 g for 20 minutes 2ml supernatant was boiled by adding 2 ml
glacial acetic acid and 2 ml ninhydrin reagent (prepared by dissolving 125 g ninhydrin in
30 ml of glacial acetic acid and 20 ml 6 M phosphoric acid) in caped test tube The tubs
were kept in boiling water bath (100oC) for 1 hour After cooling 4 ml of toluene was
added to each tube and vortex Two layers were appeared the chromophore layer of
toluene was removed and their absorbance was recorded at 590nm against reference blank
of pure toluene The proline concentrations in leaves were determined from a standard
curve prepared from extra pure proline of (Sigma Aldrich) and were calculated from the
equation and were expressed in mgg-1 of leaf dry weight
Proline (microgmL-1) = -074092 + 1660767 (OD) plusmn054031
64
22111 Soluble phenols
The dried leaf powder (01g) was milled in 3ml of 80 methanol and was centrifuged at
10000g for 15 min (Abideen et al 2015) Final volume (5ml) were adjusted by adding
80 methanol Soluble phenols were determined by using Singleton and Rossi (1965) ie
5 ml of Folin-Ciocalteu reagent (19 ratio in distilled water) and 4 ml of 75 Na2CO3
were added to 01 ml supernatant The absorbance was recorded at 765 nm after incubation
of 30 minutes at room temperature The soluble phenols concentration in leaf tissues was
determined from a standard curved prepared from Gallic acid
22112 Total soluble proteins
The protein contents were measured according to Bradford Assay reagent method against
Bovine Serum Albumin as standards (Bradford 1976) Procedure was followed as given
in chapter 1
22113 Enzymes Assay
Enzyme extract prepared as given below was used for study of enzymes mentioned in text
The juvenile and expended leaf excised was frozen in liquid nitrogen and were stored at -
20 degC These leaf samples (100mg) was firmed in liquid nitrogen and were mills in 3 ml
of ice chilled potassium phosphate buffer (pH = 7 01 M) with 1mM EDTA and 1 PVP
(wv) The homogenate was filtered through a four layers of cheesecloth and were
centrifuged at 21000 g using refrigeration centrifuge (Micro 17 TR Hanil Science
Industrial Co Ltd South Korea) at 4 degC for 20 min The supernatant was separated and
stored at -20 degC and used for investigation on following enzymes
i Superoxide dismutase (SOD)
SOD (EC 11511) antioxidant enzymeactivity was measured through Beauchamp and
Fridovich (1971) derived on the inhibition of nitroblue tetrazolium (NBT) reduction by
produced O2minus using riboflavin photo-reduction 50 mM of pH 78 phosphate buffer (with
01mM EDTA 13 mM methionine) 75 microM nitroblue tetrazolium (NBT) 2 microM riboflavin
and 100 microl of enzyme extract was added to 3ml reaction mixture Riboflavin was added at
the last before the reaction was initiated under fluorescent lamps for 10 min Exposed and
un-exposed to florescence lamp without enzyme extract were used to serve as calibration
65
standards Activity was measured at 560nm Unit of SOD activity was defined as the
amount of enzyme required for 50 inhibition of NBT conversion
ii Catalase (CAT)
CAT (EC 11116) antioxidant enzyme activity was precise according to Aebi (1984)
derived on H2O2 reduction at 240nm for 30 s (ε = 36 M-1 cm-1)100mM potassium
phosphate buffer (pH=7) with 30mM H2O2 and 50 microl of diluted enzyme extract (adding in
last) was added to 3ml reaction mixture The decrease in absorbance due to H2O2 reduction
was measured at 240 nm and expressed in micromol of H2O2 reduced m-1g-1 fresh weight at 25
degC
iii Ascorbate peroxidase (APX)
Nakano and Asada (1981) method was used for APX (EC 111111) antioxidant
enzymeactivity by measuring the decrease in ascorbate oxidation by H2O2 The reaction
mixture (3ml) contained potassium phosphate buffer (50mM pH=7) 01mM H2O2 050
mM Ascorbate and 100 microl of enzyme extract and were observed 290 nm for 1 min 25 degC
(extinction coefficient 28 mM-1cm-1)
iv Guaiacol peroxidase (GPX)
GPX (EC 11117) antioxidant enzymeactivity was estimated through Anderson et al
(1995) 3ml of 50 mM potassium phosphate buffer (pH 7) guaiacol 75 mM H2O2 10 mM
reaction mixture with 20 microl of enzyme extract adding at last Increase in absorbance was
observed due to the formation of tetra-guaiacol at 470 nm for 2 min (extinction coefficient
266 mM-1cm-1)
v Nitrate reductase (NR)
The NR activity in leaves was observed through Long and Oaks 1990 Fresh leaf samples
(01g) were placed in 5ml of 100mM potassium phosphate pH 75 (added to 10
Isopropanol and 25mM KNO3) Tubes were vacuumed for 10 min to remove air from the
mixture and were placed in water bath shaker at 33oC for 60 min in dark The tubes were
placed in hot water (100oC) for 5 min 15 mL from the reaction mixture were added in 05
mL 20 sulphanilamide (wv dissolve in 5N HCl) and 025 mL 008 N-1-Napthylene-
66
diamine dihydrochloride Final volume up to 60 ml was made by adding distilled water
Color developed over the next 20 min Absorbance was measured at 540 nm using
spectrophotometer
67
222 Observations and Results
Sole and intercropped Ziziphus mauritiana
2221 Vegetative growth
Growth of Z mauritiana in terms of shoot root and plant length and number of leaves in
two different cropping system (sole and intercrop with C cajan) in two different irrigation
intervals has been presented in Figure 21 Appendix-XII A significant increase (plt0001)
in plant length was observed in 8th day irrigation in both the cropping systems in Z
mauritiana At 4th day of irrigation interval a non-significant increase in length was
observed in intercropped plants compared to sole crop Similarly at 8th day of irrigation
plants attain almost same heights in both the cropping systems
A significant increase (plt001) in root length was observed in sole Z mauritiana
at 8th day of irrigation compared to other treatments Smallest root length revealed in plants
that were irrigated at 4th day under sole crop system
The shoot length was significantly increase (plt0001) in plants which were
irrigated at 8th day under intercropped system However shoot length remains unaffected
when comparing the different cropping system at both the irrigation intervals
A significant increase (plt0001) in number of leaves was observed in intercropped
Z mauritiana plants compared to plants cultivated according to sole system However
more increase was observed in 4th day irrigated intercropped plant as compared to 8th day
The difference in number of leaves in sole crop at both irrigating intervals remains same
i Fresh weight
Figure 22 Appendix-XII showed fresh and dry weight of stem root and leaf of Z
mauritiana plant in two different cropping system (sole and intercrop with C cajan) in
two different irrigation intervals A significant increase (plt0001) in fresh weights of leaf
stem and root was observed in intercropping (with C cajan) 4th and 8th day of irrigation
interval compared to individual cropping of Z mauritiana In 4th day of irrigation the
increment was more pronounced in fresh weights of root (7848) leaves (4130) and
stem (4047) respectively with comparison to the crop growing alone Similarly
intercropping in 8th day of irrigation showed better growth of leaves (28) stem (12)
68
and root (31) against sole crop Whereas decrease in leaves 33 (plt005) stem 70
(plt0001) and root 60 (plt0001) fresh weights were observed in 8th day of irrigation
compared to 4th day intercropping However the difference was non-significant between
two sole crops irrigated at 4th and 8th day interval
ii Dry weight
Intercropping with comparison to the sole crop showed significant (plt0001) increase in
dry weights of leaves root and stem of Z mauritiana at 4th and 8th day of irrigation (Figure
22 Appendix-XII) At 4th day of irrigation intercropping showed an increment in dry
weights of Leaves (4366) stem (4109) and root (754) compared to the sole crop
Similar increase was observed in leaves (plt0001) stem (plt0001) and root (plt0001)
weights after 8th day of irrigation However intercropping at 8th day irrigation showed an
increment in root (19) stem (11) whereas a slight decrease (1) in leaves dry weight
When comparing irrigation time an increase in stem dry weight at 4th day whereas decline
in leaves dry weight was observed Root dry weights were more or less similar at both
irrigation intervals
iii Leaf weight ratio (LWR) root weight ratio (RWR) stem weight
ratio (SWR)
Leaf weight ratio (LWR) root weight ratio (RWR) stem weight ratio (SWR) of Z
mauritiana plant grown in two different cropping system (sole and intercrop with C cajan)
in two different irrigation intervals has been presented in Figure 23 Appendix-XII An
increased in LWR and SWR was recorded at 8th day of irrigation compared to 4th day of
irrigation in both cropping systems whereas decrease in RWR was observed LWR and
SWR remained un-change in sole and inter crop system However RWR increased in
intercrop system compared to sole crop system
iv Specific shoot length (SSL) specific root length (SRL)
Specific shoot length (SSL) specific root length (SRL) of Z mauritiana plant grown in
two different cropping system (sole and intercrop with C cajan) in two different irrigation
intervals has been presented in Figure 23 Appendix-XII SSL was observed higher in 8th
day of irrigation compared to 4th day in both the cropping systems However the increase
69
in SSL was lesser in sole crop compared to intercropping Similarly SRL was recorded
lesser in 4th day of irrigation compared to 8th day of irrigation in both cropping systems
Intercropped plants showed decline in SRL compared to sole crop plants Greatest SRL
revealed in plants that were irrigated after 8th day and planted according to sole crop
system
v Plant moisture
The moisture content of Z mauritiana plant grown in two different cropping system (sole
and intercrop with C cajan) in two different irrigation intervals has been presented in
Figure 23 Appendix-XII The moisture content of plants was significantly decreased
(plt005) in sole crop while increased (plt005) in intercropping at 8th day of irrigation
compared to 4th day At 4th day moisture remained same in both cropping system
However significant increase in moisture contents was observed in inter-crop system
compared to sole crop system after 8th day of irrigation
vi Plant Succulence
Succulence of Z mauritiana plant grown in two different cropping system (sole and
intercrop with C cajan) in two different irrigation intervals has been presented in Figure
23 Appendix-XII Plant succulence in 8th day was significantly reduced in sole crop
whereas increased in intercropping system In 4th day irrigated plants decrease in
succulence was noticed compared to plants that were irrigated at 8th day under sole crop
system However significant increase (plt0001) was observed in intercropped plants
irrigated at 4th day compared to 8th day
vii Relative growth rate (RGR)
Relative growth rate (RGR) of Z mauritiana plant grown in two different cropping system
(sole and intercrop with C cajan) in two different irrigation intervals has been presented
in Figure 23 Appendix-XII Relative growth rate remains unchanged at both irrigation
times under sole crop system However decline in 8th day was observed compared to 4th
day of irrigation under intercrop system Greatest RGR was recorded in plants that were
irrigated at 4th day under intercrop system
70
2222 Photosynthetic pigments
Photosynthetic pigments including Chlorophyll a chlorophyll b total chlorophyll
Chlorophyll ab ratio and carotinoids of Z mauritiana plant grown in two different
cropping system (sole and intercrop with C cajan) in two different irrigation intervals has
been presented in Figure 24 Appendix-XII
i Chlorophyll contents
A significant increase (plt0001) in chlorophyll a b and total chlorophyll was observed in
plants growing as sole crop compared to intercropped system at both the irrigation
intervals Higher chlorophyll contents were also recorded in plants that were irrigated at
8th day compared to 4th day of irrigation The chlorophyll ab ratio increased in 4th day
while decline in 8th day in intercropped system compared to sole crop However overall
results showed non-significant changes
ii Carotinoids
A significant increase (p lt 0001) in leaf carotinoids was observed in sole crop compare
to intercropped system at both irrigation times in Z mauritiana Least carotene content
was estimated in plants that were irrigated at 4th day under intercrop system
2223 Electrolyte leakage percentage (EL)
Electrolyte leakage percentage (EL) of Z mauritiana plant grown in two different
cropping system (sole and intercrop with C cajan) in two different irrigation intervals has
been presented in Figure 25 Appendix-XII A non-significant result was observed in
electrolyte leakage in plant growing at varying cropping system and irrigating intervals
2224 Phenols
Total phenolic contents in leaves of Z mauritiana plant grown in two different cropping
system (sole and intercrop with C cajan) in two different irrigation intervals has been
presented in Figure II25 Appendix-XII A significant increase (plt001) in total phenolic
contents was observed in intercropped growing at both irrigation interval compared to sole
crop However the increase was more pronounced at 8th day of irrigation Maximum
phenolic contents were measured in plants irrigated at 8th day under intercropped plants
71
2225 Proline
Total proline contents in leaves of Z mauritiana plant grown in two different cropping
system (sole and intercrop with C cajan) in two different irrigation intervals has been
presented in Figure 25 Appendix-XII A significant decreased (plt0001) was observed
in Z mauritiana cultivated according to intercropped system in both irrigation intervals
Maximum decrease was observed in intercropped plants irrigated at 8th day whereas
highest phenolic contents were observed in plants irrigated at 4th day under sole crop
system
2226 Protein and sugars
Protein and sugar contents in leaves of Z mauritiana plant grown in two different cropping
system (sole and intercrop with C cajan) in two different irrigation intervals has been
presented in Figure 26 Appendix-XII A nonsignificant difference in total protein and
sugar contents in Z mauritiana plants was observed in two different (4th and 8th day)
irrigation intervals However the interaction with time and irrigation interval also showed
nonsignificant result
2227 Enzyme essays
Antioxidant enzymes like Catalase (CAT) Ascorbate peroxidase (APX) Guaiacol
peroxidase (GPX) Superoxide dismutase (SOD) and Nitrate reductase activity in leaf of
Z mauritiana plant grown in two different cropping system (sole and intercrop with C
cajan) in two different irrigation intervals has been presented in Figure 27 and 28
Appendix-XII
i Catalase (CAT)
A significant decreased (plt0001) in catalase activities was observed in Z mauritiana
leaves in intercropped system in both time interval with compare to sole crop at 4th day
irrigated plant However maximum decline was in sole plants irrigated at 8th day interval
However their interaction with time was nonsignificant
72
ii Ascorbate peroxidase (APX)
A significant increase (plt0001) in APX activity was observed in 8th day irrigation in both
sole and intercropped plants with compare to sole and intercropped at 4th day irrigation
interval More increase (plt0001) was observed in intercropped Z mauritiana at 8th day
Whereas nonsignificant decrease was observed in two different cropping system in 4th day
irrigation interval However interaction between time and the treatments shows significant
values
iii Guaiacol peroxidase (GPX)
A significant (plt0001) increase in GPX was observed in 8th day intercropped Z
mauritiana plant with compare to irrigation intervals as well as cropping system However
at 4th day both cropping system showed nonsignificant difference Whereas more decline
was observed in 8th day sole crop The ANOVA reflects significant (plt005) interaction
between time and the cropped system
iv Superoxide dismutase (SOD)
A nonsignificant increase in SOD was observed in intercropped at 8th day irrigation
interval Whereas there was nonsignificant differences in 4th day intercropped and at both
time intervals of sole crop However interaction between time interval and the two
cropping system shows nonsignificant result
v Nitrate and Nitrate reductase
A significant increase (plt0001) in nitrate content and activity of nitrate reductase was
observed in intercropped plants of both irrigation intervals Increase in activity was
observed (plt0001) in intercropped Z mauritiana at 4th day
73
Sole and intercropped Cajanus cajan
2228 Vegetative growth
Growth of C cajan in terms of shoot root and plant length and number of leaves was
observed in two different cropping system (sole and intercrop with Z mauritiana) in two
different irrigation intervals has been presented in Figure 21 Appendix-XIII XIV A
significant increase (plt001) in plant length was observed in intercropped C cajan
compared to sole crop at both irrigation interval Whereas sole crop at 8th day interval
showed better results as compare to sole of 4th day Similarly root length remains
unaffected and showed non-significant change in both cropping systems and even at two
different irrigation intervals While shoot length was significantly (Plt001) decreased in
sole crop compared to intercropped at 4th day irrigation Whereas non-significant
difference be observed in rest of cropping systems growing at different irrigation interval
A significant increase (plt001) in leaves number was observed in intercropped
plants compared to sole crop at 4th and 8th day irrigation interval However most
significant decrease (plt0001) was observed in sole crop at 4th day
i Fresh weight
Figure 22 Appendix-XIV showed fresh and dry weight of stem root and leaf of C cajan
plant in two different cropping system (sole and intercrop with C cajan) in two different
irrigation intervals A significant increase (plt001) in fresh weight of leaf was observed in
intercropping (with Z mauritiana) at 4th and 8th day of irrigation interval compared to
individual cropping of C cajan The increase in intercropped system compared to sole
crop was more pronounced at 4th day (42) of irrigation than the 8th day (1701) Plants
showed higher leaves fresh weights in 8th day of irrigation compared to 4th day Similarly
the interaction between cropping system and the irrigation interval was significant
(Plt005)
An insignificant difference was observed in stem at 4th (15) and 8th (12) days
fresh weights in both intercropping system at two different irrigation intervals The
interaction between cropping system and the irrigation interval also showed non-
significant result
74
A non-significant difference in root fresh weight was observed in two different
cropping systems (sole and intercropped) in 4th and 8th day of irrigation intervals However
fresh weight of crop at 8th day irrigation interval was significantly increase (plt0001) over
4th day irrigation interval Similar pattern was observed in 4th day irrigated sole and
intercropped C cajan
ii Dry weight
A significant increase in leaves (42) stem (24) and root (18) dry weights were
observed in 4th day irrigation under intercropped system compared to sole However in 8th
day of irrigation this increase of dry weights was not much prominent Under sole crop
system dry weights of leaves stem and root was increased markedly in 8th day compared
to 4th day However in intercrop system the difference in dry weights was insignificant
between 8th and 4th day of irrigation
iii Leaf weight ratio (LWR) root weight ratio (RWR) stem weight
ratio (SWR)
Leaf weight ratio (LWR) root weight ratio (RWR) stem weight ratio (SWR) of C cajan
grown in two different cropping system (sole and intercrop with Z mauritiana) in two
different irrigation intervals has been presented in Figure 23 Appendix-XIV A
significant increase (plt0001) in LWR was observed at 8th day of irrigation compared to
4th day intercropped Similar pattern was noticed in RWR however SWR showed
insignificant difference between 4th and 8th day of irrigation A slight increase in LWR was
noticed in intercropped plants compared to sole Whereas RWR declined in intercrop
compared to sole and SWR remains un-changed
iv Specific shoot (SSL) root length (SRL)
Specific shoot length (SSL) specific root length (SRL) of C cajan grown in two different
cropping system (sole and intercrop with Z mauritiana) in two different irrigation
intervals has been presented in Figure 23 Appendix-XIV SSL and SRL were observed
to increase in sole crop compared to intercrop at 4th day of irrigation However increase
SSL and SRL was recorded in intercropped compared to sole at 8th day of irrigation A
general decline in SSL and SRL was noticed in 8th day of irrigation compared to 4th day
75
v Plant moisture
The moisture content of C cajan plant grown in two different cropping system (sole and
intercrop with Z mauritiana) in two different irrigation intervals has been presented in
Figure 23 Appendix-XIV The moisture content of plants was decreased significantly
(plt005) at 8th day irrigation interval compared to 4th day in sole crop Whereas non-
significant increase was observe in intercrop plants at 8th day of water irrigation
vi Plant succulence
Succulence of C cajan plant grown in two different cropping system (sole and intercrop
with Z mauritiana) in two different irrigation intervals has been presented in Figure 23
Appendix-XIV A significant increase (plt001) was observed in intercropped plants of C
cajan compared to sole crop at both irrigation interval However succulence increased in
sole crop and decreased in intercrop plants at 8th day of irrigation compared to 4th day
vii Relative growth rate (RGR)
Relative growth rate (RGR) of C cajan plant grown in two different cropping system (sole
and intercrop with Z mauritiana) in two different irrigation intervals has been presented
in Figure 23 Appendix-XIV A significant increase in RGR was observed in 8th day
compared to 4th day in both the cropping systems Highest increase was observed in
intercropped at 8th day irrigation At 4th day irrigation intervals intercropped plants
showed better RGR compared to Sole crop
2229 Photosynthetic pigments
Photosynthetic pigments including Chlorophyll a chlorophyll b total chlorophyll
Chlorophyll ab ratio and carotinoids of C cajan plant grown in two different cropping
system (sole and intercrop with Z mauritiana) in two different irrigation intervals has been
presented in Figure 24 Appendix-XIV
i Chlorophyll contents
A significant increase (plt005) in Chlorophyll a b and total chlorophyll was observed in
intercrop plants at 8th day irrigation interval Whereas at 4th day irrigation interval Sole
76
plants showed better results as compare to intercrop plants Plants at 8th day significantly
increase chlorophyll a b and total chlorophyll compared to 4th day of irrigation
Interactions between cropping systems and irrigation intervals were found significant
(chlorophyll a (plt001) chlorophyll b (plt001) and total chlorophyll (plt0001)
respectively) However the ratio of chlorophyll ab showed non-significant values in
cropping irrigation interval and their interaction
ii Carotenoids
A significant increase (plt001) in carotinoids was observed in intercropped C cajan at 8th
day of irrigation Whereas non-significant increase was observed in sole crop at 4th day
irrigation interval with compare to intercrop However the irrigation intervals showed
significant (plt0001) difference Whereas interaction of cropping system with irrigation
time also showed significant correlation (plt0001)
22210 Electrolyte leakage percentage (EL)
Electrolyte leakage percentage (EL) of C cajan plant grown in two different cropping
system (sole and intercrop with Z mauritiana) in two different irrigation intervals has been
presented in Figure 25 Appendix-XIV A non-significant increase in EL percentage was
observed in sole crop compared to intercrop plants growing at 4th and 8th day of irrigation
No significant change was noticed between the irrigation times to C cajan The interaction
between cropping system (sole and intercropped) and irrigation interval (4th and 8th day)
also showed non-significant
22211 Phenols
Total phenolic contents in leaves of C cajan plant grown in two different cropping system
(sole and intercrop with Z mauritiana) in two different irrigation intervals has been
presented in Figure 25 Appendix-XIV A nonsignificant result was observed in total
phenolic contents of C cajan growing as sole and intercropped system at two different
irrigation intervals However the interaction between irrigation intervals with crop system
showed significant (p lt 005) results
77
22212 Proline
Total proline contents in leaves of C cajan plant grown in two different cropping system
(sole and intercrop with Z mauritiana) in two different irrigation intervals has been
presented in Figure 25 Appendix-XIV Proline contents in leaves of C cajan showed
nonsignificant increase at 4th day of irrigation interval in both sole and intercropped
system Whereas the interaction between irrigation intervals showed significant (Plt001)
results
22213 Protein and Sugars
Protein and sugar contents in leaves of C cajan plant grown in two different cropping
system (sole and intercrop with Z mauritiana) in two different irrigation intervals has been
presented in Figure 26 Appendix-XIV A less significant difference (plt005) was
observed in two different (4th and 8th day) irrigation intervals However there was
nonsignificant difference in two cropped system More decrease was observed at 4th day
intercropped plants Whereas nonsignificant increase in 8th day intercropped and 4th day
sole plants were observed However interaction between crop and time of irrigation
showed significant results (plt0001)
22214 Enzyme assay
Antioxidant enzymes like Catalase (CAT) Ascorbate peroxidase (APX) Guaiacol
peroxidase (GPX) Superoxide dismutase (SOD) and Nitrate reductase activity in leaf of
C Cajan plant grown in two different cropping system (sole and intercrop with Z
mauritiana) in two different irrigation intervals has been presented in Figure II27
Appendix-XIV
i Catalase (CAT)
A significant increase (plt001) in catalase activity was observed in intercropped C cajan
at 8th day of irrigation with compare to other irrigation time and cropped system Whereas
increase was observed in sole crop at 4th day irrigation interval with compare to 8th day
However the irrigation intervals and the interaction between cropping system with
irrigation interval also showed nonsignificant correlation
78
ii Ascorbate peroxidase (APX)
A non-significant increase in APX was observed in intercropped plant in 4th and 8th day
irrigation interval with compare to sole crops Sole crop at 8th day showed maximum
decline However the difference between cropping system and their interaction with
irrigation interval also showed nonsignificant results
iii Guaiacol peroxidase (GPX)
A significant increase (plt005) in GPX activity was observed in 8th day sole crop
However there was nonsignificant difference among intercropped at two time interval and
sole crop at 4th day irrigation Whereas interaction with time to irrigation interval also
showed less significant results
iv Superoxide dismutase (SOD)
A significant decrease (plt0001) in SOD activity was observed in intercropped at 8th day
irrigation interval with compare to 4th day Maximum decrease was observed in 8th day
intercropped Whereas sole crop at 8th day also showed better result to 4th day sole crop
However ANOVA showed significant correlation among crop system at two time interval
and 4th day irrigation
v Nitrate and Nitrate reductase
Nitrate content and activity of nitrate reductase was nonsignificant in both cropping
system using both irrigation intervals However nonsignificant increase was observed in
nitrate content and activity of nitrate reductase in intercropped Z mauritiana at 8th day
79
Sole IntercropSole Intercrop
No o
f le
aves
0
20
40
60
Len
gth
(cm
)
0
40
80
120
160
200
2404
th day
Cajanus cajan
a
RootShoot
ab
a
a
b
a
a
8th
day
Figure 21 Vegetative parameters of Z mauritiana and C cajan at grand period of growth under sole and
intercropping system at 4th and 8th day irrigation intervals (Bars represent means plusmn standard error
of each treatment and significance among the treatments was recorded at p lt 005)
Sole IntercropSole Intercrop
No of
leav
es
0
200
400
600
Len
gth
(cm
)
0
40
80
120
160
200
240
Ziziphus mauritiana
RootShoot
4th
day 8th
days
b b
a a
a
b
cc
80
Sole Intercrop
Dry
wei
ght
(g)
50
100
150
200
250
300
Fre
sh w
eight
(g)
100
200
300
400
500
Sole Intercrop
4th
day 8th
day
a
b
c
a
b b aa
b
b
c c
a
bc
a
c
ba
b
c
a
b
c
Leaf Stem Root
Ziziphus mauritiana
Sole Intercrop
Dry
wei
ght
(g)
2
4
6
8
10
12
Fre
ah w
eight
(g)
5
10
15
20
25
30
35
40
Sole Intercrop
4th
day 8th
day
aa
b
a
a
b
a
b
c
a
b
c
a
c
b
a a
b
a
b
c
a
b
c
Leaf Stem Root
Cajanus cajan
Figure 22 Fresh and dry weight of Z mauritiana and C cajan plants under sole and intercropping system
at 4th and 8th day irrigation intervals (Bars represent means plusmn standard error of each treatment
and significance among the treatments was recorded at p lt 005)
81
Figure 23 Leaf weight ratio (LWR) root weight ratio(RWR) shoot weight ratio(SWR)specific shoot
length (SSL) specific root length (SRL) plant moisture Succulence and relative growth rate (RGR) of
Zmauritiana and C cajan grow plants under sole and intercropping system at 4th and 8th
day irrigation
intervals (Bars represent means plusmn standard error of each treatment and significance among the treatments
was recorded at p lt 005)
Sole Intercrop
Mo
istu
re (
)
0
20
40
60
80
SS
L (
cm g
-1)
01
02
03
04
05
06
RW
R (
g g
-1 D
W)
005
010
015
020
LW
R (
g g
-1 D
W)
01
02
03
04
05
06
07
Sole Intercrop
Su
ccu
lan
ce
(g H
2O
g-1
DW
)00
05
10
15
20
25
RG
R
(g g
-1 d
ay-1
)
001
002
003
004
005
SR
L (
cm g
-1)
05
10
15
20
25
SW
R (
g g
-1 D
W)
02
04
06
08
10
Ziziphus mauritiana
a a
bb
b
a
bb
a
b
aa
a aa
b
a
bb
c
b
a
bb
b
aa a
ba
bc
4th day
8th day
82
(Figure 23 continuedhellip)
Sole Intercrop
Mo
istu
re (
)
0
20
40
60
80
SS
L (
cm g
-1)
2
4
6
8
10
12
RW
R (
g g
-1 D
W)
002
004
006
008
010
012
014
LW
R (
g g
-1 D
W)
01
02
03
04
05
06
07
08
Sole Intercrop
Su
ccu
lan
ce
(g H
2O
g-1
DW
)
00
05
10
15
20
25
RG
R
(g g
-1 d
ay-1
)
001
002
003
004
005
SR
L (
cm g
-1)
5
10
15
20
25
SW
R (
g g
-1 D
W)
02
04
06
08
10
Cajanus cajan
a aab
a aaa
a
bba
a
b b
c
a aab
a
bbb
abbb
aa
bc
8th day
4th day
83
Sole Intercrop
Car
oti
noid
s (m
g g
-1 F
W)
00
01
02
03
04
05
Ch
loro
phyll
(m
g g
-1 F
W)
00
03
06
09
12
15
Sole Intercrop
4th
day 8th
day
Ch
loro
phyll
ab
rat
io
00
05
10
15
20
25Chl ab
Ziziphus mauritiana
a a
bb
a
b
a
b
a ab
b
Chl aChl b
Figure 24 Leaf pigments of Zmauritiana and C cajan grow plants under sole and intercropping system at
4th and 8th
day irrigation intervals (Bars represent means plusmn standard error of each treatment and
significance among the treatments was recorded at p lt 005)
Sole Intercrop
Car
oti
noid
s (m
g g
-1 F
W)
00
01
02
03
04
05
Ch
loro
phyll
(m
g g
-1 F
W)
00
03
06
09
12
15
18
Sole Intercrop
4th
day 8th
day
ab r
atio
00
05
10
15ab
ab
Cajanus cajan
bb b
a
a
b
cc
bb b
a
84
Ele
ctro
lyte
lea
kag
e(
)
0
5
10
15
4th
day 8th
dayP
hen
ols
(m
g g
-1)
0
5
10
15
20
25
30
Sole Intercrop
Pro
line
( g g
-1)
0
10
20
30
40
Sole Intercrop
Ziziphus mauritiana
a a a
a
b b ba
a
b
c
d
Figure 25 Electrolyte leakage phenols and prolein of Z mauritiana and C cajan at grand period of growth
plants under sole and intercropping system at 4th and 8
th day irrigation intervals (Bars represent
means plusmn standard error of each treatment and significance among the treatments was recorded at
p lt 005)
85
(Figure 25 continuedhellip)
E
lect
roly
te l
eakag
e(
)
0
20
40
60
80
4th
day 8th
day
Phen
ols
(m
g g
-1)
0
2
4
6
8
10
12
Sole Intercrop
Pro
line
( g g
-1)
000
003
006
009
012
015
018
Sole Intercrop
Cajanus cajan
a aa
a
a a aa
aa a
a
86
Sole Intercrop
Sugar
s (m
g g
-1)
0
20
40
60
Sole Intercrop
Pro
tein
(m
g g
-1)
00
02
04
06
4th
day 8th
day
Ziziphus mauritiana
a aa a
a
a a a
Sole Intercrop
Sugar
s (m
g g
-1)
0
10
20
30
Sole Intercrop
Pro
tein
(m
g g
-1)
00
02
04
06
08
10
4th
day 8th
dayCajanus cajan
ab
a
c
a
b
cc
Figure 26 Total protein and sugars in leaves of Z mauritiana and C cajan plants under sole and
intercropping system at 4th and 8th
day irrigation intervals (Bars represent means plusmn standard
error of each treatment and significance among the treatments was recorded at p lt 005)
87
Sole Intercrop
SO
D (
Unit
s m
g-1
)
0
2
4
6
8
10
12
14
Sole Intercrop
Cat
alas
e (U
nit
s m
g-1
)
0
5
10
15
20
25
AP
X (
Unit
s m
g-1
)
0
20
40
60
80
GP
X (
Unit
s m
g-1
)
00
01
02
03
04
05
4th
day 8th
day
Ziziphus mauritiana
a
bc
c
a
b
cc
a
c
b
b
b bb
a
Figure 27 Enzymes activities in leaves of Z mauritiana and C cajan plants under sole and intercropping
system at 4th and 8th
day irrigation intervals (Bars represent means plusmn standard error of each
treatment and significance among the treatments was recorded at p lt 005)
88
(Figure 27 continuedhellip)
Sole Intercrop
SO
D (
Unit
s m
g-1
)
0
1
2
3
4
5
Sole Intercrop
Cat
alas
e (U
nit
s m
g-1
)
0
2
4
6
8
4th
day 8th
dayG
PX
(U
nit
s m
g-1
)
00
05
10
15
20
25
Cajanus cajan
aA
PX
(U
nit
s m
g-1
)
0
20
40
60
80
100
bb
b
aaa
b
a
bbb
a
c
a
b
89
Sole Intercrop
NO
3 (
mM
ol
g-1
)
00
02
04
06
08
10
12
14
8th
day
Sole Intercrop
Nit
rate
Red
uct
ase
(mM
ol
g-1
)
0
1
2
3
4
4th
day
Nitrate reductaseNO
3
Ziziphus mauritiana
a
b
c
cb
b
b
a
Sole Intercrop
NO
3 (
mM
ol
g-1
)
00
02
04
06
08
10
12
8th
day
Sole Intercrop
Nit
rate
Red
uct
ase
(mM
ol
g-1
)
0
2
4
6
8
10
12
4th
dayCajanas cajan
a
bb
b
aa
aa
Nitrate reductase NO3
Figure 28 Nitrate reductase activity and nitrate concentration in leaves of Z mauritiana and C cajan plants
under sole and intercropping system at 4th and 8th
dayirrigation intervals (Values represent means
plusmn standard error of each treatment and significance among the treatments was recorded at p lt
005)
90
23 Experiment No 8
Investigations of intercropping Ziziphus mauritiana with Cajanus cajan
on marginal land under field conditions
231 Materials and Methods
2311 Selection of plants
Ziziphus mautitiana and Cajanus cajan were selected for this study as described in chapter
1
2312 Experimental field
Field of Fiesta Water Park was selected to investigate intercropping of Z mauritiana with
Ccajan It is situated about 50 km from University of Karachi at super highway toward
HyderabadThe area of study has subtropical desert climate with average annual rain fall
is ~20 cmmost of which is received during the monsoon or summer seasonSince summer
temperature (April to October) are approx 30-35 degC and the winter months (November to
March) are ~20 degC Wind velocity is generally high all the year Topography of the area
was uneven with clay- loam soil having gravels Xerophytic plants are pre-dominantly
present in the area including Prosopis spp Acacia spp Euphorbia spp Caparus
deciduas etc
2313 Soil analysis
Before conducting experiment soil of Fiesta Water Park field was randomly sampled at
three locationsatone feet of depthusing soil augerThese soil samples were analyzed in
Biosaline Research Laboratory Department of Botany University of Karachi to
determine its physical and chemical properties
i Bulk density
Bulk density was determinedin accordance with Blake and Hartge (1986) by using the
following formula
Bulk density = Oven dried soil (g) volume of soil (cm3)
91
ii Soil porosity
Soil porosity was calculated in accordance with Brady and Weil (1996) by using the
following formula
Soil porosity = 1- (bulk density Particle density) times 100
Where particle density = 265 gcm3
iii Soil texture and particle size
Soil particle size was determined by Bouyoucos hydrometric method in accordance with
Gee and Or (1986)On the basis of clay silt and sand percentages soil texture was
determined by using soil texture triangle presented in Figure 31
iv Water holding capacity
Water holding capacity in percentages was calculatedaccording to George et al (2013)
v pH and Electrical conductivity of soil (ECe)
Soil saturated paste was made with de-ionized water and leave for 24 hours Soil solution
was extracted through Buckner funnel and suction pump (Rocker 300) pH of soil
solution was taken on Adwa AD1000 pHMV meter and ECe was taken on electrical
conductivity meter (4510 Jenway)
2314 Experimental design
Six months old grafted Ziziphus mauritiana saplings were carefully transported in field of
Fiesta Water Park
Three equal size plots of 100times10 sq ft were prepared for this experiment
Plot ldquoArdquo = Ziziphus mauritiana (Sole crop)
Plot ldquoBrdquo = Cajanus cajan (Sole crop)
Plot ldquoCrdquo = Ziziphus mauritiana + Cajanus cajan (intercropped)
In plot lsquoArsquo and lsquoCrsquo pits of two cubic feet depth were prepared in two parallel rows
at a distance of 10 feet (Yaragattikar amp Itnal 2003)so that the distance of pits within the
row and the distance of pits between the rows were same Each row bears nine pits
Eighteen healthy saplings of nearly equal height and vigor of Z mauritiana were
92
transplanted in the pits and were fertilized with cow-dong manure Plants were irrigated
with underground (pumped) water initially on alternate day for two weeks older leaves
fall down completely and new leaves appeared in this establishment period Later the
irrigation interval was kept fortnightly Electrical conductivity of irrigated water (ECiw)
was 24 plusmn 05 dSm-1
After establishment of Z mauritiana water soaked seeds of intercropping plant (C
cajan) were sown in plot lsquoCrsquo Three vertical lines (strips design) of equal distance were
made between the rows of Z mauritiana The distance between the line was one feet
Eleven C cajan were maintained in each line at a distance of one feet which constitute a
total of 33 C cajan in 3 lines There were 264 plants of C cajan arranged in strip pattern
as intercrop for eighteen Z mauritiana A sole crop of C cajan in plot lsquoBrsquo was arranged
with the same manner to serve as control Similarly plot lsquoArsquo was served as control of Z
mauritianaThe experiment was observed up to reproductive yield of each plant
Field diagram Theoritical model of intercropping system used in this study showing sole crop in Plot lsquoArsquo
(Z Mauritiana) and Plot lsquoBrsquo (C cajan) while Plot lsquoCrsquo represents intercropping of both
species at marginal land
Six Z mauritiana plants were randomly selected from their two rows of block lsquoCrsquo
which were facing two rows of C cajan on either sides Similarly ten plants of C cajan
facing Z mauritiana were randomly selected for further study At the same manner six Z
mauritiana from block lsquoArsquo and ten C cajan from block lsquoBrsquo grown as sole crop were
selected as control for further study
93
2315 Vegetative and reproductive growth
Vegetative growth of Z mauritiana plant was noted in terms of height volume of canopy
while height and number of branches in Ccajan bimonthly after establishment Fresh and
dry weightsof leaves stem and root were observed at final harvest in both plant species
growing as sole or intercropping
Reproductive growth of Z mauritiana such as number length and diameter fruit
weight per ten plant and average fruit yield was measured at termination of the experiment
Whereas reproductive growth in C cajan was monitored in terms of number of pods
number of seeds weight of pods and weight of seed
2316 Analyses on some biochemical parameters
Following biochemical analysis was conducted in Fully expended leavesof Z mauritiana
and C cajan growing as sole and as intercropped at grand period of growth Additionally
fruits of Z mauritiana were also analyzed for their protein soluble and insoluble sugars
and total phenolic contents
i Photosynthetic pigments
Photosynthetic pigments including chlorophyll a chlorophyll b and total chlorophyll were
estimated in leaves of Z mauritiana and C cajan according to procedure described in
chapter 1
ii Protein in leaves
Protein contents were estimated in leaves of Z mauritiana and C cajan according to
procedure described in chapter 1
iii Total soluble sugars in leaves
Total soluble sugars were estimated in leaves of Z mauritiana and C cajanaccording to
procedure described in chapter 1
94
iv Phenolic contents in leaves
Phenolic content were estimated in leaves of Z mauritiana and C cajan according to
procedure described in chapter 1
2317 Fruit analysis
i Protein in fruit
Protein content in fruit of Z mauritiana was estimated according to procedure described
in chapter 1
ii Total soluble sugars in fruits
Total soluble sugars in ripe fruits of Z mauritiana were estimated according to procedure
described in chapter 1
iii Phenolic contents in fruits
Phenolic contents in fruits of Z mauritiana were estimated according to procedure
described in chapter 1
2318 Nitrogen estimation
Nitrogen was also estimated in root zone soil as well as in fully expended leaves of Z
mauritiana and C cajan plants
Total nitrogen in leaves and soil was estimated through AOAC method 95504
(2005) One g of dried powdered sample in round bottle flask was digested in presence of
20 mL H2SO4 15 mL K2SO4 and 07g CuSO4 at 400oC heating mental After digestion 80
ml distilled water was added in digest Then distillation was done at 100oC by adding 100
mL of 45 NaOH (drop wise) in digested solution Steam was collected in 35 mL of 01M
HCl in a flask Three samples of 10 mL each steam collected solution were taken and 2-3
drops of methyl orange was added as indicator Titration was made with 01M NaOH
Changeappearance of color indicates the completion of reactionPercent nitrogen was
calculated through following equation
N = (mL of acid times molarity) ndash (mL of base times molarity) times 14007
95
2319 Land equivalent ratio and Land equivalent coefficient
The LER defined the total land area needed for sole crop system to give yield obtained
mixed crop It is mainly used to evaluate the performance of intercropping (Willey 1979)
Land equivalent ratio (LER) of two crops was estimated according to (Willey 1979) by
using formula
Whereas partial LER of Z mauritiana calculated according to
Similarly Partial LER of Ccajan were calculated as
Land equivalent coefficient (LEC) an assess of dealings the effectiveness of relationship
of two crops (Alhassan et al 2012) was calculated by using (Adetiloye et al 1983)
equation as
Yield was calculated in gram fresh weight LER and LEC of height and total chlorophyll
were also calculated by using above formula by substituting their values with yield (fruits
of Z mauritiana and seeds of C cajan) to height fruits and chlorophyll respectively
23110 Statistical analysis
Data were analyzed by using (ANOVA) and the significant differences between treatment
means wereexamined by least significant difference (Zar 2010) All statistical analysis
was performed using SPSS for windows version 14 and graphs were plotted using Sigma
plot 2000
LER= Yield of Z mauritiana + Yield of C cajan (in intercropped) + Yield of C cajan + Yield of Z mauritiana (in intercropped)
Yield of Z mauritiana (sole) Yield of C cajan (sole)
Partial LER = Yield of Z mauritiana + Yield of C cajan (in intercropped)
Yield of Z mauritiana (sole)
Partial LER = Yield of C cajan + Yield of Z mauritiana (in intercropped)
Yield of C cajan (sole)
LEC = Partial LER of Z mauritiana times Partial LER of C cajan
96
232 Observations and Results
2321 Vegetative parameters
Vegetative growth parameters of Z mauritiana include plant height volume of canopy
grown individually as well as intercropped with C cajan is presented in Figure 29
Appendix-XV A significant increase in height and canopy volume of Z mauritiana with
time (p lt 0001) and cropping system (p lt 005) was observed However the interaction
between time and cropping system showed non-significant results In general the
intercropped plants were showed higher values in all vegetative parameters than sole crop
and this increase was more pronounced after 60 days
Figure 29 Appendix-XVII showed the vegetative growth parameters of C cajan
including height and number of branches Height of C cajan was significantly increased
(plt0001) with increasing time in plants growing sole and as intercropped with Z
mauritiana The interaction with time to crop height also showed significant (plt0001)
results in both cropping systems However slight decline in height of intercropped C
cajan was noticed at 120 days compared to sole crop Number of branches was significant
increased (plt0001) in both crops with increasing time The interaction of time with
branches also showed significant (plt0001) results in both cropping systems However
number of branches was slightly increased in intercropped plants at 120 days compared to
sole crop
2322 Reproductive parameters
i Fruit number and weight (fresh and dry)
Reproductive parameters of Z mauritiana and C cajan at grand period of growth under
sole and intercropping system has been presented in Figure 210 Appendix-XVI XVIII
Individual and interactive effect of time (p lt0001) and treatment (plt001) on number and
fresh weight of fruits of Z mauritiana was showed significant results Similarly plants
grown with C cajan showed significant increase (p lt0001) in fresh weight of fruits (p
lt005) whereas fruit dry weight and circumference was non-significant in comparison to
sole crop
97
In C cajan flowers were appeared only at blooming phase (during 60 days of treatment)
and no difference in number of flowers was observed in both cropping systems (sole and
with Z mauritiana (Figure 210 XVII)
Leguminous pods were initiated soon after flowering period (during 60 days) and
last till end of the experiment (120 days) A significant increase (plt0001) in pod numbers
was observed with increasing time in both sole and intercropped system But non-
significant differences in number of pods of both cropping system and their interaction
with time were observed
Similarly number and weight of C cajan seeds were showed non-significant difference
in both cropping systems
2323 Study on some biochemical parameters
i Photosynthetic pigments
Leaf pigments of Zmauritiana and C cajan grow plants under sole and intercropping has
been presented in Figure 211 Appendix-XVI XVIII In Z muritiana leaves A significant
increase (plt005) in chlorophyll a chlorophyll b total chlorophyll and carotinoids was
observed when grown as intercrop whereas the effect on chlorophyll ab ratio was non-
significant as that of sole one
In C cajan a slight decrease (plt005) in chlorophyll lsquobrsquo and total chlorophyll
(plt001) was observed in intercropped plants compare to sole one Whereas chlorophyll
lsquoarsquo chlorophyll ab ratio and carotinoids showed nonsignificant difference between sole
and intercropped C cajan
ii Total proteins sugar phenols
Sugars protein and phenols in leaves of Z mauritianaand C cajan at grand period of
growth under sole and intercropping system is presented in Figure 212 Appendix-XVI
XVIII Total proteins and soluble and insoluble sugar content of Z mauritiana leaves was
unaffected throughout the experiment However an increase in total phenolic content
(plt001) was observed in intercropped Z mauritiana plants than grown individually
98
In C cajan total soluble sugars protein and phenols in leaves showed non-
significant differences between sole to intercropped plants
Sugars protein and phenols in fruits of Z mauritiana grown under sole and
intercropping system is presented in Figure 213 Appendix-XVI A non-significant
increase was observed in phenolic as well as in soluble insoluble and total sugar contents
in fruits of Z mauritiana plants grown with C cajan (intercrop) as compare to the fruits
of sole crop
2324 Nitrogen Contents
Nitrogen in leaves and in soil of Z mauritiana and C cajan growing under sole and
intercrop system is presented in Figure 214 Appendix-XVI XVIII ANOVA showed a
non significant effect on nitrogen content of leaf as well as root zone soil of Z mauritiana
and C cajan grown individually or as intercropping system
2225 Land equivalent ratio (LER) and land equivalent coefficient
(LEC)
Land equivalent ratio (LER) Land equivalent coefficient (LEC) of height chlorophyll and
yield of of Z 98auritiana and C cajan growing as sole and intercropping system in has
been presented in Table 22 The LER using height of both species was nearly 2 in which
PLER of Z mutitania was 48 and PLER of C cajan was 519 Whereas the calculated
values of the land equivalent coefficient (LEC) of Z mauritiana and C cajan remained
9994
The LER using yield of both species was above 2 in which PLER of Z mauritiana
was 46 Whereas PLER of C cajan was 543 However the calculated values of LEC
of both species were 100
The LER using total chlorophylls of both species were more than 25 in which
PLER of Z mauritiana was 344 and as that of PLER of C cajan was 655 Whereas
the calculated values of LEC was 999 of both the species
99
Table 21 Soil analysis data of Fiesta Water Park experimental field
Serial number Parameters Values
1 ECe (dSm-1) 4266plusmn0536
2 pH 8666plusmn0136
3 Bulk density (gcm3) 123plusmn0035
4 Porosity () 53666plusmn1333
5 Water holding capacity () 398plusmn2811
6 Soil texture Clay loam
7 Sand () 385plusmn426
8 Silt () 3096plusmn415
9 Clay () 305plusmn1
Ece is the electrical conductivity of saturated paste of soil sample
Figure 29 Soil texture triangle (Source USDA soil classification)
100
Ziziphus mauritiana
Days
0 60 120
Volu
me
(m3)
0
10
20
30
Days
0 60 120
Hei
ght
(cm
)
0
50
100
150
200
250
Sole Intercrop
a
a
bb
c c
aa
bb
c c
Cajanus cajan
Days
0 60 120
Bra
nch
es (
)
0
10
20
30
Days
0 60 120
Hei
ght
(cm
)
0
50
100
150
200
250
300
Sole Intercrop
aa
bb
c c
aa
bb
c c
Figure 210 Vegetative growth of Z mauritiana and C cajan growing under sole and intercropping
system (Bars represent means plusmn standard error of each treatment and significance among the
treatments was recorded at p lt 005)
101
Ziziphus mauritiana
Fresh Dry
Fru
it w
eig
ht
(g)
0
50
100
150
200
Days
0 60 120 180
Nu
mb
er o
f F
ruit
s
0
100
200
300
Sole Intercrop
a
b
a
b
c
c
dd
Cajanus cajan
0 60 120
Num
ber
of
Pods
0
50
100
150
200
Days
0 60 120
Num
ber
of
Flo
wer
s
0
50
100
150
Sole Intercrop
Days
aa
bb
c c
Sole Intercrop
Num
ber
of
See
ds
0
100
200
300
400
500
See
d W
eight
(g)
0
10
20
30
40
50
60Number of seedsSeed weight
Figure 211 Reproductive growth of Z mauritiana and C cajan growing under sole and intercropping
system (Bars represent means plusmn standard error of each treatment and significance among the
treatments was recorded at p lt 005)
102
Ziziphus mauritiana
Cajanus cajan
Figure 212 Leaf pigments of Zmauritiana and C cajan growing under sole and intercropping (Bars
represent means plusmn standard error of each treatment and significance among the treatments was
recorded at p lt 005)
Sole Intercrop
Car
ote
noid
s (m
g g
-1)
00
01
02
03C
hlo
rophyl
l (m
g g
-1)
00
02
04
06
08
ab r
atio
00
05
10
15
20
25
ab
ab
Sole Intercrop
Car
ote
no
ids
(mg
g-1
)
00
01
02
03
Ch
loro
ph
yll
(m
g g
-1)
00
02
04
06
08
10
ab
rat
io
0
1
2
3
4ab
ab
103
Ziziphus mauritiana
Sole Intercrop
Lea
f P
hen
ols
(m
g g
-1)
0
2
4
6
8
10
12
Lea
f P
rote
ins
(mg
g-1
)
0
2
4
6
8
Lea
f S
ug
ars
(mg
g-1
)
0
5
10
15
20
25
30
35SoluableInsoluable
Figure 213 Sugars protein and phenols in leaves of Z mauritiana and C cajan at grand period of growth under
sole and intercropping system (Bars represent means plusmn standard error of each treatment and
significance among the treatments was recorded at p lt 005)
104
(Figure 212 continuedhellip)
Cajanus cajan
Sole Intercrop
Lea
f P
hen
ols
(m
g g
-1)
0
2
4
6
8
Lea
f P
rote
ins
(mg g
-1)
00
05
10
15
20
Lea
f S
ugar
s (m
g g
-1)
0
2
4
6
8
105
Ziziphus mauritiana
Sole Intercrop
Fru
it P
hen
ols
(m
g g
-1)
0
2
4
6
8
10
12
14
Fru
it P
rote
ins
(mg g
-1)
00
02
04
06
08
10
Fru
it S
ugar
s (m
g g
-1)
0
5
10
15
20
25
30
35 SoluableInsoluable
Figure 214 Sugars protein and phenols in fruits of Z mauritiana grown under sole and intercropping
system (Bars represent means plusmn standard error of each treatment and significance among the
treatments was recorded at p lt 005)
106
Z mauritiana
Sole Intercrop
Nit
rogen
(
)
0
1
2
3
4
5
6
7 LeafSoil
Cajanus cajan
Sole Intercrop
Nit
rogen
(
)
0
1
2
3
4
5
6
7 LeafSoil
Figure 215 Nitrogen in leaves and in soil of Z mauritiana and C cajan growing under sole and intercrop
system (Bars represent means plusmn standard error of each treatment and significance among the
treatments was recorded at p lt 005)
107
Table 22 Land equivalent ratio (LER) and Land equivalent coefficient (LEC) with reference to height chlorophyll and yield of of Z mauritiana and C cajan growing
under sole and intercropping system
Plant species Parameters Formulated with
reference to Height
Formulated with
reference to Total
Chlorophyll
Formulated with reference to Yield
(fresh weight of Z mauritiana fruit
and seed of C cajan)
Z mauritiana Partial LER 1027 1666 1159
C cajan Partial LER 0950 0877 0993
Intercropped
Total LER 1977 2543 2152
Z mauritiana amp C cajan
(Sole and intercropped) LEC 0975 1461 1151
107
108
24 Discussion
Intercropping is a common practice used to obtain better yield on a limited area through
efficient utilization of given resources which may not be achieved by growing each crop
independently (Mucheru-Muna et al 2010) In this system selection of appropriate crops
planting rates and their spatial arrangement can reduce competition for light water and
nutrients (Olowe and Adeyemo 2009) In general increased growth (biomass height
volume circumference biomass succulence SSL SRL SSR LWR SWR RWR and
RGR) of each species is a good indicator of successful intercropping The SRL and SSL
measure the ratio between the lengths of root or shoot per unit dry weight of respective
tissues (Wright and Westoby 1999) The weight ratio of leaf stem and root to total plant
weight (LWR SWR and RWR) describes the allocation of biomass towards each organ to
maximize overall relative growth rate (RGR) which explains how plant responds to certain
type of condition (Reynolds and Antonio 1996) In this study height and canopy volume
of Z mauritiana and height and branches of C cajan were increased when grown together
in comparison to sole crop in field experiment (Figure 29) Whereas in drum pot culture
biomass generally the length of plant canopy volume number of leaves RGR LWR
SWR RWR SSL and SRL were either higher or unaffected in both species growing in
intercropping at 4th and 8th days intervals (Figure 21-23) Similar beneficial effects on
growth of other intercrops have also been reported under different conditions (Yamoah
1986 Atta-Krah 1990 Kass et al 1992 Singh et al 1997) Dhyani and Tripathi (1998)
observed increased height stem diameter crown width and timber volume of three
intercropped species than sole crop Bhat et al (2013) also revealed significant
improvement in annual extension height and spread in apple plants intercropped with
leguminous plants
The increased growth of both intercropped plants of this study was well reflected
by their biochemical parameters Leaf pigments like chlorophyll a chlorophyll b and total
chlorophyll were either higher or remained unaffected (Figure 211) in both intercropped
plants than sole crops of field experiments Whereas in drum pot culture chlorophyll
content (Figure 24) was higher only in intercropped C cajan (specially in 8th days) Bhatt
et al(2008) and Massimo and Mucciarelli (2003) also reported the increased accumulation
of chlorophyll a b and total chlorophylls in leaves of soybean and peppermint when
109
grown with their respective intercrops Our results are also in agreement with Liu et al
(2014) and Otusanya et al (2008) reported similar results in Lycopersican esculentum and
later in Capsicum annum as well Some other reports are also available which shows non-
significant effect on leaf pigments in both cropping systems (Shi-dan 2012 Luiz-Neto-
Neto et al 2014)The synthesis and activity of chlorophyll depends on severity and type
of applied stress it generally increase in low saline mediums (Locy et al 1996) or
remained unaffected however sometimes stimulated (Kurban et al 1999 Parida et al
2004 Rajesh et al 1998)
Proteins and carbohydrates (sugars) perform vast array of functions which are
necessary for plant growth and reproduction (Copeland and McDonald 2012) Variation
in their contents helps to predict plant health which is usually decreased with applied stress
(Arbona et al 2013) Both are also the compulsory factors of animals diet since they
cannot manufacture sugars and some of the components of proteins which must be
obtained from food (Bailey 2012) In our experiment protein content was either remained
unchanged or increased which indicated a good coordination of both intercrops in field
and drum pot experiments (Figure 26 and 212) Liu et al (2014) also found that protein
and sugars were not affected in tomatogarlic intercrops In another experiment similar
results were found when corn was grown with and without intercropping (Borghi et al
2013)
Reactive oxygen species (ROS) are produced as a spinoff of regular metabolism
however under stress the overproduction of ROS may lead to oxidative damage (Baxter et
al 2014) In low concentrations ROS worked as messengers to regulate several plant
processes and also helps to improve tolerance to various biotic and abiotic stresses (Miller
et al 2009 Nishimura and Dangl 2010 Suzuki et al 2011) but when the concentration
goes beyond the critical limit ROS would become self-threatening at every level of
organization (Foreman et al 2003) To maintain a proper workable redox state an
efficient scavenging system of enzymatic (SOD CAT GPX and APX) andor non-
enzymatic (polyphenols sugars glutathione and ascorbic acid) antioxidants is required
which would be of critical importance when plant undergoes stress (Sharma et al 2012)
Among these enzymes SOD is a first line of defense which converts dangerous superoxide
radicals into less toxic product (H2O2) In further CAT APX and GPX worked in
association to get rid off from the excessive load of other oxygen radicals or ions (H2O2
110
OH- ROO etc) In this study antioxidant enzymes (SOD CAT GPX and APX) were
found to work in harmony which was not affected during 4th day treatment in both species
in comparison to sole crop (Fig 27) showing strong antioxidant defense which was not
compromised by cropping system When comparing in 8th day treatment a significant
general increase in all enzyme activities were observed in both species except for SOD
and GPX of C cajan (Fig 27) These results displayed relatively better performance and
tight control over the excessive generation of ROS which would be predicted in this case
due to less availability of water than in 4th day treatment (Karatas et al 2014 Doupis et
al 2013) Similarly by coping oxidative burst and maintaining cellular redox equilibrium
plants were able to improve growth performance especially in Z mauritiana (Fig 21)
Water deficit affect stomatal conductance which could bring about changes in
photosynthetic performance hence overproduction of ROS is usually found among
different crops (Moriana et al 2002 Miller et al 2010) As a response tolerant plants
overcome this situation by increased activity of antioxidant enzymes which was evident in
Wheat Rice olive etc (Zhang and Kirkham 1994 Sharma and Dubey 2005 Guo et al
2006 Sofo et al 2005)
Phenolic compounds despite their role in physiological plant processes are
involved in adsorbing and neutralizing reactive oxygen species (ROS Ashraf and Harris
2004) The overproduction of ROS may cause several plant disorders Plants produce
secondary compounds like polyphenols to maintain balance between ROS generation and
detoxification (Posmyk et al 2009) Increased synthesis and accumulation of phenolic
compounds is reported to safeguard cellular structures and molecules especially under
biotic abiotic constraints (Ksouri et al 2007 Oueslati et al 2010) In this study
intercropped Z mauritiana of field and both species in drum pot culture showed higher
phenolic content than individual crop (Figure 25 and 212) which may be attributed to
adaptive mechanism for scavenging free radicals to prevent cellular damage (Rice-Evans
1996)
In terms of fruit yield we observed that Z mauritiana is suitable for intercropping
as suggested by Yang et al (1992) Number of flowers fruits and fruit fresh weight of
both species either increased considerably or no-affected in intercropped plants compared
to individual ones (Figure 210) Moreover fruit quality of Z mauritiana includes proteins
phenols and soluble extractable and total sugars were also higher in intercropped plants
111
(Figure 213) Results of this study are better than other experiments reported by
Sharma (2004) Kumar and Chaubey (2008) and Kumar et al (2013) who did not find
influence of other understory forage crops (like Aonla) on the yield of Z mauritiana
However in other case the yield of intercropped ber was some time higher (Liu 2002)
Singh et al 2013 found no adverse effects on the yield of pigeonpea when intercropped
with mungbean however it improved the grain yield of associated species
A leguminous plant C cajan is used in this experiment as secondary crop which
can supplement Z mauritiana by improving soil fertility Results of both experiments
showed that the nitrogen was higheror un-affected (Figure 214) in soils of intercropped
plants which supports our hypothesis that leguminous intercrop increase N supply This
can be achieved by acquisition of limited resources to manage rootrhizosphere
interactions which can improve resource-use efficiency (Zhang et al 2010
Shen et al 2013 White et al 2013b Ehrmann and Ritz 2014 Li et al 2014) As a
consequence it impact on overall plant performance which starts from high photosynthetic
activity by increasing chlorophyll results in more availability of photoassimilate for
growth and reproductive allocation (Eghball and Power 1999) Use of C cajan in tree
intercropping proved beneficial for producing high yield crops and for the environment
(Gilbert 2012 Glover et al 2012)
Land equivalent ratio (LER) is commonly used to evaluate the effectiveness of
intercropping by using the resources of same environment compared with sole crop
(Vandermeer 1992 Rao et al 1990 1991 Cao et al 2012) It is the ratio of area for sole
crop to intercrop required to produce the equal amount of yield at the same management
level (Mead and Willey 1980 Dhima et al 2007) On the other hand land equivalent
coefficient (LEC) describe an association that concern with the strength of relationship It
is the proportion of biomassyield of one crop explained by the presence of the other crop
The LER 1 or more indicate a beneficial effect of both species on each other which increase
the yield of both crops as compare to single one (Zada et al 1988) In this experiment all
LER values were about 2 or more than 2 while LEC values were around 1 or more than
one in ZizyphusCajnus intercropping Both LER and LEC values were in descending
order of chlorophylls gt yield gt height (Table 22) However the partial LER was higher in
Zizyphus than Cajanus in all cases These results describe the superiority of intercropping
over sole cropping where LER values are even gt2 Some other studies reported LER from
112
09-14 (Bests 1976) 12-15 (Cunard 1976) and up to 2 (Andrews and Kassam 1976)
Similar results were reported in poplarsoybean system (Rivest et al 2010) black
locustMedicago sativa (Gruenewald et al 2007) wheatjujube (Zhang et al 2013)
Acacia salignasorghum (Droppelmann et al 2000 Raddad and Luukkanen 2007) The
high LER values in our system indicating a harmony in resource utilization in both species
which was also corroborated with their respective LEC values The greater LEC values (gt
025) suggesting an inbuilt tendency of studied crops to give yield advantage (Kheroar and
Patra 2013) Experiments based on traditional practices of growing legumes with cereals
demonstrated greater and continuous cash returns than individual-crops (Baker 1978) In
addition the same authors found further increase in cash returns by increasing the
proportion of cereal and incorporating maize with sorghum and millet In agreement with
our findings similar reports are also available from different intercropping systems
including sesamegreengram (Mandal and Pramanick 2014) maizeurdbean (Naveena et
al 2014) and pegionpeasorghum (Egbe and Bar-Anyam 2010)
After detailed investigations of both species using two different experiment designs
(drum pot and field) it is evident that intercropping had beneficial effects on growth
physiology biochemisty and yield of both species Furthermore by using this system
higher outcome interms of edible biomass and green fodder using marginal lands can be
obtained in a same time using same land and water resources which can help to eliminate
poverty and uplift socio-economic conditions
113
3 Chapter 3
Investigations on rang of salt tolerance in Carissa carandas
(varn karonda) for determining possibility of growing at waste
saline land
31 Introduction
Carissa carandas commonly known as Karonda or lsquoChrist thornrsquo belonging to family
Apocynaceae shows capability of growing under haloxeric conditions It is an important
plant which has established well at tropical and subtropical arid zone under high
temperatures It is large evergreen shrub and having short stem It has fork thorn and hence
used as hedges or fence around fields The leaves are oval or elliptic 25 to 75 cm long
dark green leathery and secrete white milk if detached The fruits are oblong broad- ovoid
or round 125- 25 cm long It has thin but tough epicarp Fruits are in clusters of 3-10
Young fruits are pinkish white and become red or dark purple on maturation
The plant is propagated through seed in August and September Budding and cutting
could also be undertaken Planting is started after first shower of monsoon Plants raised
from seeds are able to flower within two years Flowering starts in March and fruit ripen
from July to September (Kumar et al 2007) The fruit possess good amount of pectin and
acidity hence used in prickle jelly jam squash syrup and in chutney by the commercial
name lsquoNakal cherryrsquo (Mandal et al 1992) They are rich in vitamin C and good source
of Anthocyanin (Lindsey et al 2000) Its fruits also are one of the richest source of iron
(391 mg 100gm) (Tyagi et al 1999) Juice of its root is also used to treat various
microbial diseases such as diarrhea dysentery and skin disease (Taylor et al 1996)
Hence its range of salt and suitability for cultivation at waste saline land or with saline
water irrigation is being undertaken for commercial exploitation by preparing jams jellies
and prickles (Kumar 2014) Investigations on its growth and development at higher range
of salinities are being undertaken with an interest to cultivate it if profitable at highly saline
waste land
114
32 Experiment No 9
Investigation on the effect of higher range of salinities on growth of
Carissa carandas (varn karonda) created by irrigation of different
dilutions of sea salt
321 Materials and methods
3211 Drum Pot Culture
Drum pot culture as recommended by Boyko (1966) and modified by Ahmed and
Abdullah (1982) was used for the present investigation which was been already described
in Chapter 1 earlier
3212 Plant material
About six months old sapling of Carissa carandas (varn Karonda) having almost equal
height and volume poted in polythene bag in 3kg of soil fertilized with cow-dong manure
were purchased from the Noor nursery Gulshan-e-Iqbal Karachi Sindh and were
transported to the Biosaline research field department of Botany University of Karachi
3213 Experimental setup
Plants were transplanted in drum pot (Homemade lysimeter) filled with sandy loam mixed
with cow dung manure (91) Each drum pot was irrigated weekly during summer and
fortnightly during winter months with 20 liters tap water (Eciw= 0 6 dSm-1) or water of
sea salt concentrations of various ie 03 (Eciw = 42 dSm-1) 04 (Eciw =61 dSm-1)
06 (Eciw = 99 dSm-1) and 08 (Eciw = 129 dSm-1) The plants were established initially
by irrigation with tap water for two weeks and later salinity was gradually increased till
desired percentage is achieved for different treatments by dessolving of sea salt in
irrigation water Three replicates were maintained for each treatment Urea DAP and
KNO3 were the source of NPK provided in the ratio 312 50g granules Osmocot (Scotts-
Sierra Horticulture Products) and 50g Mericle-Gro (Scotts Miracle-Gro Products Inc)
were dissolved in irrigation water per drum after six months at six monthly intervals
Height and volume of canopy of these plants were recorded prior to the starting the
experiment and then after every six months interval
115
Since the vegetative growth performance in plants irrigated with 03 sea salt (Eciw = 42
dSm-1) was found comparatively better than control and only 26 decrease was noticed
in volume of canopy at plant irrigated with 04 sea salt (Eciw = 61 dSm-1) (Table III41)
the onward investigations were focused at higher salinity levels and plants were irrigated
with 06 (Eciw = 99 dSm-1) and 08 (Eciw = 129 dSm-1) sea salt in rest of experiment
3214 Vegetative parameters
Vegetative growth on the basis of plant height and volume were recorded while
reproductive growth was observed on the basis of number of flowers and number and
weight of fruits per plant Length and diameter of fruit were also recorded in ten randomly
selected fruits
3215 Analysis on some biochemical parameters
Following biochemical analysis of leaves was performed at grand period of growth (onset
of flowers)
i Photosynthetic pigments
Fresh fully expended leaves (01g) was crushed in 80 chilled acetone Further procedure
was followed described in chapter 1
ii Soluble sugars
Dry leaf samples (01g) were milled in 5mL of 80 ethanol and were centrifuged at 4000
g for 10 minutes Same procedure was followed as described in chapter 1
iii Protein content
The protein contents were measured according to Bradford Assay reagent method against
Bovine Serum Albumin which was taken for standard (Bradford 1976) as described in
chapter 1
iv Soluble phenols
The dried leaf powder (01g) was milled in 3ml of 80 methanol and was centrifuged at
10000g for 15 min Further procedure has been described in chapter 2
116
3216 Mineral Analysis
Estimation of Na+ and K+ were made according to Chapman and Pratt (1961) Oven dried
grinded Leaves (1g) furnace at 550ordmC for 6 hours and were digested in 5 ml of 2N HCl
Diluted and filtered solution was used to estimated Na+ and K+ in flame photometer
(Petracourt PFP I) The concentration of these ions was calculated against the following
standard curve equations
Na+ (ppm) = 0016135x1879824
K+ (ppm) = 0244346x1314603
117
322 Observations and Result
3221 Vegetative parameters
Vegetative growth in terms of height and volume of canopy of C carandas growing under
salinities created by irrigation of different dilutions of sea salt is presented in Table 32
Appendix-XIX A significant increase (plt0001) in plant height and volume of canopy
was observed with increasing time but the increase was rapid at early period of growth
However there was significant (plt0001) reduction under salinity stress The interaction
of time and salinity also showed significant (plt001) effect on plant parameters but the
increase in height and volume of canopy at Eciw= 42dSm-1of sea salt salinity was more
than control Plants irrigated with Eciw= 61 dSm-1 and Eciw= 99 dSm-1sea salt solution
showed decrease in height with respect to control but the difference between their
treatments was insignificantly higher decrease was observed in Eciw= 129 dSm-1 sea salt
irrigated plants
3222 Reproductive parameters
Reproductive growth in terms of flowers and fruits numbers flower shedding percentage
fresh and dry weight of ten fruit their length and diameter under salinities created by
irrigation of different dilutions of sea salt is presented in Table 33 Appendix-XX Number
of flowers and fruits significantly (plt0001) decreased with increasing salinity treatment
Difference in flower initiation seems non-significant at early growth period in controls and
salinity treatments However drastic decrease was observed in plants irrigated beyond
Eciw= 99 dSm-1 with increase in salinity
Flowers shedding percentage (Table 33 Appendix-XX) show an increase directly
proportional with increase in salinity however the difference in number of flowers
between the plants irrigated with Eciw= 99 dSm-1 and Eciw= 129 dSm-1 sea salt solution
is of little significance level (plt001)
Fresh and dry weight of average fruits (plt001) and their diameter (plt001) showed
decrease with increasing salinity whereas diameter and length of fruits showed non-
significant difference
118
3224 Study on some biochemical parameters
i Photosynthetic Pigments
Photosynthetic Pigments including Chlorophyll a chlorophyll b total chlorophyll
chlorophyll a b ratio and carotenoids of C carandas growing under salinities created by
irrigation of different dilutions of sea salt is presented in Figure 31 Appendix-XX The
chlorophyll contents of leaves significantly decreased (plt0001) over control with
increasing salinity however Chlorophyll rsquobrsquo at Eciw= 99 dSm-1salinity shows significant
increase (plt0001) over control Similarly Carotenoids at Eciw= 99 dSm-1 salinity show a
bit less significant increase (plt001) compare to control while at higher salinity (Eciw=
129 dSm-1) the decline is observed at all above mentioned parameters
iii Protein Sugars and phenols
Some biochemical parameters including Protein sugars and phenolic contents of C
carandas growing under salinities created by irrigation of different dilutions of sea salt is
presented in Figure 31 Appendix-XX Soluble proteins in leaves show non-significant
decrease at Eciw= 99 dSm-1salinity as compared with controls but a significant decrease
(plt005) was noted at Eciw= 129 dSm-1 salinity Sugars also showed non-significant
decrease at both the salinity whereas on contrary soluble phenols showed significant
increase (plt0001) with increasing salinity
3225 Mineral analysis
Mineral analysis including Na and K ions performed in leaves of C carandas growing
under salinities created by irrigation of different dilutions of sea salt is presented in Figure
32 Appendix-XX Sodium significantly increased (plt0001) all the way with increasing
salinity of growth medium Whereas significant decrease (plt0001) was observed in
Potassium with increasing salinity K+Na+ ratio show continuous increase with increasing
salinity
119
Table 31 Electrical conductivities of different sea salt concentration used for determining
their effect on growth of C carandas
Treatment
Sea salt ()
ECiw of irrigation water (dSm-1) ECe of soil saturated paste
(dSm-1)
Non-saline control 06 09
03 42 48
04 61 68
06 99 112
08 129 142
Whereas ECiw and ECe are the electrical conductivities of irrigation water and soil saturated past measured in deci semen per meter
120
Table 32Vegetative growth in terms of height and volume of canopy of C carandas growing under salinities created by irrigation of different dilutions of
sea salt
Treatment
Sea salt
(ECiw dSm-1)
Initial values prior to
starting saline water
irrigation
Growth at different salinities after 06 months
Height Volume Height Volume of canopy
cm m3 cm
increase
over initial
values
increase
decrease over
control
m3 increase over
initial values
increase
decrease
over control
Control 3734plusmn455 0029plusmn0001 8227plusmn4919 5363plusmn830 - 014plusmn0015 7952plusmn269 -
42 3674plusmn1415 0026plusmn0003 9930plusmn6142 6280plusmn205 +1710 019plusmn0017 8593plusmn098 +806
61 3752plusmn1243 0026plusmn0001 6490plusmn5799 4132plusmn485 -2305 012plusmn0010 7740plusmn117 -282
99 3819plusmn4499 0028plusmn0005 5793plusmn5821 3123plusmn1446 -4185 009plusmn0008 6759plusmn377 -1499
129 3676plusmn3114 0026plusmn0008 5250plusmn4849 2775plusmn1276 -4836 006plusmn0005 5690plusmn1110 -2844
LSD0 05
Salinity
Time Fisherrsquos least significant difference
91
172
002
0005
Values represent means plusmn standard error of each treatment and significance among the treatments was recorded at p lt 005
120
121
Table 33 Vegetative growth in terms of height and volume of canopy of C carandas growing under salinities
created by irrigation of different dilutions of sea salt
Treatment
Sea salt
(ECiw dSm-1)
Growth at different salinities after 12 months
Height Volume of canopy
cm
increase
over initial
values
increase
decrease over
control
m3
increase
over initial
values
increase
decrease over
control
Control 16214 plusmn633 7674plusmn307 - 077plusmn012 9689plusmn449 -
99 9736plusmn1048 6056plusmn561 -2109 034plusmn006 9367plusmn412 -333
129 6942plusmn565 4741plusmn480 -3822 022plusmn002 9064plusmn623 -645
Table 33 continuedhellip
Treatment
Sea salt
(ECiw= dSm-1)
Growth at different salinities after 18 months
Height Volume of canopy
Cm
increase
over initial
values
increase
decrease over
control
m3
increase
over initial
values
increase
decrease over
control
Control 1676plusmn1135 7776plusmn756 - 094plusmn011 9701plusmn578 -
99 10547plusmn842 6351plusmn666 -1833 045plusmn010 9445plusmn1024 -264
129 7581plusmn593 5154plusmn716 -3372 030plusmn003 9318plusmn580 -395
Table 33 continuedhellip
122
Table 33 continuedhellip
Treatment
Sea salt
(ECiw= dSm-1)
Growth at different salinities after 24 months
Height Volume of canopy
Cm
increase
over initial
values
increase
decrease over
control
m3
increase
over initial
values
increase
decrease over
control
Control 1911plusmn6
05 8055plusmn941 - 121plusmn015 9837plusmn522 -
99 1110plusmn5
31 6557plusmn543 -1859 053plusmn002 9509plusmn1032 -334
129 8754plusmn10
67 5990plusmn801 -2564 040plusmn008 9287plusmn745 -560
Table 33 continuedhellip
Treatment
Sea salt
(ECiw= dSm-1)
Growth at different salinities after 30 months
Height Volume of canopy
Cm
increase
over initial
values
increase
decrease over
control
m3
increase
over initial
values
increase
decrease over
control
Control 2052plusmn1126 8182plusmn676 - 146plusmn029 9873plusmn729 -
99 11700plusmn816 6743plusmn610 -1759 070plusmn011 9565plusmn850 -312
129 9628plusmn552 6189plusmn573 -2436 050plusmn004 9417plusmn1011 -462
LSD0 05 Salinity 77 007
Time 168 016
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and significance among the treatments was recorded at p lt 005
123
Table 34 Reproductive growth in terms of flowers and fruits numbers flower shedding percentage fresh and dry weight of ten fruit and their totals
perplant fruit length and diameter of C carandas growing under salinities created by irrigation of different dilutions of sea salt
Treatment
Sea salt
(ECiw= dSm-1)
Flower Fruits Flower
shedding
Weight of
Ten
fruit(fresh)
Weight of
Ten
fruit(dry)
Weight of
total fruitplant
(fresh)
Weight of
total fruitplant
(dry)
length
fruit
diameter
fruit
Numbers Numbers g g g g mm mm
Control 19467plusmn203 16600plusmn231 1468plusmn208 2282plusmn022 605plusmn009 37891plusmn891 10047plusmn283 1800plusmn003 1423plusmn006
99 12050plusmn202 7267plusmn491 3980plusmn307 1880plusmn035 530plusmn029 13695plusmn1174 3880plusmn469 1732plusmn037 1297plusmn011
129 12567plusmn549 6967plusmn203 4449plusmn082 1541plusmn023 435plusmn026 10742plusmn470 3041plusmn268 1711plusmn015 1233plusmn038
LSD0 05 Salinity 1514 1417 929 115 097 3785 1494 0971 097
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and significance among the treatments was recorded at p lt 005
123
124
Sea Salt (ECiw
= dSm-1
)
Cont 99 129
Car
ote
nio
ds
(mg
g-1
)
00
01
02
03
04
Ch
loro
ph
yll
(m
g g
-1)
00
01
02
03
04
05
06
ab
rat
io
00
05
10
15
20
25
30
35
ab
Chl a Chl b
a
a
a a
b
bcbc
a
b
c
a a
b
Figure 31 Chlorophyll a chlorophyll b total chlorophyll chlorophyll a b ratio carotenoids contents of C
carandas growing under salinities created by irrigation of different dilutions of sea salt (Bars
represent means plusmn standard error of each treatment and significance among the treatments was
recorded at p lt 005)
125
Sea Salt (ECiw
= dSm-1
)
Cont 99 129
Ph
eno
ls (
mg
g-1
)
0
5
10
15
20
Pro
tein
s (m
g g
-1)
0
1
2
3
4
Su
gar
s (m
g g
-1)
0
30
60
90
120
150Soluble Insoluble
a
a
a
a
a
a
b
b
b
c
ab
a
a
b
Figure 32 Total protein sugars and phenolic contents of C carandas growing under salinities created by
irrigation of different dilutions of sea salt (Bars represent means plusmn standard error of each treatment
and significance among the treatments was recorded at p lt 005)
126
Sea Salt (ECiw
= dSm-1
)
Cont 99 129
Ions
(mg
g-1
DW
)
0
20
40
60
80
100
120
KN
a ra
tio
00
01
02
03
04
05
06
07
Na K KNa
c
a
b
b
a
c
a
b
c
Figure 33 Mineral analysis including Na and K ions was done on leaves of C carandas growing under salinities
created by irrigation of different dilutions of sea salt (Bars represent means plusmn standard error of each
treatment and significance among the treatments was recorded at p lt 005)
127
33 Discussion
The volume and height of plants were increased per unit time under saline conditions This
increase was observed after six months in 03 sea salt (ECiw = 42 dSm-1) treated plants in
comparison to control (Table 32) Slight decrease was observed at 04 sea salt
(ECiw=61dSm-1) irrigation after which (Eciw= 99 dSm-1 and Eciw = 129 dSm-1sea salt) the
growth was significantly inhibited (Table 33) Noble and Rogers (1994) also noticed a general
decrease in growth of some of the glycophytes Humaira and Ahmad (2004) and Rivelli et al
(2004) also reported a proportional decrease in height of canola with increasing salinity
Cotton plants irrigated with saline water or those grown at saline soil are reported to increase
Na+ content in leaves accompanied by significant reduction in vegetative biomass (Meloni et
al 2001) Bayuelo-Jimenez et al (2003) observed salt induced growth inhibition of tomato
plant which was higher in shoot than root
Reproductive growth in terms of number of flowers number of fruits fruit length and
diameter were decreased and percent flower shedding increased with increasing salinity
(Table 34) These effects were higher at Eciw= 99 dSm-1and then maintained with further
salinity increment However weight of fruits (fresh and dry) and total fruits per plant were
linearly decreased with increasing medium salt concentrations A decrease in different phases
of reproductive growth like flowering fertilization fruit setting yield and quality of seeds etc
are reported to be seriously affected at different level of salinity by various workers (Lumis et
al 1973 Waisel 1991 Shannon et al 1994 Tayyab et al 2016) Cole and Mclead (1985)
and Howie and Lloyd (1989) reported severe effects of different salinity treatments on
flowering intensity fruit setting and number of fruits of Citrus senensis Walker et al (1979)
also reported reduction in the fruit weight during early ripening stage of Psidium guajava
Decrease in fruit diameter of strawberries (Fragaria times ananassa) has been reported with
salinity (Ehlig and Bernstein 1958)
In this study photosynthetic pigments of C carandas were decreased with salinity and
this decrease was more sever at Eciw = 129 dSm-1sea salt salinity (Figure 31) Such a decline
in amount of leaf pigments across different salinity regimes was also reported in cotton
(Ahmed and Abdullah 1979) Pea (Hernandez et al 1995 and Hernandez et al 1999) Vicia
128
faba (Gadallah 1999) Mulberry genotype (Agastian et al 2000) and B parviflora (Parida et
al 2004)
Leaf sugars and protein were decreased in both salinity levels (Figure 32) which could
be attributed to inhibition in transport of photosynthetic product (Levit 1980) Decrease
synthesis and mobilization of glucose fructose and sucrose has been demonstrated in number
of plants growing under salt stress (Kerepesi and Galiba 2000) Inhibition in the protein and
nucleic acid synthesis in Pisum sativum and Tamarix tetragyna plants were also reported by
Bar-Nun and Poljahoff-Mayber (1977) Melander and Harvath (1977) suggested that salt
induced reduction in protein is due to increase in protein hydrolysis
A significant increase in leaves phenol with increase in salinity (Figure 32) was
observed in present investigation was also demonstrated previously in Achilleacollina (Giorgi
et al 2009) Lactuca sativa (Kim et al 2008) and B parviflora (Parida et al 2004)
Inspite of over irrigation of saline water and maintaining leaching fraction of about
40 in drum pots accumulation of salts in rhizosphere soil was not completely avoided which
was evident in the differences between ECiw and ECe values (Table 31) Deposition of salts
in rhizosphere soil interferer absorption of minerals in plants For instance leaf Na+ content
of C carandas was significantly increased while K+ decreased with increasing soil salinity
(Figure 33) Over accumulation of toxic ions disturbed plant water status which directly
affects plant growth (Flowers et al 1977 Greenway and Munns 1980) A negative
relationship between Na+ and K+ concentration in roots and leaves of guava was also reported
by Ferreira et al (2001) Increase in Na+ content decreased K+ availability and K+Na+ ratio
in Vicia taba (Gadallah 1999) and also affect the uptake of other essential minerals in
Casurina equsetifolia (Dutt et al 1991)
Carissa carandas found to be a good tolerant to salinity and drought and it can produce
edible fruits from marginal lands of arid areas Fruits of this species can be consumed in a raw
form as well as in industrial products like pickles jams jellies and marmalades
129
4 Conclusions
In the light of above mentioned investigations it appears that pre-soaking treatment of Cajanus
cajan seeds has initiated metabolic processes at faster rate earlier which has helped seeds to
start germinative metabolism prior to be effected by toxic Na+ ions at higher salinities Cajanus
cajan and Ziziphus mauritiana were found to be the good companions for intercropping These
species synergistically enhanced the growth and biochemical performance of each other by
improving fertility of marginal land and maintaining harmony among different physiological
parameters which was missing in their sole crop Their intercropping could produce fodder
and delicious fruits even from under moderately saline substrate up to profitable extant
Carissa carandas also tolerated low and moderately salinities well by adjusting proper
regulation of physiological and biochemical parameters of growth It can provide protein rich
edible fruits jams jellies and pickles of commercial importance for benefit of poor farmer
from moderately saline barren land
130
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167
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168
6 THESIS APENDECES
Appendix-I One way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution of seed germination of pre-soaked seeds of C cajan in non-saline water prior to germination under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Mean
germination rate
(GR)
Salinity treatment 4422 20 221133 21015 0000
Error 441949 42 10522
Total 4864 62
Mean germination
velocity (GV)
Salinity treatment 418813 20 20941 51836 0000
Error 169671 42 40398
Total 588484 62
Mean
germination
time (GT)
Salinity treatment 0271 20 0013 8922 0000
Error 0064 42 0002
Total 0335 62
Mean germination
Index (GI)
Salinity treatment 4422 20 221133 21015 0000
Error 441949 42 10523
Total 4864607 62
Final
germination
(FG)
Salinity treatment 32107 20 1605397 25285 0000
Error 2666 42 63492
Total 34774 62
Appendix-II Two way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution of seed germination of pre-soaked seeds of C cajan in non-saline water prior to germination under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Germination percentage per
day
Salinity treatment 509583 20 25479 19187 0000
Time 53156 9 5906 4663 0002
Salinity treatment times time 251743 180 1398576 1053 ns
Error 531130 400 1327825
Total 1375283 629
Germination
rate per day
Salinity treatment
Time 761502 9 84611 83129 0000
Salinity treatment times time 442265 20 22113 24630 0000
Error 359117 400 0898
Total 2108622 629
Appendix-III One way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution of seed
germination of pre-soaked seeds of C cajan in respective saline water prior to germination under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Final mean germination
velocity (GV)
Salinity treatment 0538 6 0089 35585 0000
Error 0035 14 0003
Total 0573
Final mean
germination time (GT)
Salinity treatment 20862 6 3477 26256 0000
Error 1854 14 0132
Total 22716 20
Final mean germination
index (GI)
Salinity treatment 110514 6 18419 190215 0000
Error 1356 14 0097
Total 111869 20
Final
germination percentage (GP)
Salinity treatment 6857 6 1142857 40 0000
Error 400 14 28571
Total 7257 20
Appendix-IV Two way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution of seed
germination of pre-soaked seeds of C cajan in respective saline water prior to germination under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Germination percentage per
day
Salinity treatment 86644 6 14440816 505428 0000
Time 23378 6 3896 136373 0000
Salinity treatment times time 2717 36 75472 2641 0001
Error 2800 98 28571
Total 115540 146
Germination rate
per day
Salinity treatment 117386 6 19564 360762 0000
Time 128408 6 21401 394636 0000
Salinity treatment times time 58747 36 1632 30091 0000
Error 5314 98 0054
Total 309855 146
169
Appendix-V One way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution on seedling
emergence and height of germinating seeds of C cajan under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Seedling height of C cajan
Salinity treatment 200822 5 40056 169666 0000
Error 2833 12 0236
Total 203115 17
Seedling
emergence of C cajan
Salinity treatment 24805 6 4134 6381 000
Error 9070 14 647867
Total 33875 20
Appendix-VI Two way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution on growth and
development of C cajan in lysemeter (Drum pot) under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Plant height of
C cajan
Salinity treatment 261079 5 52215 720259 0000
Time 126015 8 15751 132488 0000
Salinity treatment times time 76778 40 1919 16144 0000
Error 11413 96 118893
Total 477028 161
Appendix-VII One way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution on growth
and development of C cajan in lysemeter (Drum pot) under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Number of
Flowers of C
cajan
Salinity treatment 3932 3 131075 39719 0000
Error 264 8 33
Total 419625 11
Number of pods
of C cajan
Salinity treatment 1473 3 491 23105 0000
Error 170 8 2125
Total 1643 11
Number of
seedspod of C cajan
Salinity treatment 3 3 1
Error 0 8 0
Total 3 11
Number of seeds plant of
C cajan
Salinity treatment 19332 3 6444 45621 0000
Error 1130 8 14125
Total 20462 11
Weight of
seeds plant of C cajan
Salinity treatment 592976 3 197658 85572 0000
Error 18478 8 2309
Total 611455 11
Chlorophyll a
of C cajan
Salinity treatment 0117 3 0039 81241 0000
Error 0004 8 0000
Total 0121 11
Chlorophyll b
of C cajan
Salinity treatment 0004 3 0001 15222 0001
Error 0001 8 0000
Total 0005 11
Total chlorophyll of
C cajan
Salinity treatment 0160 3 0053 164401 0000
Error 0002 8 0000
Total 0162 11
Chlorophyll a b
ratio of C cajan
Salinity treatment 242 3 0806 9327 0005
Error 0692 8 0086
Total 3112 11
Carotenoids of
C cajan
Salinity treatment 0015 3 0005 4510 0039
Error 0009 8 0001
Total 0025 11
Soluble sugars
of C cajan
Salinity treatment 0043 3 0014 6515 0015
Error 00178 8 0002
Total 0061 11
Insoluble
sugars of C
cajan
Salinity treatment 0118 3 0039 36262 0000
Error 0008 8 0001
Total 0127 11
Total sugars of
C cajan
Salinity treatment 0019 3 0006 4239 0045
Error 0012 8 0001
Total 0031 11
Protein of C cajan
Salinity treatment 0212 3 0070 15735 0001
Error 0036 8 0004
Total 0248 11
170
Appendix-VIII One way ANOVA for completely randomized design for range of salt tolerance of nitrogen fixing symbiotic bacteria
associated with root of C cajan
Variables Source Sum of Squares df Mean Square F-value P
Nodule
associated
Rhizobial colonies of C
cajan
Salinity treatment 35927 2 17963 229402 0000
Error 1409 18 0078
Total 37337 20
Appendix-IX Two way ANOVA for completely randomized design for growth and development of Z mauritiana in large size clay pot being irrigated with water of two different sea salt concentration
Variables Source Sum of Squares df Mean Square F-value P
Height of
Z mauritiana
Time 91030 2 45515 839 0000
Salinity treatment 3268 2 1634 10 0000
Time times Salinity treatment 1533 4 383 238 ns
Error 6751 42 161
Total 104554 71
Number of
branches of
Z mauritiana
Time 25525 2 127625 25333 0000
Salinity treatment 86333 2 43166 11038 0000
Time times Salinity treatment 27416 4 6854 1752 ns
Error 16425 42 3910
Total 6575 71
Number of
flowers of
Z mauritiana
Time 73506 2 36753 167777 0000
Salinity treatment 12133 2 6066 25061 0000
Time times Salinity treatment 27824 4 6956 28736 0000
Error 10166 42 242063
Total 127759 71
Fresh weight of
Shoot of
Z mauritiana
Time 3056862 2 1528431 340777 0000
Salinity treatment 107829 2 53914 12020 0000
Time times Salinity treatment 51303 4 12825 2859 0031
Error 251167 56 4485
Total 3515820 71
Dry weight of Shoot of
Z mauritiana
Time 784079 2 392039 338932 0000
Salinity treatment 26344 2 13172 11387 0000
Time times Salinity treatment 13042 4 3260 2818 0033
Error 64774 56 1156690
Total 913855 71
Succulence of
Z mauritiana
Time 0002 2 0001 0214 ns
Salinity treatment 0006 2 0003 0682 ns
Time times Salinity treatment 0007 4 0002 0406 ns
Error 0199 45 0004
Total 51705 54
Spacific shoot
length of Z mauritiana
Time 0000 2 914 0176 0000
Salinity treatment 0002 2 0001 2096 ns
Time times Salinity treatment 0003 4 0001 1445 ns
Error 0023 45 0001
Total 6413 54
Moisture
contents of Z mauritiana
Time 1264 2 0632 0243 ns
Salinity treatment 3603 2 1801 0691 ns
Time times Salinity treatment 4172 4 1043 0400 ns
Error 117146 45 2603
Total 131675 54
Relative growth
rate of Z mauritiana
Time 1584206 1 1584206 532968 ns
Salinity treatment 18921 2 9460 3183 ns
Time times Salinity treatment 61624 2 30812 10366 0000
Error 89172 30 2972
Total 4034 36
Appendix-X One way ANOVA for completely randomized design for growth and development of Z mauritiana in large size clay pot
being irrigated with water of two different sea salt concentration
Variables Source Sum of Squares df Mean Square F-value P
Chlorophyll a
of Z mauritiana
Salinity treatment 0004 2 0002 7546 0003
Error 0006 21 0000
Total 0010 23
Chlorophyll b of Z mauritiana
Salinity treatment 0037 2 0018 4892 0018
Error 0080 21 0003
Total 0117 23
171
Total
chlorophyll of
Z mauritiana
Salinity treatment 0144 2 0072 39317 0000
Error 0038 21 0002
Total 0182 23
Chlorophyll ab ratio of
Z mauritiana
Salinity treatment 1499 2 0749 33416 0000
Error 0471 21 0022
Total 1969 23
Total soluble
sugars of
Z mauritiana
Salinity treatment 378271 2 189135 36792 0000
Error 107952 21 5140
Total 486223 23
Total protein contents of
Z mauritiana
Salinity treatment 133006 2 66502 5861 0009
Error 238268 21 11346
Total 371274 23
Appendix-XI Three way ANOVA for split-split plot design for physiological investigations on growth of Z mauritiana and C cajan in
drum pot being irrigated with water of sea salt concentration at two irrigation intervals
Variables Source Sum of Squares df Mean Square F-value P
Height of
Z mauritiana
Time 4499 2 2249 28888 0004
Crop 448028 1 448028 2208 ns
Irrigation intervals 2523 1 2523 2774 ns
Time times Crop 928088 2 464044 2288 ns
Time times irrigation interval 1120400 2 560200 0615 ns
Crop times irrigation interval 2690151 1 2690 2957 ns
Time times Crop times irrigation interval 171927 2 85963 0094 ns
Error 10916 12 909732
Total 35
Canopy volume of Z mauritiana
Time 7943 2 3971 6554 ns
Crop 0382 1 0382 0579 ns
Irrigation intervals 0068 1 0069 0103 ns
Time times Crop 0265 2 0133 0201 ns
Time times irrigation interval 1142 2 0571 0852 ns
Crop times irrigation interval 0722 1 0722 1077 ns
Time times Crop times irrigation interval 1998 2 0999 1491 ns
Error 8043 12 0670
Total 29439 35
Appendix-XII Two way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C
cajan in drum pot being irrigated with water of sea salt concentration at two irrigation intervals
Variables Source Sum of Squares df Mean Square F-value P
Plant length of
Z mauritiana
Crop 2986 1 2986 75322 0000
Irrigation interval 2986 1 2986 75322 0000
Crop times Irrigation interval 15336 1 153367 3868 ns
Error 317166 8 39645
Total 292428 12
Shoot length of
Z mauritiana
Crop 1069741 1 1069741 30890 0000
Irrigation interval 1069741 1 1069741 30890 0000
Crop times Irrigation interval 253001 1 253001 73058 0026
Error 27704 8 3463
Total 103376 12
Root length of
Z mauritiana
Crop 19763 1 19763 2671 ns
Irrigation interval 481333 1 481333 65059 0000
Crop times Irrigation interval 800333 1 800333 108177 0000
Error 59186 8 7398
Total 49165 12
Main branches
of Z mauritiana
Crop 33333 1 33333 5797 0042
Irrigation interval 48 1 48 8347 0020
Crop times Irrigation interval 0333 1 0333 0057 ns
Error 46 8 575
Total 2888 12
Lateral
branches of Z mauritiana
Crop 1344083 1 1344083 41356 0000
Irrigation interval 54675 1 54675 16823 0000
Crop times Irrigation interval 784083 1 784083 24125 0000
Error 26 8 325
Total 22465 12
Leaf numbers of
Z mauritiana
Crop 22465 12 98283 96482 0000
Irrigation interval 25025 1 25025 24566 0001
Crop times Irrigation interval 11907 1 11907 11688 0009
Error 8149 8 1018667
172
Total 2037850 12
Shootroot ratio
of Z mauritiana
Crop 0027 1 0027 1842 ns
Irrigation interval 0001 1 0001 0097 ns
Crop times Irrigation interval 0825 1 0825 54909 0000
Error 0120 8 0015
Total 27776 12
Plant fresh
weight of Z mauritiana
Crop 398107 1 398107 577818 0000
Irrigation interval 139514 1 139514 20249 0000
Crop times Irrigation interval 146898 1 146898 21321 0000
Error 5511 8 688982
Total 7248659 12
Plant dry weight of Z mauritiana
Crop 87808 1 87808 471436 0000
Irrigation interval 57893 1 57893 31082 0000
Crop times Irrigation interval 61132 1 61132 32821 0000
Error 14900 8 186257
Total 1875710 12
Stem fresh
weight of
Z mauritiana
Crop 46687 1 46687 227539 0000
Irrigation interval 17933 1 17933 87402 0000
Crop times Irrigation interval 20180 1 20180 98351 0000
Error 16414 8 205185
Total 1718530 12
Root fresh weight of
Z mauritiana
Crop 58450 1 58450 2295 0000
Irrigation interval 42186 1 42186 165641 0000
Crop times Irrigation interval 37307 1 37307 146487 0000
Error 203746 8 25468
Total 357145 12
Leaf fresh weight of
Z mauritiana
Crop 29970 1 29970 19089 0000
Irrigation interval 117018 1 1170187 7453 0025
Crop times Irrigation interval 2310 1 2310 14714 0004
Error 125596 8 15699
Total 699711 12
Stem dry weight
of Z mauritiana
Crop 13587 1 13587 216591 0000
Irrigation interval 11856 1 11856 18899 0000
Crop times Irrigation interval 6787763 1 6787 108197 0000
Error 50188 8 62735
Total 4689795 12
Root dry weight
of Z mauritiana
Crop 1358787 1 13587 216591 0000
Irrigation interval 1497427 1 14974 118615 0000
Crop times Irrigation interval 128773 1 12877 1020052 0000
Error 100993 8 12624
Total 124421 12
Leaf dry weight
of Z mauritiana
Crop 2374 1 2374 135380 0000
Irrigation interval 8748 1 8748 4987 ns
Crop times Irrigation interval 26403 1 2640 150539 0000
Error 140313 8 17539
Total 127170 12
Plant moisture of Z mauritiana
Crop 22082 1 22082 5608 0045
Irrigation interval 38702 1 38702 9830 0013
Crop times Irrigation interval 44406 1 44406 11279 0009
Error 31496 8 3937
Total 29872 12
Stem moisture of Z mauritiana
Crop 0005 1 0005 0000 ns
Irrigation interval 110663 1 110663 12023 0008
Crop times Irrigation interval 0897 1 0897 0097 ns
Error 73633 8 9204
Total 28532 12
Root moisture of Z mauritiana
Crop 235266 1 235266 16502 0003
Irrigation interval 3923 1 3923 0275 ns
Crop times Irrigation interval 0856 1 0856 0060 ns
Error 114051 8 14256
Total 17572 12
Leaf moisture
of Z mauritiana
Crop 130413 1 130413 47746 0000
Irrigation interval 22256 1 22256 8148 0021
Crop times Irrigation interval 210662 1 210662 77127 0000
Error 21850 8 2731
Total 38888 12
173
Relative growth
rate of Z mauritiana
Crop 0000 1 0000 287467 0000
Irrigation interval 0000 1 0000 164217 0000
Crop times Irrigation interval 0000 1 0000 179626 0000
Error 0000 8 0000
Total 0009 12
Relative water
contents of Z
mauritiana
Crop 37381 1 37381 1380 ns
Irrigation interval 49871 1 49871 1841 ns
Crop times Irrigation interval 13496 1 13496 0498 ns
Error 216649 8 27081
Total 50855 12
Chlorophyll a of Z mauritiana
Crop 0103 1 0103 32466 0000
Irrigation interval 0003 1 0003 1075 ns
Crop times Irrigation interval 0000 1 0000 0187 ns
Error 0025 8 0003
Total 1498 12
Chlorophyll b
of Z mauritiana
Crop 0027 1 0027 196164 0000
Irrigation interval 0002 1 0002 15656 0004
Crop times Irrigation interval 0006 1 0006 45063 0000
Error 0001 8 0000
Total 0456 12
Total chlorophyll
of Z mauritiana
Crop 0257 1 0257 53469 0000
Irrigation interval 0001 1 0001 0315 ns
Crop times Irrigation interval 0002 1 0002 0442 ns
Error 0038 8 0004
Total 3736 12
Chlorophyll a b ratio of
Z mauritiana
Crop 0002 1 0002 0028 ns
Irrigation interval 0169 1 0169 1696 ns
Crop times Irrigation interval 1064 1 1064 10643 0011
Error 0799 8 0099
Total 43067 12
Carotenoids of
Z mauritiana
Crop 0018 1 0018 42747 0000
Irrigation interval 0002 1 0002 5298 0050
Crop times Irrigation interval 0003 1 0003 8118 0021
Error 0003 8 0000
Total 0451 12
Phenol of
Z mauritiana
Crop 24641 1 24641 13168 000
Irrigation interval 5078 1 5078 2714 ns
Crop times Irrigation interval 10339 1 10339 5525 0046
Error 14969 8 1871
Total 6289 12
Proline of Z mauritiana
Crop 0001 1 0001 52288 0000
Irrigation interval 0000 1 0000 6972 0029
Crop times Irrigation interval 0000 1 0000 0358 ns
Error 0000 8 0000
Total 0005 12
Protein of Z mauritiana
Crop 200001 1 200001 296 ns
Irrigation interval 69264 1 69264 102 ns
Crop times Irrigation interval 4453 1 4453 006 ns
Error 540367 8 67545
Total 814086 11
CAT enzyme of
Z mauritiana
Crop 74171 1 74171 11404 0009
Irrigation interval 299930 1 299930 46117 0000
Crop times Irrigation interval 15336 1 15336 2358 ns
Error 52029 8 65036
Total 441467 11
APX enzyme of
Z mauritiana
Crop 191918 1 191918 6693 0032
Irrigation interval 4665 1 4665 162723 0000
Crop times Irrigation interval 336912 1 336912 11750 0009
Error 229383 8 28672
Total 5423 11
GPX enzyme of
Z mauritiana
Crop 0000 1 0000 0020 ns
Irrigation interval 0103 1 0103 5893 0041
Crop times Irrigation interval 0109 1 0109 6220 0037
Error 0140 8 0017
Total 0353 11
SOD enzyme Crop 8471 1 8471 1364 ns
174
of
Z mauritiana
Irrigation interval 6220 1 6220 1001 ns
Crop times Irrigation interval 21142 1 21142 3405 ns
Error 49664 8 6208
Total 85498 11
NR enzyme of
Z mauritiana
Crop 7520 1 75208333333 37253364154 0003
Irrigation interval 1360 1 1360 6737 0318
Crop times Irrigation interval 0016 1 0016 0079 ns
Error 1615 8 0201
Total 10512 11
Nitrate of
Z mauritiana
Crop 003 1 003 3028 ns
Irrigation interval 0018 1 0018 1831 ns
Crop times Irrigation interval 0003 1 0003 0336 ns
Error 0079 8 0009
Total 0130 11
Appendix-XIII Three way ANOVA for split-split design for physiological investigations on growth of Z mauritiana and C cajan in drum
pot being irrigated with water of sea salt concentration at two irrigation intervals
Variables Source Sum of Squares df Mean Square F-value P
Height of
C cajan
Time 14990 2 7495 235059 0000
Crop 7848 1 7848 42235 0000
Irrigation intervals 749056 1 749056 9676 0009
Time times Crop 2638 2 1319140 7098 00262
Time times irrigation interval 309932 2 154966 2001 ns
Crop times irrigation interval 9127 1 9127 0117 ns
Time times Crop times irrigation interval 31974 2 15987 0206 ns
Error 928935 12 77411
Total 29065 35
Apendix-XIV Two way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C
cajan in drum pot being irrigated with water of sea salt concentration at two irrigation intervals
Variables Source Sum of Squares df Mean Square F-value P
Plant length of C cajan
Crop 1056563 1 1056563 12331 0007
Irrigation interval 21675 1 21675 2529 ns
Crop times Irrigation interval 137363 1 137363 1603 ns
Error 68544 8 8568
Total 334030 12
Shoot length of C cajan
Crop 808520 1 808520 36580 0000
Irrigation interval 165020 1 165020 7466 0025
Crop times Irrigation interval 285187 1 285187 12902 0007
Error 17682 8 22102
Total 224013 12
Root length of C cajan
Crop 16567 1 16567 0674 ns
Irrigation interval 3520 1 3520 0143 ns
Crop times Irrigation interval 26700 1 26700 1087 ns
Error 196453 8 24556
Total 11133 12
Main branches
of C cajan
Crop 80083 1 80083 64066 0000
Irrigation interval 10083 1 10083 8066 0021
Crop times Irrigation interval 075 1 075 06 ns
Error 10 8 125
Total 335 12
Letral branches
of C cajan
Crop 0 1 0
Irrigation interval 0 1 0
Crop times Irrigation interval 0 1 0
Error 0 8 0
Total 0 12
Leaf numbers
of C cajan
Crop 1776333 1 1776333 16679 0003
Irrigation interval 972 1 972 9126 0016
Crop times Irrigation interval 176333 1 17633 1655 0234
Error 852 8 1065
Total 22342 12
Shootroot ratio of C cajan
Crop 0385 1 0385 0638 0447
Irrigation interval 0007 1 0007 0011 0916
Crop times Irrigation interval 2669 1 2669 4424 0068
Error 4825 8 0603
Total 264061 12
Crop 76816 1 76816 7494853 0025
175
Plant fresh
weight of
C cajan
Irrigation interval 730236 1 730236 7124832 0028
Crop times Irrigation interval 266869 1 266869 2603812 0145
Error 81993 8 102491
Total 25941 12
Plant dry weight of C cajan
Crop 38270 1 38270 1150145 0009
Irrigation interval 53046 1 53046 15942 0003
Crop times Irrigation interval 20202 1 20202 6071 0039
Error 26619 8 3327
Total 4150 12
Stem fresh weight of
C cajan
Crop 16100 1 16100 1462 ns
Irrigation interval 9900 1 9900 0899 ns
Crop times Irrigation interval 00675 1 0067 0006 ns
Error 8806 8 11007
Total 3318 12
Root fresh weight of
C cajan
Crop 0190 1 0190 0248 ns
Irrigation interval 27331 1 27331 35753 0000
Crop times Irrigation interval 2698 1 2698 3529 0097
Error 6115 8 0764
Total 432050 12
Leaf fresh
weight of C cajan
Crop 541363 1 541363 13825 0005
Irrigation interval 347763 1 347763 8881 0017
Crop times Irrigation interval 208333 1 208333 5320 0049
Error 313246 8 39155
Total 7236 12
Stem dry weight
of C cajan
Crop 10323 1 10323 11530 0009
Irrigation interval 0452 1 0452 0505 ns
Crop times Irrigation interval 0232 1 0232 0259 ns
Error 7162 8 0895
Total 125151 12
Root dry weight
of C cajan
Crop 0007 1 0007 012 ns
Irrigation interval 0607 1 0607 972 0014
Crop times Irrigation interval 0367 1 0367 588 0041
Error 05 8 0062
Total 3515 12
Leaf dry weight
of C cajan
Crop 9363 1 9363 15649 0004
Irrigation interval 34003 1 3400 5683 0000
Crop times Irrigation interval 11603 1 11603 19392 0002
Error 4786 8 0598
Total 95072 12
Plant moisture of C cajan
Crop 199182 1 19918 6011 0039
Irrigation interval 272215 1 27221 8215 0020
Crop times Irrigation interval 76654 1 76654 2313 0166755
Error 265079 8 33134
Total 38272 12
Stem moisture
of C cajan
Crop 100814 1 10081 3290 0107246
Irrigation interval 53460 1 53460 1744 0223065
Crop times Irrigation interval 19778 1 1977 0645 0444938
Error 245119 8 30639
Total 31036 12
Root moisture
of C cajan
Crop 26266 1 26266 1389 ns
Irrigation interval 223809 1 223809 11836 0008
Crop times Irrigation interval 0097 1 0097 0005 ns
Error 151272 8 18909
Total 58346 12
Leaf moisture
of C cajan
Crop 2623 1 2623 39350 0000
Irrigation interval 1765 1 1765 26477 0000
Crop times Irrigation interval 1425 1 1425452 21378 0001
Error 533411 8 66676
Total 36263 12
Relative growth
rate of C cajan
Crop 0000 1 0000 17924 0002
Irrigation interval 0000 1 0000 21296 0001
Crop times Irrigation interval 0000 1 0000 88141 0017
Error 0000 8 0000
Total
Crop 256935 1 256935 1560 ns
Irrigation interval 268827 1 26882 1633 ns
176
Electrolyte
leakage of C
cajan
Crop times Irrigation interval 30379 1 30379 0184 ns
Error 1316923 8 16461
Total 50381 12
Chlorophyll a
of C cajan
Crop 0101 1 0101 7957 0022
Irrigation interval 0062 1 0062 4893 ns
Crop times Irrigation interval 0199 1 0199 15600 0004
Error 0102 8 0012
Total 5060 12
Chlorophyll b
of C cajan
Crop 0017 1 0017 7758 0023
Irrigation interval 0027 1 0027 12389 0007
Crop times Irrigation interval 0056 1 0056 25313 0001
Error 0017 8 0002
Total 1727 12
Total
chlorophyll of C cajan
Crop 0178 1 0178 14819 0004
Irrigation interval 0198 1 0198 16520 0003
Crop times Irrigation interval 0509 1 0509 42379 0000
Error 0096 8 0012
Total 13217 12
Chlorophyll a b
ratio of C cajan
Crop 0065 1 0065 0691 ns
Irrigation interval 0033 1 0033 0357 ns
Crop times Irrigation interval 0016 1 0016 0173 ns
Error 0756 8 0094
Total 35143 12
Carotenoids of C cajan
Crop 0021 1 0021 19599 0002
Irrigation interval 0028 1 0028 26616 0000
Crop times Irrigation interval 0041 1 0041 38531 0000
Error 0008 8 0001
Total 1443 12
Phenol of C cajan
Crop 0799 1 0799 3171 ns
Irrigation interval 0040 1 0040 0159 ns
Crop times Irrigation interval 0911 1 0911 3617 ns
Error 2016 8 0252
Total 970313 12
Proline of C cajan
Crop 0008 1 0008 14867 0004
Irrigation interval 0019 1 0019 34536 0000
Crop times Irrigation interval 0008 1 0008 14969 0004
Error 0004 8 0000
Total 0155 12
Protein of C
cajan
Crop 116376 1 116376 3990 ns
Irrigation interval 434523 1 434524 14899 0048
Crop times Irrigation interval 33166 1 33166 1137 ns
Error 233303 8 29163
Total 817371 11
CAT enzyme
of C cajan
Crop 0249 1 0249 0121 ns
Irrigation interval 2803 1 2803 13702 ns
Crop times Irrigation interval 92392 1 9239 4517 ns
Error 16362 8 2045
Total 28654 11
APX enzyme
of C cajan
Crop 855939 1 855939 4073 ns
Irrigation interval 1078226 1 1078226 5130 ns
Crop times Irrigation interval 13522 1 13522 64349 000
Error 1681112 8 210139
Total 17137 11
GPX enzyme
of C cajan
Crop 0965 1 0965 9265 0160
Irrigation interval 1167 1 1167 11195 0101
Crop times Irrigation interval 0887 1 0887 8514 0194
Error 0833 8 0104
Total 3854 11
SOD enzyme
of C cajan
Crop 4125 1 4125 9731 0142
Irrigation interval 4865 1 4865 11477 0095
Crop times Irrigation interval 20421 1 20421 48172 0001
Error 3391 8 0423
Total 32804 11
Nitrate
reductase
enzyme
Crop 0053 1 0053 0034 ns
Irrigation interval 0001 1 0001 0000 ns
Crop times Irrigation interval 10329 1 10329 6650 0327
177
of C cajan Error 12424 8 1553
Total 22808 11
Nitrate of
C cajan
Crop 0039 1 0039 0576 ns
Irrigation interval 0083 1 0083 1222 ns
Crop times Irrigation interval 0003 1 0003 0005 ns
Error 0545 8 0068
Total 0668 11
Appendix-XV Two way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C
cajan intercropped on marginal land under field condition
Variables Source Sum of Squares df Mean Square F-value P
Height of Z mauritiana
Time 79704 3 26568 77303 0000
Treatment 979209 1 979209 4702 0455
Time times Treatment 756019 3 252006 1210 3381 ns
Error 3332 16 208259
Total 90366 39
Canopy volume of Z mauritiana
Time 1049 3 3498 115444 0000
Treatment 3509 1 3509 5966 0266
Time times Treatment 3374 3 1124 1911 1684 ns
Error 9413 16 5883
Total 1284 39
flowers numbers of Z
mauritiana
Time 1794893 3 598297 770043 0000
Treatment 19980 1 19980 10152 0057
Time times Treatment 21017 3 7005 3559 0381
Error 31488 16 1968
Total 1882468 39
Fruits numbers
of Z mauritiana
Time 324096 3 108032 297941 0000
Treatment 10824 1 10824 64081 0000
Time times Treatment 7141 3 2380 14093 0001
Error 2702 16 168913
Total 351833 39
Appendix-XVI One way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C cajan intercropped on marginal land under field condition
Variables Source Sum of Squares df Mean Square F-value P
Weight of ten
fruits (FW) of
Z mauritiana
Treatment 557113 1 557113 6663 0032
Error 668923 8 83615
Total 1226036 9
Weight of ten fruits (DW) of
Z mauritiana
Treatment 4356 1 4356 0321 ns
Error 10862 8 13577
Total 112976 9
diameter of fruit of Zmauritiana
Treatment 0534 1 0534 0946 ns
Error 4514 8 0564
Total 5048 9
Fruit weight per plant of
Z mauritiana
Treatment 0739 1 0739 4022 ns
Error 1471 8 0184
Total 2211 9
Fruit sugar
(soluble) of
Z mauritiana
Treatment 5041 1 5041 0081 ns
Error 497328 8 62166
Total 502369 9
Fruit sugar (extractable) of
Z mauritiana
Treatment 32041 1 32041 0424 ns
Error 604384 8 75548
Total 636425 9
Total fruit
sugars of Z mauritiana
Treatment 16 1 16 0780 ns
Error 164 8 205
Total 18 9
Chlorophyll a of
Z mauritiana
Treatment 0082 1 0082 1384 0020
Error 0024 4 0006
Total 0105 5
Chlorophyll b
of Z mauritiana
Treatment 0011 1 0011 8469 0043
Error 0005 4 0001
Total 0016 5
Total chlorophyll of
Z mauritiana
Treatment 0152 1 0152 11927 0025
Error 0051 4 0013
Total 0203 5
Treatment 0015 1 0015 0867 ns
Error 0067 4 0017
178
Chlorophyll a b
ratio of Z mauritiana
Total 0082 5
Carotinoids of Z mauritiana
Treatment 0011 1 0011 9719 0035
Error 0004 4 0001
Total 0015 5
Leaf protein of
Z mauritiana
Treatment 0106 1 0106 4 ns
Error 0106 4 0027
Total 0213 5
Leaf sugars
(soluble) of
Z mauritiana
Treatment 054 1 054 0025 ns
Error 848 4 212
Total 8534 5
Leaf sugars
(Extractable) of Z mauritiana
Treatment 486 1 486 8055 0046
Error 2413 4 0603
Total 7273 5
Total sugars in
leaf of Z
mauritiana
Treatment 216 1 216 0104 ns
Error 83333 4 20833
Total 85493 5
Leaf phenols of
Z mauritiana
Treatment 8166 1 8166 5665 ns
Error 5766 4 1442
Total 13933 5
Leaf nitrogen of Z mauritiana
Treatment 15 1 15 1939 ns
Error 3093 4 0773333
Total 4593 5
Soil nitrogen of
Z mauritiana
Treatment 0375 1 0375 21634 ns
Error 0693 4 0173
Total 1069 5
Appendix-XVII Two way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C
cajan intercropped on marginal land under field condition
Variables Source Sum of Squares df Mean Square F-value P
Height of Ccajan
Time 700196 2 350098 2716 0000
Treatment 594405 1 594405 16017 0000
Time times Treatment 488829 2 244415 6586 0004
Error 1001996 27 37111
Total 705495 59
Number of branches of
Ccajan
Time 8353 2 4176 1050050 0000
Treatment 24066 1 24066 18672 0000
Time times Treatment 24133 2 12066 9362 0000
Error 348 27 1288
Total 8572 59
Number of flowers of
Ccajan
Time 289297 2 144648 301277 0000
Treatment 365066 1 365066 0701 ns
Time times Treatment 730133 2 365066 0701 ns
Error 14059 27 520733
Total 317415 59
Number of pods
of Ccajan
Time 347682 2 173841 70559 0000
Treatment 159135 1 159135 1558 ns
Time times Treatment 8167 2 40835 0399 ns
Error 27574 27 1021276
Total 447407 59
Appendix-XVIII One way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C
cajan intercropped on marginal land under field condition
Variables Source Sum of Squares df Mean Square F-value P
Shoot weight
(FW) of
Ccajan
Treatment 0 1 0 0 ns
Error 87444 4 21861
Total 87444 5
Shoot weight
(RW) of Ccajan
Treatment 0 1 0 0 ns
Error 13808 4 3452
Total 13808 5
Number of
seeds of
Ccajan
Treatment 245 1 245 0005 ns
Error 940182 18 52232
Total 940427 19
Weight of seeds
of Ccajan
Treatment 02 1 02 0000 ns
Error 7585 18 421406
Total 7585 19
179
Chlorophyll a of
Ccajan
Treatment 0001 1 0001 5442 ns
Error 0001 4 0000
Total 0002 5
Chlorophyll b
of Ccajan
Treatment 0006 1 0006 9079 0039
Error 0002 4 0001
Total 0008 5
Total
chlorophyll of
Ccajan
Treatment 0017 1 0017 51558 0001
Error 0001 4 0000
Total 0019 5
Chlorophyll a b ratio of
Ccajan
Treatment 0183 1 0183 5532 ns
Error 0132 4 0033
Total 0316 5
Leaf protein of Ccajan
Treatment 0001 1 0001 0017 ns
Error 0228 4 0057
Total 0228 5
Leaf sugars of
Ccajan
Treatment 0015 1 0015 0003 ns
Error 1624 4 406
Total 16255 5
Leaf phenols of
Ccajan
Treatment 0201 1 0201 0140 ns
Error 5746 4 1436
Total 5948 5
Leaf nitrogen
of Ccajan
Treatment 1306 1 1306 3062 ns
Error 1706 4 04266
Total 3013 5
Appendix-XIX Two way ANOVA for completely randomized design for investigations on determining range of salt tolerance in Carissa
carandas
Variables Source Sum of Squares df Mean Square F-value P
Height of C carandas
Time 72042 5 14408 55957 0000
Salinity treatment 49345 2 24672 196775 0000
Time times Salinity treatment 16679 10 1667920 13302 000
Error 3009 24 125385
Total 143777 53
Volume of
canopy of
C carandas
Time 3329 4 0832 38126 000
Salinity treatment 1393 2 0696 67129 000
Time times Salinity treatment 0813 8 0102 9792 000
Error 0207 20 0010
Total 5969 44
Appendix-XX One way ANOVA for completely randomized design for investigations on determining range of salt tolerance in Carissa carandas
Variables Source Sum of Squares df Mean Square F-value P
Number of
flowers of C carandas
Salinity treatment 10288 2 5144194 1342937 0000
Error 229833 6 38305
Total 10518 8
Number of fruits of
C carandas
Salinity treatment 18000 2 9000 268215 0000
Error 201333 6 33555
Total 18201 8
Flower shedding
percentage of C carandas
Salinity treatment 1541647 2 770823 53455 0000
Error 86519 6 144199
Total 1628166 8
Weight of ten fruits (FW) of
C carandas
Salinity treatment 82632 2 41316 187678 0000
Error 1321 6 0220
Total 83953 8
Weight of ten
fruits (DW) of
C carandas
Salinity treatment 4355 2 2177 13753 0005
Error 095 6 0158
Total 5305 8
Fruits per plant
(FW) of
C carandas
Salinity treatment 133127 2 66563 278148 0000
Error 1435861 6 239310
Total 134563 8
Fruits per plant
(DW) of C carandas
Salinity treatment 8782 2 439117 117790 0000
Error 223677 6 37279
Total 9006 8
Size of fruits of C carandas
Salinity treatment 1301 2 0651 4125 ns
Error 0946 6 0158
Total 2248 8
Salinity treatment 5607 2 2804 17592 0003
180
Diameter of fruit
of C carandas
Error 0956 6 0159
Total 6563 8
Chlorophyll a of C carandas
Salinity treatment 0112 2 0056 119786 0000
Error 0003 6 0000
Total 0115 8
Chlorophyll b of
C carandas
Salinity treatment 0005 2 0002 434 0000
Error 0000 6 0000
Total 0005 8
Total chlorophyll of C carandas
Salinity treatment 0159 2 0079 104188 0000
Error 0005 6 0001
Total 0164 8
Chlorophyll a b
ratio of C carandas
Salinity treatment 9661 2 4831 324691 0000
Error 0089 6 0015
Total 9751 8
Carotenoids of C carandas
Salinity treatment 0029 2 0014 28822 0000
Error 0003 6 0001
Total 0032 8
Leaf Protein of
C carandas
Salinity treatment 2722 2 1361 98 0012
Error 0833 6 0138
Total 3555 8
Soluble sugar of
C carandas
Salinity treatment 234889 2 117444 12735 0006
Error 55333 6 9222
Total 290222 8
In soluble sugars
of C carandas
Salinity treatment 595395 2 297698 39094 0000
Error 45689 6 7615
Total 641085 8
Total sugar of
C carandas
Salinity treatment 1576898 2 788448 39201 0000
Error 120676 6 20113
Total 1697574 8
Phenols of C carandas
Salinity treatment 14675 2 7338 74202 0000
Error 0593 6 0099
Total 15268 8
Leaf Na+ of
C carandas
Salinity treatment 1346 2 673 673 0000
Error 6 6 1
Total 1352 8
Leaf K+ of C carandas
Salinity treatment 798 2 399 133 0000
Error 18 6 3
Total 816 8
Leaf K+ Na+
ratio of C carandas
Salinity treatment 0305 2 0153 654333 0000
Error 0001 6 0000
Total 0307 8
181
7 Publications
ii
Investigation on intercropping of Ziziphus mauritiana with Cajanus
cajan for fruit and fodder at marginal land and cultivation of Carissa
carandas for fruits through saline water irrigation
PhD Thesis
Submitted to the Board of advance Studies and Research in fulfillment of
the Degree of Doctor of Philosophy in the Department of Botany
University of Karachi
TAYYAB
DEPARTMENT OF BOTANY
UNIVERSITY OF KARACHI
2015
iii
Investigation on intercropping of Ziziphus mauritiana with Cajanus
cajan for fruit and fodder at marginal land and cultivation of Carissa
carandas for fruits through saline water irrigation
Thesis Approved
RESEARCH SUPERVISOR EXTERNAL EXAMINER
PROF DR RAFIQ AHMAD
FPAS FTWAS
Professor (Retd) Botany (Plant Physiology)
PI Biosaline Research Projects
Department of Botany
University of Karachi
iv
CERTIFICATE
It is hereby certified that this thesis is based on the results of the experimental work carried
out by Mr TAYYAB SO MUHAMMAD HANIF under my supervision on the topic
ldquoInvestigation on intercropping of Ziziphus mauritiana with Cajanus cajan for fruit
and fodder at marginal land and cultivation of Carissa carandas for fruits through
saline water irrigationrdquo
Mr TAYYAB had been enrolled under my guidance for the award of PhD in
Department of Botany University of Karachi I have personally checked all the research
work reported in the thesis and certify its accuracyvalidity It is further certified that the
materials included in this thesis have not been used partially or fully in a manuscript
already submitted or in the process of submission in partialcomplete fulfillment for award
of any other degree from any other university Mr TAYYAB has fulfilled requirements of
the University of Karachi for the submission of this dissertation and I endorse its
evaluation for the award of PhD Degree
RESEARCH SUPERVISOR
PROF DR RAFIQ AHMAD
FPAS FTWAS
Professor (Retd) Botany (Plant Physiology)
PI Biosaline Research Projects
Department of Botany
University of Karachi
Karachi-75270 Pakistan
v
DEDICATED TO MY FAMILY
MUHAMMAD HANIF (MY FATHER)
MRS ARIFA (LATE)
(MY BELOVED MOTHER)
SHAHEEN TAYYAB (MY WIFE)
vi
ACKNOWLEDGMENTS
All the praises for almighty Allah and all respects for Prophet Muhammad (Peace be Upon
Him) who has shown me the straight path
I am grateful to my supervisor Prof Dr Rafiq Ahmad for his keen interest
patronage and guidance during this research work which made successful submission of
this thesis
I also obliged to Prof Dr Ehtesham Ul Haque and Prof Dr Javed Zaki (Present
and Former Chairmen Department of Botany respectively) for providing me all the
necessary facilities and administrative support
Being employed as lecturer in Department of Botany Govt Islamia Science
College Karachi I am also thankful to Education and literacy Department Govt of Sindh
(Pakistan) for providing me facilities to perform this study
Thanks are due to Dr D Khan in assessing statistical data analysis and colleague
of Biosaline lab Dr M Azeem Dr Naeem Ahmed and M Wajahat Ali Khan for their
cooperation throughout the course of study
I am also gratefully acknowledged to Mr Noushad Raheem and Mr Noor Uddin
of Fiesta Water Park for providing field plot and facilities to perform this study I am also
thankful to Pakistan Metrological Department for providing environmental data
I am also obliged to Dr M Qasim and Dr M Waseem Abbasi for their suggestions
and support in writing this thesis
Assistance of Abbul Hassan (Lab attendant) Tajwar Khan (Biosaline field
Attendant) and Mr Wahid (Plant Physiology Lab Assistant) is also acknowledged
Thanks are also due to my friends Dr Rafat Saeed Dr Kabir Ahmad Dr Zia Ur
Rehman Farooqi Dr Noor Dr M Yousuf Adnan Asif Bashir Dr A Rauf A Hai Faiz
Ahmed MA Rasheed Jallal Uddin Saadi Ahsan Shaikh Saima Fehmi A Mubeen
Khan Dr Noor Ul Haq Saima Ahmad S Safder Raza SM Akber and my college
colleagues for giving me encouragement during this research work
vii
I can never forget the support and encouragement and good wishes of Mr M
Wilayat Ali Khan Mrs Shahnaz Rukhsana Mr Mansoor Mrs Rabia Mansoor Mrs
Chand Bibi and Mrs Saeeda Anwar
In the last I am highly grateful to my beloved father Muhammad Hanif my loving
mother Arifa (when she alive) my caring wife Shaheen and sweet childrenrsquos Sara and
Sarim my supportive brothers and sisters and all family members for their prayers love
sacrifices and encouragements provided during course of this research work
viii
TABLE OF CONTENTS
No Title Page no
Acknowledgement vi
Summary xix
Urdu translation of summary xxi
General introduction 1
Layout of thesis 11
1 Chapter 1 13
11 Introduction 13
12 Experiment No 1 15
121 Materials and methods 15
1211 Seed collection 15
1212 Experimental Design 15
122 Observations and Results 17
13 Experiment No 2 22
131 Materials and methods 22
1311 Seed germination 22
132 Observations and Results 23
14 Experiment No 3 28
141 Materials and methods 28
1411 Seedling establishment 28
142 Observations and Results 29
1421 Seedling establishment 29
1422 Shoot height 29
15 Experiment No 4 31
151 Materials and methods 31
1511 Drum pot culture 31
1512 Experimental design 31
1513 Vegetative and Reproductive growth 32
1514 Analysis on some biochemical parameters 32
152 Observations and Results 34
1521 Vegetative and Reproductive growth 34
ix
No Title Page no
1522 Study on some biochemical parameters 34
16 Experiment No 5 41
161 Materials and methods 41
1611 Isolation Identification and purification of bacteria 41
1612 Preparation of bacterial cell suspension 41
1613 Study of salt tolerance of Rhizobium isolated from root
nodules of C cajan
41
162 Observations and Results 42
17 Experiment No 6 44
171 Materials and methods 44
1711 Experimental design 44
1712 Vegetative and reproductive growth 45
1713 Analysis on some biochemical parameters 45
172 Observations and Results 46
1721 Vegetative and Reproductive growth 46
1722 Study on some biochemical parameters 46
18 Discussion (Chapter 1) 51
2 Chapter 2 59
21 Introduction 59
22 Experiment No 7 60
221 Materials and Methods 60
2211 Growth and Development 60
2212 Drum pot culture 60
2213 Experimental Design 60
2214 Irrigation Intervals 61
2215 Estimation of Nitrate content 62
2216 Relative Water content (RWC) 62
2217 Electrolyte leakage percentage (EL) 62
2218 Photosynthetic pigments 63
2219 Total soluble sugars 63
22110 Proline content 63
22111 Soluble phenols 64
x
No Title Page no
22112 Total soluble proteins 64
22113 Enzymes Assay 64
222 Observations and Results 67
2221 Vegetative growth 67
2222 Photosynthetic pigments 70
2223 Electrolyte leakage percentage (EL) 70
2224 Phenols 70
2225 Proline 71
2226 Protein and sugars 71
2227 Enzyme essays 71
2228 Vegetative growth 73
2229 Photosynthetic pigments 75
22210 Electrolyte leakage percentage (EL) 76
22211 Phenols 76
22212 Proline 77
22213 Protein and Sugars 77
22214 Enzyme assay 77
23 Experiment No8 90
231 Materials and Methods 90
2311 Selection of plants 90
2312 Experimental field 90
2313 Soil analysis 90
2314 Experimental design 91
2315 Vegetative and reproductive growth 93
2316 Analysis on some biochemical parameters 93
2317 Fruit analysis 94
2318 Nitrogen estimation 94
2319 Land equivalent ratio and Land equivalent coefficient 95
23110 Statistical analysis 95
232 Observations and Results 96
2321 Vegetative parameters 96
2322 Reproductive parameters 96
xi
No Title Page no
2323 Study on some biochemical parameters 97
2324 Nitrogen Contents 98
2325 Land equivalent ratio land equivalent coefficient 98
24 Discussion (Chapter 2) 108
3 Chapter 3 113
31 Introduction 113
32 Experiment No 9 114
321 Materials and methods 114
3211 Drum Pot Culture 114
3212 Plant material 114
3213 Experimental setup 114
3214 Vegetative parameters 115
3215 Analysis on some biochemical parameters 115
3216 Mineral Analysis 116
322 Observations and Result 117
3221 Vegetative parameters 117
3222 Reproductive parameters 117
3223 Study on some biochemical parameters 118
3224 Mineral analysis 118
33 Discussion (Chapter 3) 127
4 Conclusion 129
5 References 130
6 Appendices 168
7 Publications 181
xii
LIST OF FIGURES
Figure Title Page no
11 Effect of irrigation water of different sea salt solutions on seed
germination indices of C cajan
27
12 Effect of irrigating water of different sea salt solutions on
seedling emergence (A) and shoot length (B) of C cajan
30
13 Environmental data of study area during experimental period
(July-November 2009)
36
14 Effect of salinity using irrigation water of different sea salt
concentrations on height of C cajan during 18 weeks treatment
36
15 Effect of salinity using irrigation water of different sea salt
concentrations on initial and final biomass (fresh and dry) of C
cajan
37
16 Percent change in moisture succulence relative growth rate
(RGR) and specific shoot length (SSL) of C cajan under
increasing salinity using irrigating water of different sea salt
concentrations
37
17 Effect of irrigating water of different sea salt solutions on
reproductive growth parameters including number of flowers
pod seeds and seed weight of C cajan
38
18 Effect of irrigating water of different sea salt solutions on leaf
pigments including chlorophyll a chlorophyll b total
chlorophyll and carotenoids of C cajan
39
19 Effect of irrigating water of different sea salt solutions on total
proteins soluble insoluble and total sugars in leaves of C cajan
40
110 Growth of nitrogen fixing bacteria associated with root of C
cajan under different NaCl concentrations
42
111 Photographs showing growth of Rhizobium isolated from the
nodules of C cajan in vitro on YEM agar supplemented with
different concentrations of NaCl
43
xiii
Figure Title Page no
112 Effect of salinity using irrigation water of different sea salt
concentrations on height number of branches fresh weight and
dry weight of shoot of Z mauritiana after 60 and 120 days of
treatment
47
113 Effect of salinity using irrigation water of different sea salt
concentrations on succulence specific shoot length (SSL)
moisture and relative growth rate (RGR) of Z mauritiana
48
114 Effect of salinity using irrigation water of different sea salt
concentrations on number of flowers of Z mauritiana
49
115 Effect of salinity using irrigation water of different sea salt
concentrations on leaf pigments including chlorophyll a
chlorophyll b total chlorophyll and chlorophyll ab ratio of Z
mauritiana
49
116 Effect of salinity using irrigation water of different sea salt
concentrations on total sugars and protein in leaves of Z
mauritiana
50
21 Vegetative parameters of Z mauritiana and C cajan at grand
period of growth under sole and intercropping system at two
irrigation intervals
79
22 Fresh and dry weight of Z mauritiana and C cajan plants under
sole and intercropping system at 4th and 8th day irrigation
intervals
80
23 Leaf weight ratio (LWR) root weight ratio (RWR) shoot weight
ratio (SWR)specific shoot length (SSL) specific root length
(SRL) plant moisture Succulence and relative growth rate
(RGR) of Z mauritiana and C cajan grow plants under sole and
intercropping system at 4th and 8th day irrigation intervals
81
24 Leaf pigments of Z mauritiana and C cajan grow plants under
sole and intercropping system at 4th and 8th day irrigation
intervals
83
xiv
Figure Title Page no
25 Electrolyte leakage phenols and proline of Z mauritiana and C
cajan at grand period of growth plants under sole and
intercropping system at 4th and 8th day irrigation intervals
84
26 Total protein in leaves of Z mauritiana and C cajan plants
under sole and intercropping system at 4th and 8th day irrigation
intervals
86
27 Enzymes activities in leaves of Z mauritiana and C cajan plants
under sole and intercropping system at 4th and 8th day irrigation
intervals
87
28 Nitrate reductase activity and nitrate concentration in leaves of
Z mauritiana and C cajan plants under sole and intercropping
system at 4th and 8th day irrigation intervals
89
29 Soil texture triangle (Source USDA soil classification) 99
210 Vegetative growth of Z mauritiana and C cajan growing under
sole and intercropping system
100
211 Reproductive growth of Z mauritiana and C cajan growing
under sole and intercropping system
101
212 Leaf pigments of Z mauritiana and C cajan growing under sole
and intercropping
102
213 Sugars protein and phenols in leaves of Z mauritiana and C
cajan at grand period of growth under sole and intercropping
system
103
214 Sugars protein and phenols in fruits of Z mauritiana grown
under sole and intercropping system
105
215 Nitrogen in leaves and in soil of Z mauritiana and C cajan
growing under sole and intercrop system
106
31 Chlorophyll a chlorophyll b total chlorophyll chlorophyll a b
ratio carotenoids contents of C carandas growing under
salinities created by irrigation of different dilutions of sea salt
124
xv
Figure Title Page no
32 Total protein sugars and phenolic contents of C carandas
growing under salinities created by irrigation of different
dilutions of sea salt
125
33 Mineral analysis including Na and K ions was done on leaves of
C carandas growing under salinities created by irrigation of
different dilutions of sea salt
126
xvi
LIST OF TABLES
Table Title Page no
11 Electrical conductivities of different sea salt solutions
used in germination of C cajan
18
12 Effect of irrigation water of different sea salt solutions
on germination percentage (GP) per day of C cajan
seeds pre-soaked in non-saline water prior to
germination with duration of time under various salinity
regimes
19
13 Effect of irrigation water of different sea salt solutions
on germination rate (GR) per day of seeds C cajan pre-
soaked in non-saline water prior to germination with
duration of time under various salinity regimes
20
14 Effect of irrigation water of different sea salt solutions
on mean germination rate (GR) coefficient of
germination velocity (GV) mean germination time
(GT) mean germination index (GI) and final
germination (FG) of C cajan seeds pre-soaked in non-
saline water prior to germination under various salinity
regimes
21
15 Electrical conductivities of different sea salt solutions
used in germination of C cajan
24
16 Effect of irrigation water of different sea salt solutions
on germination percentage (GP) per day of C cajan
seeds pre-soaked in respective sea salt concentrations
with duration of time
25
17 Effect of irrigation water of different sea salt solutions
on germination rate (GR) per day of C cajan seeds pre-
soaked in respective sea salt concentrations with
duration of time
26
xvii
Table Title Page no
18 Electrical conductivities of different Sea salt
concentrations and ECe of soil saturated paste at the end
of experiment
30
21 Soil analysis data of Fiesta Water Park experimental
field
99
22 Land equivalent ratio (LER) and Land equivalent
coefficient (LEC) with reference to height chlorophyll
and yield of Z mauritiana and C cajan growing under
sole and intercropping system
107
31 Electrical conductivities of different sea salt
concentration used for determining their effect on
growth of C carandas
119
32 Vegetative growth in terms of height and volume of
canopy of C carandas growing under salinities created
by irrigation of different dilutions of sea salt
120
33 Vegetative growth in terms of height and volume of
canopy of C carandas growing under salinities created
by irrigation of different dilutions of sea salt
121
34 Reproductive growth in terms of flowers and fruits
numbers flower shedding percentage fresh and dry
weight of ten fruit and their totals per plant fruit length
and diameter of C carandas growing under salinities
created by irrigation of different dilutions of sea salt
123
xviii
LIST OF ABBREVIATIONS
APX Ascorbate peroxidase
CAT Catalase
DAP Diammonium Phosphate (fertilizer)
dSm-1 Deci Siemens per meter
ECe Electrical conductivity of the Soil saturated extract
ECiw Electrical conductivity of the irrigation water
GPX Guaiacol Peroxidase
GR Glutathione reductase
GSH Reduced glutathione
LEC Land equivalent coefficient
LER Land equivalent ratio
NPK Nitrogen Phosphate Potash (fertilizer)
NR Nitrate reductase
RGR Relative growth rate
ROS Reactive oxygen species
RWR Root weight ratio
SOD Superoxide dismutase
SRL Specific Root Length
SSL Specific Shoot Length
SWR Shoot weight ratio
xix
Summary
Salinity is a growing threat to crop production which affects sustainability of agriculture
in aridsemiarid areas Growth responses of plant to salinity vary considerably among
species Cajanus cajan Ziziphus mauritiana and Carissa carandas are sub-tropical crops
grown worldwide particularly in Asian subcontinent for edible and fodder purposes but
not much is known about their salinity tolerance and intercropping
Effect of salinity has been initially studied in present work at germination of C cajan
under different sea salt salinities using presoaked seeds with water and respective salt
solutions Seed germination decreased with increasing salinity and it was more sever in
presoaking under water of different salinities The 50 threshold reduction started at
ECiw= 35 dSm-1 sea salt in presoaking treatments However this threshold was decreased
up to ECiw= 168 dSm-1 sea salt at further seedling establishment stage Growth experiment
of C cajan in drum pot culture (Lysimeter) also showed a salt induced growth reduction
in which plant tolerate salinity up to 42 dSm-1 At this salinity leaf pigments (chlorophylls
and carotenoids) proteins and insoluble sugars decreased up to 50 whereas soluble
sugars were increased (~25) Reproductive growth was also affected at this salinity in
which at least 70 reduction in flowers pods and seeds were observed
Salt tolerance of symbiotic nitrogen fixing bacteria associated with root of C cajan
showed salinity tolerance up to ECw= 366 dSm-1 NaCl salinity invitro environment For
intercropping experiments Ziziphus mauritiana (grafted variety) was selected with C
cajan Preliminary investigations showed a growth promotion in Z mauritiana at low
salinity (ECe= 72 dSm-1) and growth was remained unaffected up to ECe= 111 dSm-1
Intercropping of C cajan with Z mauritiana was primarily done in drum pot (Lysimeter)
culture Result showed better growth responses of both species when growing together as
intercrops than sole in which encouraging results were found in 8th day irrigation interval
rather than of 4th day Biochemical parameters eg photosynthetic pigments protein
phenols electrolyte leakage and sugars of these species displayed increase or decrease
according to their growth responses Increased activity of antioxidant enzymes and that of
nitrate reductase and its substrate (NO3) also contributed in enhancement of growth
Field experiment of intercropping of above mentioned plants at marginal land
irrigated with underground water (Eciw= 28 dSm-1) showed better vegetative growth of
xx
both species than sole crop The overall reproductive growth remained unaffected
although the numbers size and weight of fruit were better in intercropping system
Photosynthetic pigments were mostly increased whereas leaf protein and sugars remained
unchanged In addition higher values of LER and LEC (gt 1) indicated the success of
intercropping system
Experiment on salinity tolerance of Carissa carandas (varn karonda) using drum
pots culture showed improvement at low salinity (up to ECiw= 42 dSm-1 sea salt) whereas
higher salinity (ECiw= 129 dSm-1 sea salt) adversely affected vegetative and reproductive
growth Plant managed to tolerate up to ECiw= 99 dSm-1 sea salt Salinity severely affected
biochemical parameters including photosynthetic pigments proteins and sugars whereas
leaf phenolics were increased Leaf accumulated high amount of Na+ whereas affect
absorption of essential minerals like K+ was decreased
In the light of above mentioned investigations it appears that C cajan can be
propagated in saline soils with good presoaking techniques in non-saline water which
would helped to grow at moderately saline conditions It could be a good option used as
intercrop species because of its ability to improve soil fertility even under water deficit
conditions The proposed Cajanus-Ziziphus intercropping system could help poor farmers
to generate income from unproductive soils by obtaining sufficient fodder from C cajan
for their cattle and producing delicious edible fruits from Z mauritiana for commercial
purposes Carissa carandas could also be introduced as new crop for producing fruits from
moderate saline waste lands and used for preparing prickle jam and jelly for industrial
purposes
xxi
لاصہ خ
کا عمل ے ں ب ڑھئ لف پ ودوں می ی ےمخ طرہ ہ
وا خ ا ہ ے ب ڑھی لئ داوار کے ی ں زرعی ب وں می
ر علاق ج
ن ی م ب
ر و ب ج ن کھاری پ ن کھاری پ ن ب
دا کروت ی ر اور ر ب ے ارہ ا ہ وت لف ہ ی ی مخ کاف ں ودگی می اص Subtropical کی موج ا اور خ ی و پ وری دب ں ج ی ں ہ صلی
کی ف طے
خ
وراک و ں ج می
ی ملکوں
ائ ی ش کھاکر ای کی ی ان پ ودوں کم لوگ ہ ہت کن ب ں لی ی ی ہ
وئ عمال ہ
ارے کے طور ب ر است ری پ ن سے خ
ں ی ے ہ ں علم رکھئ ارے می ے عمل کے ت گئ ے گائ
کر ا ھ ملا
ی سات ک ہ رواداری اور ات
وں ج ن ر کےب ے ارہ
ھگوئ ہلے سے ت ں ب کاز والے محلول می لف ارت ی
مک کے مخ
دری ں ں سمی ی مطالعہ می
دائ ی کھاری اب کا
کہ پ ن کے و ی ج وئ ع ہ
کمی واف ں ی ت می ب
کی طن وں ج ن
ھ ب ہ کے سات
اف ں اض کھاری پ ن می ا گی ا کی دہ اہ کا مش رات
iwEC =اب
1-35 dSm می خ ی کہ ت ی ج مک کے ب راب ررہ
دری ں زی سمی کا
ہ ارت ں ی ام می ی ت صدی dSm= iwEC 168-1پ ودوں کے ق
ق
ی ک رہ ں Lysemeterت ے والے پ ودوں می ڑھئ ں ب روان چ می 1-dSm 24 ں جوضلہ مک محلول می
دری ں زی سمی کا
ارت
ں کر می ر خل ب زب ر س ی
ات اور غ روز مادوں لمخی
گ اف الت ف کے رت ی ت
ائ ی ں ض کھاری پ ن می ی اس
گئ کھی
ت ت د زا ب رداش
ت صدی 05اف
ق
ی ش کم وب ں کر می ی کہ خل ب زب ر س ں 50کمی ج وں می ج ن
ھلی اورب ھول ت ں ت ن می ری ج دی ب ڑھوب ولی
ا پ ا رہ مات
ہ ں اف ت صدی اض
05ق
ی گئ کھی
ت ح طور د
کمی واض ت صدی
ق
ی وی شلک سہب ڑ سے می کی چ ر مک (Symbiotic)ارہ
کی ں ا رت ی
کٹ ی ے والے ب
کرئ مد خ
ن من روج ی
اب سے (NaCl)ت
ی ر کے سا dSmwEC 366 =-1رواداری ں ب ری ہ می ج ے عمل کے ت گئ ے
گائ
کر ا ھ ملا
ی سات ک ہ یات
گئ کھی
ت ک د ر ت ھ ارہ
ت
بی ق کے ب
حق ی ت دائ ی ا اب گی ا ی
کھاری پ ن کو ج کم ں ے می ج ں dSme (Ec 72 =-1(ن ی کہ می ری ج ں ب ڑھوب ی ر می e (Ec =ب
)1-111 dSm ہل ہلے ب ے عمل ب گئ ے
گائ
کر ا ھ ملا
ی سات ک ہ کو ات ر ی ر اور ب ی ارہ
ر رہ اب ر می ی
ک غ کی خد ت
Lysemeter ج ب رآم ت ا ی زا ب ی کے جوضلہ اف
اش ی ے سے آب
ف ف ھ دن کے و
سی ت آت
کی ی ار دن ی خ
گئ کی ں ں دمی ن می ے ج
وئ ہ
ے عمل گئ ے گائ
کر ا ھ ملا
ی سات ک ہ سی ت ات
کی ی ے پ ودوں
گائ
ن ہا ا کی پ ودوں ب شام
وں اق
ے دوپ ج گئ
ت ا ی زا ب ادہ جوضلہ اف ں زت می
ی ول ب ات ف روزمادوں لخمی
گ اف الت ف کے رت ی ت
ائ ی ضلاات می درخ ی می
ائ کی می ی
ائ ےجی
وئ Electrolyteب رآمد ہ
Leakage کی کر ں س ی وں می ب ی ان پ ودوںاور ب
ی ش کمی ب ں دار می ی دپ ں مق
ں دکھائ ر می
اظ ی ری کے ب
کے ب ڑھوب
xxii
Antioxidant ی ظرح سے ہ اور اس ہ اف ں اض کی سرگرمی وں می امروں
اور اس کے Nitrate Reeducatesخ
Substrate )3(NO ا ی کا سی ب ب ہ اف ں اض ما می وں
ش ھی ی
ت
ےdSmiw(Ec 28 =-1(معمولی گئ ے ئ کب راب ں سی ی می ائ ہ ت والے ت درج ں می ری ہ می ج
ی ت ئ ن ہا زمب کی ب الا پ ودوں
ے عمل گئ ے گائ
کر ا ھ ملا
ی سات ک ہ سی ت ات
کی ی ے پ ودوں
ادوں ب ر لگائ ی
ب ما ب وں
ش دی ی ولی
ے پ
وئ ج خاضل ہ
ت ا ی ر بہی ادہ ب ں زت می
ےض ر رہ ہی ں ب ام می ط ے ت گئ ے
گائ
کر ا ھ ملا
ی سات ک ہ شامت اور وزن ات عداد ج
کی ت ھلوں ی کہ ت ی ج ر رہ اب ر می ی
الت ف ی غ ی ت
ائ
ی وئ ں ہ ہی
ع ب ی دت لی واف ی ب
کوئ ں دار می کی مق کر
ات اور س ں لمخی ی وں می ب ی کہ ب ہ ج
اف ا اض مات
ں ں روزمادوں می
گ اف د کے رت LER مزت
ے LEC (gt1)اور ی ہ کرئ ارہ کی ظرف اس ی ائ کامی کی ام
ط ے ت گئ ے
گائ
کر ا ھ ملا
ی سات ری ات ک ہ
کی ب ڑھوب
ک دا کروت ں ری ہ می ج کھاری پ ن ) Lysemeterو کھاری پ ن روداری کے ت ا کم گی ا ں اگات iwEC = 142می
1-dSm ( کھاری پ ن ادہ ی کہ زت ی ج وئ ری ہ ہی ں ب مک( می
دری ں زی سمی کا
زی dSm= iwEC 129-1 ارت کا دری ارت سمی
ی وئ ر ہ
اب ری ب ری ظرح می
دی ب ڑھوب ولی
ی اور پ
ائ علی
ں ف مک( می
ی کہ ں ک dSm9= iw(Ec 9-1(ج مک ت
دری ں زی سمی کا
ارت
ت کب رداش ات اور س روز مادوں لخمی گ اف الت ف کے رت ی ت
ائ ی ضلاات می درخ ی می
ائ کی می ی
ائ ےجی اب رہ کامی ں ےمی
ر ب ری ظرح کرئ
ں ی وں می ب وا ب ہ ہ
اف ں اض ی ول می ب
ں ف ی وں می
ب ی کہ ب ں ج ی
وب ر ہ اب می
+Na ہ سے کی وج مع ی ج اف رلز کے K+اض روری می
ی سے ض ج
ی وئ ر ہ
اب کی ضلاجی ت می ے
کرئ زب چ
ا ت ق حق الا ت ہ ت درج ے ظر می
وئ ےہ
ھگوئ ں ت ی می
ائ ہلے سے ت کہ ب ی
ے آئ مئ ں ی ہ ت ات سا ی می
ئ کی روش ر ت ہ سے ارہ کی وج ے
ت ف
ھی مدد دے س ں ت ے می گئ ں ا ن می ن زمی مکی دل ں وکہ معی ے ج ا ہ اسکی ا خ ھی لگات
ں ت ن خالات می مکی کو ں وں ج ن
وزہ کے ب ے مج ا ہ کی
داواری ی ر ب ی ے عمل غ گئ ے
گائ
کر ا ھ ملا
ی سات ک ہ ی ر ات ر اور ب ی ضلاجی ت والی ارہ
اف ے اض لئ وروں کے
اپ کی صور ت خ ر ن ارہ زمی
ھی دا ت کروت ے ا ہ وسکی ت ہ اب کا ذرت عہ ت ے ی ب ڑھائ
کی آمدئ وں
کشاپ ی صورت
ارئ ح کی ت ل
ھ ی ت وردئ دار ج ی ر سے مزت ارہ اور ب ی خ
عئصت
صل کے طور ب ی ف ئے ب لئ ے کے
کرئ دا ی ھل ب ن سے ت کارآمد زمی ر ی
ن اور غ مکی
دل ں ے معی
لئ اضد کے ے رمق ا ہ اسکی ا خ کی ی ش ب
1
General Introduction
Intercropping is a major resource conservation technique for sustainable agriculture under
various climatic conditions (Zhang et al 2010 Li et al 2014) It can reduced operational
cost for the production of multiple crops with maintained or even higher level of
productivity (Vandermeer 2010 Perfecto and Vandermeer 2010) It can enhance the
water use efficiency by saving 20 to 40 irrigation water with improved fertilizer
management (Fahong et al 2004 Jat et al 2005 Jani et al 2008) Intercropping system
is more suitable in marginal areas with lower mechanization and cultivation input by
farmers on small tracts of farmlands (Ngwira et al 2012) It can enhance the cumulative
production per unit area and protect the small farmers against market fluctuations or crop
failure ensure the income improve soil fertility and food demands (Rusinamhodzi et al
2012) In this system dominating more compatible and productive species are selected or
replaced in which complementarity effects and beneficial interactions resulting enhanced
yield as compared to monoculture (Huston 1997 Loreau and Hector 2001) It was
estimated that in species diverse systems biomass production is 17 times higher as
compared to monoculture (Cardinale et al 2007)
It is suggested that intercropping is the best suitable cropping system which can
improve the resource-use efficiency by procurement of limiting resources enhanced
phyto-availability and effective plants interactions (Marschner 2012 White and
Greenwood 2013 Ehrmann and Ritz 2014) It is widespread in many areas of world
particularly in latin America it is estimated about 70-90 by small farmers which mainly
grow maiz potatoes beans and other crops under this system whereas intercropping of
maiz with different crops is estimated about 60 (Francis 1986) Additionally
agroforestry is more than 1 billion ha in this area (Zomer et al 2009) The land used for
intercropping system of various crops is greatly varied from 17 in India to 98 in Africa
(Vandermeer 1989 1992 Dupraz and Liagre 2011)
In intercropping system two or more crops or genotypes coexist and growing
together at a same time on a similar habitat (Li et al 2013) It may be divided into various
types such as in mixed intercropping system two or more crops simultaneously growing
without or with limited distinct arrangements whereas in relay intercropping system
second crop is planted when the first is matured while in strip intercropping both the crops
2
are simultaneously growing in strips which can facilitate the cultivation and crop
interactions (Ram et al 2005 Sayre and Hobbs 2004)
Several less-conventional fruit tress including Manilkara zapota (Chicko)
Ziziphus mauritiana (Jujubar) Carissa carndas (Karanda) Annona squamosa (Sugar
apple) and Grewia asiatica (Falsa) has been reported with high nutritional value with
capability to grow at marginal lands (Mass and hoffman 1997) Qureshi and Barrett-
Lennard (1998) suggested few grafted plants that can widely use to improve the quality
and productivity of fruits Grafting is also used to induce stress tolerance in plants against
various abiotic and biotic stresses including salinity stress (Rivero et al 2003) Both root
stocks and shoot stocks contribute to increase the tolerance level of plants Root stocks
represent the first part of defense to control the uptake and translocation of nutrients and
salts throughout the plant (Munns 2002 Santa-cruz et al 2002 Zrig et al 2011) while
shoot stocks develops physiological and biochemical changes to promote plant growth
under stress conditions (Moya et al 2002 Chen et al 2003)
Ziziphus mauritiana Lamk (varn grafted ber) belongs to the family Rhamnaceae
grows widely in most of the dry tropical and subtropical regions around the world Various
grafting methods are used for their propagation including wedge and whip or tongue
methods (Nerd and Mizrahi 1998) Intercropping of these grafted fruit trees with various
leguminous crops is also being successfully practiced in many countries thought the world
Leguminous crops are considered excellent symbiotic nitrogen fixing crops It can
effectively improve soil fertility and offset the critical problems of sub-tropical areas to
fight against desertification and soil degradation These plants are considered as an
excellent source of proteins for humans and animals They can fix the 90 of atmospheric
nitrogen and contribute 40 nitrogen to the soil thus increase the soil fertility (Peoples et
al 1995) However most of the leguminous plants are not salt tolerant while some
species are better drought tolerant and effectively contribute in marginal lands (Zahran
1999)
Among the leguminous plants Pigeon pea (Cajanus cajan (L) Millspaugh) of the
family Fabaceae is widely grown for food fodder and fuel production particularly in
semiarid areas The salinity tolerance of this specie is not well documented both at
germination and seedling stages This crop is still underexploited due to its edible and
3
economic importance While limited investigations has been made to uncover its
nutritional quality medicinal uses and drought tolerance
The identical physiological traits are important in both the mono and intercropping
systems to maximize the resource acquisition The exploitation of best possible
combination of traits of different plants in intercropping system is very important to
maximize the overall performance in intercropping system It depends on the above ground
beneficial plant interactions for light space and optimal temperatures (Wojtkowski 2006
Zhang et al 2010 Shen et al 2013 Ehrmann and Ritz 2014) as well as the
complementary below ground plant interactions with soil biotic factors (Bennett et al
2013 Li et al 2014)
Water is also a major limiting factor intercropping can enhanced the acquisition
of water by root architecture and distribution in the soil profile for effective utilization of
rainfall (Zegada-Lizarazu et al 2006 De Barros et al 2007) and enhanced the water use
efficiency for effective hydraulic redistribution by deep rooted crops and water stored in
the soil profile (Morris and Garrity 1993 Xu et al 2008) Mycorrhizal networks around
the roots of intercrop plants also enhanced the availability of water and available resources
and reduced the surface runoff (Caldwell et al 1998 Van-Duivenbooden et al 2000
Prieto et al 2012)
Intercropping with leguminous plants can enhanced the agricultural productivity in
less productive soils due to enhanced nitrogen availability and also improved the soil
fertility by effective nitrogen fixation (Seran and Brintha 2010 Altieri et al 2012) Due
to weaker soil nitrogen competition intercropping with legumes enhanced the nitrogen
availability to the non-leguminous intercrop which also absorbs the additional nitrogen
released in the soil or root nodules of the leguminous plant (Li et al 2013 White et al
2013a) The use of legumes in many intercropping systems is pivotal According to the
listing of Hauggaard-Nielsen and Jensen (2005) seven out of ten are the legumes among
the most frequently used intercrops around the world
The ecological range of adaptability of legumes reaches from the inner tropics to
arctic regions with individual species expressing tolerance to drought temperature
nutrient deficiency in soil water logging salinity and other environmental conditions
(Craig et al 1990 Hansen 1996) The woody perennial leguminous plants have a number
4
of purposes they can be used to reclaim degraded wastelands retard erosion and provide
shade fuel wood timber and green manure (Giller and Wilson 1991)
Trees with nitrogen fixing capability play an important role to offset the critical
problems of tropical and sub-tropical regions in their fight against desert encroachment
and soil impoverishment These plants are capable to live in N-poor soils through their
association with Rhizobium that fix atmospheric nitrogen Nitrogen fixing activity in the
field depends both on their N2-fixing potential and on their tolerance to existing
environmental stresses (Galiana et al 2002) Symbiotic N2 fixation in leguminous plants
can mainly be considered an excellent source of protein supply for human and animal
consumption They range from extensive pasture legumes to intensive grain legumes and
are estimated to contribution up to 40 of their nitrogen to the soil (Simpson 1987)
The traits in the monocropping system in the selected crop extensively exploit the
acquisition of limiting resources in the environment and continuously focused on the
availably of similar resources for the successful crop production (White et al 2013 ab)
whereas in intercropping with different crops cycling of resources can be optimized to
the complementarity or facilitation traits (Costanzo and Barberi 2014) to overcome
resource limitations during the growing season (Hill 1996 George et al 2014)
For the long term sustainable agriculture and food production in resource limiting
areas with lower input Intercropping systems have the potential to increase the
productivity With efficient mechanization cultural practices and optimized nutrient
management rapid improvements are also possible through this system In future
perspective intercrops with higher resource use efficiency through plant breeding and
genetics is likely to be the most effective option for sustainable agriculture and
development
Increase of world population and demand of additional food production
The demand and production gap of food fodder fuel wood and livestock products is
increasing day by day due to global population which will increase from about 7 billion
(FAO 2014) to 9 billion by 2050 (Haub 2013) The increasing urbanization further
intensifies the problem which will increase from 54 to 66 expected in 2050 (UN
2014) Majority of this rise in urbanization will occur in developing countries around the
5
globe The major problem is to meet the challenge of increasing food demand for this ever
growing population up to 70 more food crops to feed the additional 23 billion population
worldwide by 2050 (FAO 2010 2011) Hence there is great need to increase the re-
vegetation for fuel wood and fodder production (Thomson 1987) An increase in
production could be envisaged through increasing the yield of already productive land or
through more extensive use of unproductive land The high concentration of salts in soil
or water does not let the conventional crops grow and give feasible economic return
Hence it is necessary to search for unconventional crops for foods fodder and fuel which
could give profitable yield under saline conditions (Ahmad and Ismail 1993) Reclamation
of this land through chemical and engineering treatments is very expensive The most
appropriate use of saline wasteland is the production of high yielding salt tolerance fuel
wood timber and forage species (Qureshi et al 1993) Therefore the most attractive
option is to screen a range of species and identify those which have potential of being
commercially valuable for the degraded environments (Ismail et al 1993)
Pakistan is in semi-arid region and the 6th most populated county of the world
Population drastically increased in Pakistan which was 80 million in 1980 and annual
increase in population is about 4 million (UNDES 2011) This is continuously
overburdened and it is estimated that in 2025 it will reach to 250 million and 335 million
in 2050 which decrease the available water per capita to less than 600 m3 resulting 32
shortfall of water requirements causing an alarming condition particularly for Pakistan
Furthermore this shortfall in 2050 leading to severe food shortage upto 70 million tones
which indicates the further development and serious measures for the new resources
(ADB 2002) Subsequent severe food and fodder crises along with all the resource
limitations with continuous increase in urbanization from the current 35 to 52 in 2025
will further intensity the agriculture production and demand
Shortage of good quality irrigation water
On earth surface the major resources of available fresh water is deposited in the form of
ponds lakes rivers ice sheets and caps streams and glaciers whereas underground water
as underground streams and aquifers With the drastic increase in population the water
consumption rise as the twice of the speed of population growth The scarcity of water is
widespread to many countries of different regions Majority of population in developing
countries suffering from seasonal or year round water shortage which will increase with
6
expected climatic changes Currently almost 50 countries around the globe are facing
moderate to severe shortage of water
Due to the greenhouse effect it is estimated that since the start of 20th century 14
degF temperature is already risen which will likely rise at least another 2degF and over the next
100 years it is estimated about more than 11degF due to the consequences of biogenic gases
(El-Sharkawy 2014) This is mainly due to the product of human activities including
industrial malpractices excess fossil fuel consumption deforestation poor land use and
cultural practices
Rising in atmospheric CO2 concentration which probably reached 700 μmol (CO2)
molminus1 resulting severe climatic changes It will accelerate the melting of ice and glacier
resulting the rising rainfall and storms in tropics and high latitude consequently 06 to 1
meter rise in sea level on the expense of costal lowlands across the continents After this
initial high flows the decrease in inflow was very terrifying Due to these climatic changes
humans suffering from socioeconomic changes including degradation of lands with lower
agricultural output and degradation of natural resources will further enhanced the poverty
and hunger resulting dislocation and human migrations (Randalls 2010)
In the mean while scarcity of good quality water is increasing day by day with the
demands of water for domestic agricultural and industrial utilization which will further
increase up to 10 of the total available resources as estimated by 2025 which needs
serious water managements (Bhutta 1999) It is very challenging for the modern
agriculture to ensure the increasing demand of more arable and overburdened population
with the limiting resources including the unavailability of good quality water and
deterioration of even previously productive land (Du et al 2015)
In Pakistan Indus River basin is the back bone of agriculture and socioeconomic
development which contributes 65 of the total river flows and 90 for the food
production with a share of 25 to the GDP It is estimated that about 30-40 of its surface
storage capacity will reduce by 2025 due to siltation of reservoirs and climatic changes It
will impose serious threat to irrigated agriculture in near future consequently with
decreases in groundwater resources resulting shortage of fresh water and 15-20
reduction in grain yield in Pakistan (World Bank 2006)
7
Spread of saline soil and reduction in agricultural yield
Along with scarcity of water soil salinity is one of the major environmental stresses which
severely threaten the agriculture The damages of salinity is widespread around the world
which is so far effected the more than 800 million hectare (more than 6) of land
worldwide including 397 million ha by salinity associated with 434 million ha by sodicity
(FAO 2010) The out of total 230 million hactares of irrigated land more than 45 million
hactares (20) is so far effected by salinity which is about the 15 of total cultivated land
(Munns and Tester 2008)
In Pakistan out of 2036 million hectares of cultivated land more than 6 million
hectares is affected by salinity and water logging of various degrees (Qureshi et al 2004)
About 16 million hectares of tropical arid plains which have been put under crop
cultivation depend extensively on canal irrigation network This area (about 60) is now
seriously affected by water logging and salinity (Qureshi et al 2004) The rise of subsoil
water levels accompanied by its subsequent decline due to irrigation combined with
insufficient drainage has led to salinization of valuable agricultural land in arid zones all
over the world (Ahmad and Abdullah 1982) The dominated cation in salt-affected soil is
Na+ followed by Ca2+ and Mg2+ while the anions Cl and SO4 are almost equal in
occurrence (Qureshi et al 1993) Salt content varies in different regions of the salt-
affected areas but at certain sites could reach up to an ECe of 90-102 dSm-1 (Ahmad and
Ismail 1993)
Salinity is a chief anxiety to meet the ever growing demands of food crops Salinity
adversely affects the plant growth and productivity Plants differentially respond to salt
stress and categories into four classes Salt sensitive moderately salt sensitive moderately
salt tolerant and highly salt tolerant plants on the basis of their tolerance limits Whereas
mainly plants are divided into halophytes (salt tolerant) and glycophytes (salt sensitive) on
the basis of adaptive evolution (Flowers 2004 Munns and Tester 2008) Unfortunately
majority of cultivated crops are not able to withstand in higher salinity regimes and
eventually die under higher saline conditions which proposed serious attentions to manage
the dissemination of salinity (James et al 2011 Rozema and Flowers 2008)
Excessive accumulation of salts in rhizosphere initially reduced the water
absorption capacity of roots leading to hyperosmotic stress followed by specific ion
8
toxicity (Munns 2008 Rahnama et al 2010) Plants initially manage the overloaded salt
by various excluding and avoidance mechanisms depending on their tolerance levels The
management of salt inside the cytosol is depends on the compartmentalization capacity of
plants followed by osmotic adjustments and efficient antioxidant defense mechanisms
Whereas higher salt beyond the tolerance impose injurious effects on various
physiological mechanisms These are including disruption of membrane integrity
increased membrane injuries nutrient ion imbalances osmotic disturbance
overproduction of reactive oxygen species (ROS) compromised photosynthesis and
respiration due to stomatal closure and damages of enzymatic machinery (Munns and
Tester 2008) In specific ion toxicity Na+ and Cl- are the chief contributors in
physiological disorders Excessive Na+ in rhizosphere antagonize the uptake of K+
resulting lower growth and productivity (James et al 2011) Salt load in the cytosol trigger
the overproduction of ROS including H2O2 OH- super oxides and singlet oxygen They
are involved in sever oxidative damages to various vital cellular components including
DNA RNA lipids and proteins (Apel and Hirt 2004 Ahmad and Umar 2011)
Strategies to cope up the salinity problem
The development and cultivation of highly salt tolerant crop varieties for salt affected areas
is the major necessity to meet the future demands of food production whereas the majority
of available food crops are glycophytes Therefore it is an emergent need of crop
improvement methods which are more efficient cost effective and grow on limiting
resource The use of poor quality water for irrigation is also very important under the
proposed shortage of fresh water in near future For the development of salt tolerant
varieties more understanding of stress mechanisms are required at whole plant molecular
and cellular levels
The variability in stress tolerance of salt sensitive genotypes (glycophytes) and
highly salt tolerant plants (halophytes) showed genetic basis of salt tolerance It indicate
that salt tolerance is a multigenic trait which involves variety of gene expressions and
related mechanisms Salt stress induces both the qualitative and quantitative changes in
gene expression (Manchanda and Garg 2008) These multigenetic expressions play a key
role in upregulation of various proteins and metabolites responsible for the management
of anti-stress mechanisms (Bhatnagar-Mathur et al 2008) Plant breeding and transgenic
strategies are intensively used for decades to improve the crop performance under salinity
9
and aridity conditions Few stress tolerant varieties are so far released for commercial
production whereas in natural condition where plant exposed to variety of climatic
conditions the overall performance of plant have changed as compared to controlled in
invitro conditions (Schubert et al 2009 and Dodd and Perez-Alfocea 2012) The success
stories about transgenic approaches for crop improvement under stressful environments
are still very scanty because of the insufficient understanding about the sophisticated
mechanisms of stress tolerance (Joseph and Jini 2010) It indicates that there is less
correlation between the assessment of stress tolerance in invitro and invivo conditions
Although there have been some achievement in this connection in some model plants
including rice tobacco and Arabidopsis (Grover et al 2003) which proposed the
possibilities of success in other crops in future Variety of technicalities and associated
financial challenges are still associated with this strategy
In conventional cultivation practices continuous irrigation with poor quality water
can enhanced the salinization due to evapotranspiration leading to increased saline andor
sodic soils This problem can be cope up by intercropping system in which high salt
tolerant or salt accumulator plants are intercropped with salt sensitive crops which can
accumulate salt thus can reduce the risk of salt increment in soil Additionally better
cultivation practices including the micro-jet or drip irrigation and partial root zone drying
technique is also very fruitful to optimize the water requirements and avoid the risks
associated with conventional flooding irrigation system
In dry land agriculture plantation of deep rooted perennials during off season or
annuals can reduced the risk of salinization They continuously grown and utilize excess
amount of water create a balance between water utilization and rail fall Thus prevent the
chance of salt accumulation on soil surface due to increased water table and
evapotranspiration (Manchanda and Garg 2008) The efficient irrigation and
intercropping strategy is seemed quite attractive cost effective and very beneficial in less
mechanized poor marginal areas It can ameliorate the injurious effects of salinity and
increased production per unit area thus ensure the sustainable agriculture in semi-arid or
marginal lands (Venkateswarlu and Shanker 2009)
A number of plant species are available that are highly compatible with saline
sodic and marginal lands The cultivation of these species with proposed intercropping
system is economically feasible to grow in marginal soil Some plants including Carissa
10
carandus Ziziphus mauritiana and Cajanus cajan was selected to revealed their potential
for intercropping under saline marginal lands These are important plants which can
established well at tropical and subtropical arid zone under high temperatures Hence their
range of salt tolerance and suitability for cultivation at waste saline land or with saline
water irrigation is being undertaken for commercial exploitation
Objective of present investigation
The plan of present investigation has been worked out to look into possibility of increasing
production of an unconventional salt tolerant fruit tree (Z mauritiana) by intercropping
with a legume ( C cajan) which apart from increasing fertility of soil could be able to
provide fodder for grazing animals from salt effected waste land Possibility of making
use of saline water for irrigation has also been considered for growing leguminous plant
(C cajan) and salt tolerant unconventional fruit tree (Crissa carandas) under saline
condition
11
LAYOUT OF THESIS
Chapter 1 Monoculture of Cajanus cajan (Vern Arhar) and Ziziphus mauritiana
(Varn Ber) under different range of salinities created by irrigation of
various sea salt concentrations
A Experiments on Cajanus cajan
Following experiments were performed under A
Experiment No 1 Effect of Pre-soaked seeds of C cajan in distilled water for
germination in water of different sea salt concentrations
Experiment No 2 Effect of Pre-soaked seeds of C cajan in various dilutions of sea salt
for germination in water of respective sea salt concentrations
Experiment No 3 Seedling establishment experiment of C cajan on soil irrigated with
sea salt of different concentrations
Experiment No 4 Growth and development of C cajan in Lysimeter (Drum pot culture)
being irrigated with water of different sea salt concentrations
Experiment No 5 Range of salt tolerance of nitrogen fixing symbiotic bacteria
associated with root of C cajan
B Experiments on Ziziphus mauritiana
Experiment No 6 Growth and development of Z mauritiana in large size clay pot being
irrigated with water of two different sea salt concentrations
Discussion (Chapter 1)
Chapter 2 Intercropping of Ziziphus mauritiana with Cajanus cajan
Experiment No 7 Physiological investigations on Growth of Ziziphus mauritiana and
Cajanus cajan intercropped in drum pot (Lysimeter) culture being
irrigated with water of sea salt concentration at two irrigation
intervals
Experiment No 8 Investigations of intercropping Ziziphus mauritiana with Cajanus
cajan on marginal land under field conditions
12
Discussion (Chapter 2)
Chapter 3 Investigations on rang of salt tolerance in Carissa carandas (varn
karonda) for determining possibility of growing at waste saline land
Experiment No 9 Investigation on the effect of higher range of salinities on growth of
Carissa carandas (varn karonda) created by irrigation of different
dilutions of sea salt
Discussion (Chapter 3)
13
1 Chapter 1
Monoculture of Cajanus cajan (Vern Arhar) and Ziziphus mauritiana
(Varn Ber) under different range of salinity created by irrigation of
various sea salt concentrations
11 Introduction
Scarcity of good quality water enforced the growers to irrigate the crops with
lowmoderately saline water at marginal lands which ultimately enhance soil salinity due
to high evapo-transpiration (Azeem and Ahmad 2011) To overcome this situation people
are now focusing on less-conventional plants which can grow on resource limited areas
and can produce edible biomass for human and animal consumption
Ziziphus mauritiana (varn grafted ber) is salt and drought tolerant plant which can
grow on marginal and degraded land (Morton 1987) It has wide spread crown and a short
bole fast growing tree with average bearing life of 25 years The ripe fruit (drupe) is juicy
hard or soft sweet-tasting pulp has high sugar content vitamins A amp C carotene
phosphorus and calcium (Nyanga et al 2013 2008 Pareek 2013) The leaves contain 6
digestible crude protein and an excellent source of ascorbic acid and carotenoids The
leaves are used as forage for cattlesheepgoats and also palatable for human consumption
(Sharma et al 1982 Bal and Mann 1978 Agrawal et al 2013) The timber is very hard
can be worked to make boats charcoal and poles for house building Roots bark leaves
wood seeds and fruits are reputed to have medicinal properties The tree also used as a
source of tannins dyes silk (via silkworm fodder) shellac and nectar (Dahiru et al 2006
Chrovatia et al 1993 Gupta 1993)
Some atmospherics nitrogen fixing bacterial associated deep rooted drought
tolerent leguminious plants like Cajanus cajan can fix up to 200 Kg nitrogen ha-1 year-1
due to symbiotic association of Rhizobium with its deep penetrating roots (Bhattacharyya
et al 1995) Total cultivated area of Pigeon pea is about 622 million hectare and global
annual crop production is around 474 million tonnes whereas total seed production of
this crop is about 015 million tonnes (FAOSTAT 2013) Its seeds are an excellent source
of good quality protein (up to 24) and foliage is used as animal fodder with high
nutritional value (Pandey et al 2014) Besides being used as food and fodder this plant
14
also have therapeutic value and it is used against diabetes fever dysentery hepatitis and
measles (Grover et al 2002) It also use traditionally as a laxative and was identified as
an anti-malarial remedy beside other medicinal species (Ajaiyeoba et al 2013 Qasim et
al 2010 2011 2014)
Following experiments were conducted to evaluate the seed germination seedling
establishment and growth of C cajan as well as grafted sapling of Z mauritiana under
various salinity regimes Investigations were also undertaken to find-out of their
intercropping has any beneficial effect on growth at marginal saline land saline
environment
15
12 Experiment No 1
Effect of Pre-soaked seeds of Cajanus cajan in distilled water for
germination in water of different sea salt concentrations
121 Materials and methods
1211 Seed collection
Seeds of C cajan were purchased from local seed market Mirpurkhas Sindh and were
tested to determine the effect of salinity on germination at the biosaline laboratory Botany
department Karachi University Karachi The best lot of healthy seeds having 100
germination was selected for further experiments
1212 Experimental Design
Seeds of C cajan were surface sterilized with 01 sodium hypochlorite solution for 2-3
minutes washed in running tap water then soaked in sterilized distilled water for one hour
(Saeed et al 2014) Sterilized glass petri plates (9cm) lined with filter paper were moist
with 10 ml of distilled water at different saline water of different sea salt concentrations
and their germination percentage was observed Their electrical conductivities on these
sea salt dilutions are mentioned in Table 11 Three replicates were used for each treatment
Ten seed were placed in each petri plate which were kept in temperature controlled
incubator (EYELA LTI-1000 Japan) at 28 plusmn 1ordmC in dark Experiment was continued for 7
days Data were recorded on daily bases Analyses of varience by using repeated measures
and the significant differences between treatment means were examined by least
significant difference (Zar 2010) All statistical analysis was performed using SPSS for
windows version 14 and graphs were plotted using Sigma plot 2000
Germination percentage of C cajan was recorded every 24 hours per seedling
evaluation procedure up to 07 days The final percent germination related with salinity in
accordance with Maas and Hoffman (1977) The percent germination was calculated using
the following formula (Cokkizgin and Cokkizgin 2010)
16
Germination index for C cajan was recorded according to AOSA (1990) by using
following formula
Where Gt is the number of germinated seed on day t and Dt is the total number of
days (1 - 7)
Coefficient of germination velocity of C cajan was calculated described by Maguire
(1962)
Where G represents the number of germinated seeds counted per day till the end of
experiment
Mean germination time of C cajan was calculated by Ellis and Roberts (1981) by
using following formula
Where lsquonrsquo is the number of germinated seeds in day d whereas Σn is the total
germinated seeds during experimental period
Germination rate was of C cajan determined according to following formula
(Shipley and Parent 1991)
Where numbers of germinated seeds were recorded from 1 to 7
17
122 Observations and Results
Cajanus cajan (imbibed in distilled water) grown at different salinity regimes showed 50
reduction at 16 salt concentration corresponding ECiw 168 dSm-1 (Table 1 2 Appendix
I)
Rate of germination was inversely correlated with sea salt concentration It was
significantly (p lt 0001) decreased from first day to final (day 7) of observation Higher
germination rate was recorded in control and at lower concentrations of sea salt in early
days of seed incubation with contrast to higher concentrations of sea salt which was
reduced with increasing day of incubation (Table 13 Appendix I)
A significant decrease (p lt 0001) in coefficient of germination velocity was
observed with increasing salinity (Table 14 Appendix I)
A significantly increase (p lt 0001) in mean germination time of seeds was observed
with increasing sea salt concentrations However the difference was insignificant at lower
salinities (Table 14 Appendix I)
A significant decrease (p lt 0001) in mean germination index was observed with
increasing salt concentrations except lower salinities More reduction was observed
byhond 16 and onward sea salt concentration (Table 14 Appendix I)
18
Table 11 Electrical conductivities of different sea salt solutions used in germination of C cajan
Sea salt () ECiw (dSm-1)
Non saline control 06
01 09
02 16
03 35
04 42
05 58
06 62
07 79
08 88
09 99
10 101
11 112
12 128
13 131
14 145
15 159
16 168
ECiw is the electrical conductivity of irrigation water measured in deci semen per meter
19
Table 12 Effect of irrigation water of different sea salt solutions on germination percentage (GP) per day
of C cajan seeds pre-soaked in non-saline water prior to germination with duration of time under
various salinity regimes
Sea Salt
(ECiw= dSm-1)
GP
1st day
GP
2nd day
GP
3rd day
GP
4th day
GP
5th day
GP
6th day
GP
7th day
Control 8333plusmn667 90plusmn00 9333plusmn333 9333plusmn333 9333plusmn333 9333plusmn333 9333plusmn333
09 8667plusmn333 9333plusmn333 9667plusmn333 9667plusmn333 100plusmn00 100plusmn00 100plusmn00
16 7667plusmn667 80plusmn10 8333plusmn882 8333plusmn882 8333plusmn882 8333plusmn882 8667plusmn667
35 6667plusmn333 9333plusmn333 9333plusmn333 9333plusmn333 9333plusmn333 9333plusmn333 9333plusmn333
42 70plusmn00 8667plusmn333 90 plusmn00 90 plusmn00 90 plusmn00 90 plusmn00 90 plusmn00
58 6333plusmn667 7333plusmn333 8333plusmn333 90 plusmn00 90 plusmn00 90 plusmn00 90 plusmn00
62 5667plusmn667 80plusmn577 90 plusmn00 90plusmn00 90 plusmn00 90 plusmn00 90plusmn00
79 5333plusmn333 70plusmn00 8333plusmn333 8333plusmn333 8333plusmn333 8333plusmn333 8333plusmn333
88 4000plusmn00 6667plusmn667 8333plusmn333 8333plusmn333 8333plusmn333 8333plusmn333 8333plusmn333
99 2667plusmn333 60 plusmn00 90 plusmn00 90plusmn00 90 plusmn00 90 plusmn00 90 plusmn00
101 2333plusmn333 70plusmn577 7333plusmn333 7333plusmn333 7333plusmn333 7333plusmn333 7333plusmn333
112 70plusmn577 7667plusmn333 80plusmn00 8333plusmn333 8333plusmn333 8333plusmn333 8333plusmn333
128 6667plusmn333 6667plusmn333 6667plusmn333 6667plusmn333 6667plusmn333 6667plusmn333 6667plusmn333
131 3333plusmn882 50plusmn00 5333plusmn333 5333plusmn333 5333plusmn333 5333plusmn333 5667plusmn333
145 3333plusmn667 40 plusmn00 50 plusmn577 50plusmn577 50 plusmn577 5333plusmn333 5333plusmn333
156 3667plusmn667 40plusmn577 4667plusmn882 4667plusmn882 50plusmn577 50plusmn577 5333plusmn667
168 1667plusmn882 3333plusmn333 3333plusmn333 3333plusmn333 3667plusmn333 3667plusmn333 4333plusmn333
LSD 005 Salinity 18496
Time (days) 13322
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and LSD among the treatments is recorded at p lt 005
20
Table 13 Effect of irrigation water of different sea salt solutions on germination rate (GR) per day
of seeds C cajan pre-soaked in non-saline water prior to germination with duration of
time under various salinity regimes
Sea Salt
(ECiw= dSm-1)
GR
1st day
GR
2nd day
GR
3rd day
GR
4th day
GR
5th day
GR
6th day
GR
7th day
Control 833plusmn067 450plusmn00 311plusmn011 233plusmn008 187plusmn007 156plusmn006 133plusmn005
09 867plusmn033 467plusmn017 322plusmn011 242plusmn008 200plusmn00 167plusmn00 143plusmn00
16 767plusmn067 400plusmn050 278plusmn029 208plusmn022 167plusmn018 139plusmn015 124plusmn010
35 667plusmn033 467plusmn017 311plusmn011 233plusmn008 187plusmn007 156plusmn006 133plusmn005
42 700plusmn00 433plusmn017 300plusmn00 975plusmn750 180plusmn00 150plusmn00 129plusmn00
58 633plusmn067 367plusmn017 278plusmn011 225plusmn00 180plusmn00 150plusmn00 129plusmn00
62 567plusmn067 400plusmn029 300plusmn00 225plusmn00 180plusmn00 150plusmn00 129plusmn00
79 533plusmn033 350plusmn00 278plusmn011 208plusmn008 167plusmn007 139plusmn006 119plusmn005
88 400plusmn00 333plusmn033 278plusmn011 208plusmn008 167plusmn007 139plusmn006 119plusmn005
99 267plusmn033 300plusmn00 300plusmn00 225plusmn00 180plusmn00 150plusmn00 129plusmn00
101 233plusmn033 350plusmn029 244plusmn011 183plusmn008 147plusmn007 122plusmn006 105plusmn005
112 700plusmn058 383plusmn017 267plusmn00 208plusmn008 167plusmn007 139plusmn006 119plusmn005
128 667plusmn033 333plusmn017 222plusmn011 167plusmn008 133plusmn007 111plusmn006 095plusmn005
131 333plusmn088 250plusmn00 178plusmn011 133plusmn008 107plusmn007 089plusmn006 081plusmn005
145 333plusmn067 200plusmn00 167plusmn019 125plusmn014 100plusmn012 089plusmn006 076plusmn005
156 367plusmn067 200plusmn029 156plusmn029 117plusmn022 100plusmn012 083plusmn010 076plusmn010
168 167plusmn088 167plusmn017 111plusmn011 083plusmn008 073plusmn007 061plusmn006 062plusmn005
LSD 005 Salinity 0481
Time (days) 0378
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and LSD among the treatments is recorded at p lt 005
21
Table 14 Effect of irrigation water of different sea salt solutions on mean germination rate (GR)
coefficient of germination velocity (GV) mean germination time (GT) mean
germination index (GI) and final germination (FG) of C cajan seeds pre-soaked in non-
saline water prior to germination under various salinity regimes
Sea Salt
(ECiw= dSm-1) GR GV GT GI FG
Control 2624plusmn100 369plusmn005 027plusmn00 2624plusmn100 9667plusmn333
09 2743plusmn063 365plusmn009 027plusmn001 2743plusmn063 100plusmn00
16 2398plusmn218 423plusmn036 024plusmn002 2398plusmn218 8333plusmn882
35 2467plusmn086 378plusmn005 026plusmn00 2467plusmn086 9333plusmn333
42 3169plusmn733 311plusmn058 035plusmn008 3169plusmn733 9333plusmn333
58 2264plusmn081 399plusmn015 025plusmn001 2264plusmn081 90plusmn00
62 2253plusmn073 400plusmn013 025plusmn001 2253plusmn073 9333plusmn333
79 2074plusmn081 402plusmn00 025plusmn00 2074plusmn081 8333plusmn333
88 1927plusmn043 449plusmn008 022plusmn00 1927plusmn043 90plusmn577
99 1853plusmn033 486plusmn009 021plusmn00 1853plusmn033 90plusmn00
101 1635plusmn056 470plusmn022 021plusmn001 1635plusmn056 8667plusmn882
112 2263plusmn042 369plusmn020 027plusmn001 2263plusmn042 9667plusmn333
128 1953plusmn098 341plusmn00 029plusmn00 1953plusmn098 9667plusmn333
131 1368plusmn059 440plusmn018 023plusmn001 1368plusmn059 6667plusmn333
145 1276plusmn099 446plusmn019 023plusmn001 1276plusmn099 60plusmn577
156 1289plusmn153 447plusmn030 023plusmn002 1289plusmn153 8000plusmn100
168 876plusmn104 589plusmn078 018plusmn002 876plusmn104 8667plusmn333
LSD005 5344 3312 0064 5344 1313
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and LSD among the treatments is recorded at p lt 005
22
13 Experiment No 2
Effect of Pre-soaked seeds of Cajanus cajan in various dilutions of sea
salt for germination in water of respective sea salt concentrations
131 Materials and methods
1311 Seed germination
Procedure of seed germination has been mentioned in Experiment No 1 earlier The seeds
were pre-soaked in various sea salt concentrations instead of non-saline water and
germinated in respective sea salt concentrations Their electrical conductivities mentioned
in Table 15 Data were calculated and analysed according to formulas given in Experiment
No 1
Since these pre-soaked seeds in different sea salt concentration showed 50
germination at 03 equivalent to ECiw= 42dSm-1 sea salt solution any further work
beyond ECiw= 42dSm-1was not continued
132 Observations and Results
The final percent germination related with salinity in accordance with Maas and
Hoffman (1977) linear relative threshold response model as follows
Relative Final Germination = 100-200 (Ke ndash 005)
Where threshold salt concentration was 005 and Ke is the concentration of salts
at which relative final germination may be predicted This model indicated 50
declined in final germination at 030 salt concentration corresponding to ECiw= 42
dSm-1 (Table 16 Appendix II)
Rate of germination was significantly decreased (p lt 0001) from first day to final
(day 07) of observation and it was inversely correlated with sea salt concentration High
germination rate was recorded in control and low sea salt concentrations in early days of
seed incubation compared to higher sea salt concentrations but the difference in rate was
reduced (Table 17 Appendix II)
23
A progressive decline (p lt 0001) in coefficient of germination velocity was
observed with increasing salinity and fifty percent reduction was observed at 021 sea
salt concentration (ECiw = 319 dSm-1 Figure 11 Appendix II)
Final germination percentage was decreased significantly with increasing sea salt
concentrations However the difference was insignificant at lower (ECiw = 16 dSm-1)
salinity (Figure 11 Appendix II)
Mean germination time of seeds was increased significantly (p lt 0001) with
increasing sea salt concentrations However the difference was insignificant at lowest
(ECiw = 09 dSm-1) salinity (Figure 11 Appendix II)
Mean germination index was also significantly decreased (plt0001) with
increasing salt concentrations except for ECiw = 09 dSm-1 salinity Fifty percent reduction
in mean germination index was observed at 0188 sea salt concentration (ECiw = 289
dSm-1 Figure 11 Appendix II)
24
Table 15 Electrical conductivities of different sea salt solutions used in germination of C cajan
Sea salt () ECiw (dSm-1)
0 04
005 09
01 16
015 24
02 32
025 39
03 42
ECiw is the electrical conductivity of irrigation water measured in deci semen per meter
25
Table 16 Effect of irrigation water of different sea salt solutions on germination percentage (GP) per day of C cajan seeds pre-soaked in respective sea salt concentrations
with duration of time
Sea salt
ECiw (dSm-1)
GP
1st day
GP
2nd day
GP
3rd day
GP
4th day
GP
5th day
GP
6th day
GP
7th day
Control 6667plusmn333 8667plusmn333 10000plusmn000 10000plusmn000 10000plusmn000 10000plusmn000 10000plusmn000
09 7000plusmn000 7667plusmn333 9000plusmn000 10000plusmn000 10000plusmn000 10000plusmn000 10000plusmn000
16 4667plusmn333 6000plusmn000 7333plusmn333 8000plusmn000 8667plusmn333 8667plusmn333 9000plusmn577
24 4333plusmn333 5000plusmn000 6000plusmn577 6667plusmn333 7333plusmn333 7333plusmn333 8000plusmn000
32 3000plusmn000 3333plusmn333 3667plusmn333 4333plusmn333 5000plusmn577 6000plusmn577 7000plusmn577
39 1667plusmn333 2333plusmn333 2333plusmn333 4000plusmn577 4333plusmn333 5000plusmn000 6000plusmn000
42 667plusmn333 1333plusmn333 2333plusmn333 2333plusmn333 3333plusmn333 3667plusmn333 5000plusmn000
LSD 005 Salinity 327 Time 327
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and LSD among the treatments was recorded at p lt 005
25
26
Table 17 Effect of irrigation water of different sea salt solutions on germination rate (GR) per day of Ccajan
seeds pre-soaked in respective sea salt concentrations with duration of time
Sea salt
(ECiw= dSm-1)
GR
1st day
GR
2nd day
GR
3rd day
GR
4th day
GR
5th day
GR
6th day
GR
7th day
Control 667plusmn033 433plusmn017 333plusmn000 250plusmn000 200plusmn000 167plusmn000 143plusmn000
09 700plusmn000 383plusmn017 300plusmn000 250plusmn000 200plusmn000 167plusmn000 143plusmn000
16 467plusmn033 300plusmn000 244plusmn011 200plusmn000 173plusmn007 144plusmn006 129plusmn008
24 433plusmn033 250plusmn000 200plusmn019 167plusmn008 147plusmn007 122plusmn006 114plusmn000
32 300plusmn000 167plusmn017 122plusmn011 108plusmn008 100plusmn012 100plusmn010 100plusmn008
39 167plusmn033 117plusmn017 078plusmn011 100plusmn014 087plusmn007 083plusmn000 086plusmn000
42 067plusmn033 067plusmn017 078plusmn011 058plusmn008 067plusmn007 061plusmn006 071plusmn000
LSD 005 Salinity 014
Time 014 Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and LSD among the treatments is recorded at p lt 005)
27
Sea salt (ECiw = dSm-1
)
Contr
ol
09
16
24
32
39
42
Germ
ination Index(s
eedd
ays
-1)
0
2
4
6
8
Fin
al germ
ination (
)
0
20
40
60
80
100
Coeff
icie
nt of
germ
ination v
elo
city
(seedd
ays
-1)
00
01
02
03
04
05
06
07
Sea salt (ECiw = dSm-1
)
Contr
ol
09
16
24
32
39
42G
erm
ination tim
e (
Days
)
0
1
2
3
4
LSD005 = 0086
a = 0664 b = 1572
R2 = 0905 n =21
LSD005 = 062
a = 1239
b = 9836
R2 = 0894 n=21
LSD005 = 053
a = 8560b = -2272
R2 = 0969 n=21
RGF = 100-200 (Ke -005) Ke = 030
Figure 11 Effect of irrigation water of different sea salt solutions on seed germination indices of C cajan
(Bars represent means plusmn standard error of each treatment and significance among the treatments
was recorded at p lt 005)
28
14 Experiment No 3
Seedling establishment experiment of Cajanus cajan on soil irrigated with
sea salt of different concentrations
141 Materials and methods
1411 Seedling establishment
Seedling establishment experiment was carried out in Biosaline research field Department
of Botany University of Karachi Surface sterilized seeds pre-soaked were sown in small
plastic pots filled with 15 Kg sandy loam soil provided with farm manure at 91 ratio (30
water holding capacity) Sea salt solutions of different concentrations mentioned above
were used for irrigation The electrical conductivity of soil saturated paste (ECe) was also
determined at the end of the experiment (Table 18) Data on seedlings emergence was
recorded and their height were measured after 14 days of salinity treatment EC of the soil
(ECe) was initially 054 dSm-1 Statistical analyses were done according to the procedures
given in Experiment No 1
Since germination percentage of seeds pre-soaked in non-saline water was found
better under different concentrations of sea salt the seeds sown in soil for taking for
seedling establishment were pre-soaked in distilled water
29
142 Observations and Results
1421 Seedling establishment
Seedling emergence from soil was reduced significantly (p lt 0001) with increasing salt
concentration of irrigation water Not a single seedling emerged from soil in ge ECiw= 39
dSm-1 saline water irrigation However lower salinities (ECiw= 09 16 dSm-1) showed
slight decrease in seedling emergence with respect to controls Seedling emergence related
with salinity in accordance with a quadratic model as follows
Equation for seedling emergence () = 977751+ 44344 salt ndash 22215238 (salt)2 plusmn
6578 r = 09810 F = 15358 (p lt 00001)
Fifty percent reduction in seedling emergence was noticed at 016 sea salt
concentration (ECiw = 241 dSm-1 Figure 12 Appendix III)
1422 Shoot height
Shoot height was measured after fourteen days of irrigation Shoot length was
significantly decreased (p lt 0001) with increasing salinity A lower decrease was
observed in low sea salt salinity (ECiw= 09 and 16 dSm-1) compared to controls while
higher decrease in shoot height was noticed from ECiw= 2 dSm-1sea salt concentration
Shoot height related with salinity as follows
Equation for shoot height (cm) = 9116714 ndash 3420286 salt plusmn 09221 r = 0968 F =
128893 (p lt 0001)
Fifty percent reduction in shoot height was estimated at 013 sea salt concentration
(ECiw = 210 dSm-1) (Figure 12 Appendix III)
30
Table 18 Electrical conductivities of different Sea salt concentrations and ECe of soil saturated paste at the
end of experiment (ECe = 0447 + 1204 (salt ) plusmn 02797 R = 0987 F = 72301 (p lt
000001)
Sea salt () ECiw (dSm-1) ECe (dSm-1)
0 04 05
005 09 161
01 16 278
015 24 354
02 32 433
025 39 483
03 42 552
Electrical conductivity of soil saturated paste determined after 14 days of saline water irrigation in pots
Figure 12 Effect of irrigating water of different sea salt solutions on seedling emergence (A) and shoot
length (B) of C cajan (Bars represent means plusmn standard error of each treatment where similar
letters are not significantly different at p lt 005)
e f
Sea salt (ECiw = dSm-1
)
Contr
ol
16
27
8
35
4
43
3
48
3
Shoot le
ngth
(cm
)
0
2
4
6
8
10ab
c
de
Contr
ol
16
27
8
35
4
43
3
48
3Seedlin
g e
merg
ence (
)
0
20
40
60
80
100a
bb
c
d
A B
31
15 Experiment No 4
Growth and development of Cajanus cajan in Lysimeter (Drum pot
culture) being irrigated with water of different sea salt concentrations
151 Materials and methods
1511 Drum pot culture
A modified drum pot culture (lysimeter) installed by Ahmad amp Abdullah (1982) at
Biosaline research field (Department of Botany University of Karachi) was used in
present experiment Each drum pot (60 cm diameter 90 cm depth) was filled with 200 kg
of sandy loam mixed with cow-dung manure (91) having 28 water holding capacity
They are fixed at cemented platform at slanting position with basal hole to ensure rapid
drain Over irrigation was practiced to avoid the accumulation of salt in the root zone
1511 Experimental design
Growth and development of C cajan in drum pots was carried out in six different drum
pot sets (each in triplicate) and irrigated with sea salt of following concentrations
Drum pot Sets Sea salt
()
ECiw ( dSm-1) of
irrigation water
Resultant ECe (dSm-1) after
end of experiment
Set I Non saline (C) 04 05
Set II 005 sea salt 09 16
Set III 001 sea salt 16 28
Set IV 015 sea salt 24 35
Set V 02 sea salt 28 38
Set VI 025 sea salt 34 43
Note ECiw is the electrical conductivity of irrigation water and ECe is the electrical conductivity of the saturated soil extract taken after
eighteen weeks at the end of experiment
Ten surface sterilized seeds with 01 sodium hypochlorite were sowed in each
drum pot and were thinned to three healthy and equal size seedlings after two weeks of
establishment in their respective sea salt concentration Each drum pot was irrigated with
15 liters non-saline or respective sea salt solution at weekly intervals Electrical
conductivity of soil was measured by EC meter (Jenway 4510) using saturated soil paste
32
at the end of experiment Experiment was conducted for a period of 18 weeks (July to
November 2009) during which environmental data which includes average humidity
(midnight 76 and noon 54) temperature (low 23oC and high 33oC) wind velocity (14
kmph) and rainfall (~4 cm) was recorded (Pakistan Metrological Department Karachi) is
given in Figure 13Statistics were analysed according to the procedures given in
Experiment No 1
1512 Vegetative and Reproductive growth
Shoot height was measured at every two week interval after seedling establishment Fresh
and dry weight of shoot was recorded at final harvest (18th week when pods were fully
matured) Leaf succulence (dry weight basis Abideen et al 2014) Specific shoot length
(SSL Panuccio et al 2014) and relative growth rate (RGR Moinuddin et al 2014) were
measured using following equations
Succulence (g H2O gminus1 DW) = (FW minus DW) DW
SSL = shoot length shoot dry weight
RGR (g gminus1 dayminus1) = (lnW2 - lnW1) (t2 - t1)
Whereas FW fresh weight DW dry weight W1 and W2 initial and final dry weights and
t1 and t2 initial and final time of harvest in days
Reproductive data in terms of number of flowers number of pods number of seeds
and seed weight per plants was recorded during reproductive period
1513 Analysis on some biochemical parameters
Biochemical analysis of leaves was carried out at grand period of growth Following
investigations was undertaken at different biochemical parameters
i Photosynthetic pigments
Fresh and fully expended leaves (at 2nd3rd nodal part) samples (01g) were crushed in 80
chilled acetone and were centrifuged at 3000rpm for 10 minutes Supernatant were
separated and adjusted to 5ml final volume The absorbance was recorded at 663nm and
645 nm on spectrophotometer (Janway 6305 UVVis) for chlorophyll content while 480
33
and 510 nm for carotenoids Chlorophyll ab ratio was calculated after the amount
estimated The chlorophyll and carotenoid contents were determined according to Strain
et al (1971) and Duxbury and Yentsch (1956) respectively
Chlorophyll a (microgml) = 1163 (A665) ndash 239 (A649)
Chlorophyll b (microgml) = 2011 (A649) ndash 518 (A665)
Total Chlorophylls (microgml) = 645 (A665) + 1772 (A649)
Carotenoids (microgml) = 76 (A480) ndash 263 (A510)
ii Total soluble sugars
Dry leaf samples (01g) were homogenized in 5mL of 80 ethanol and were centrifuged
at 4000 g for 10 minutes 10 mL diluted supernatant in 5mL Anthronrsquos reagent was kept
to boil in 100oC water bath for 30 minutes and were cooled in running tap water Optical
density was taken at 620nm for the determination of soluble carbohydrates according to
Fales (1951)Total soluble carbohydrates was estimated against glucose as standard and
was calculated from the equation mentioned and expressed in mgg-1 dry weight
Total carbohydrates (microgmL-1) = 228462 OD 097275 plusmn004455
iii Protein content
Fresh and fully expended leaves at 2nd3rd nodal part were taken for protein estimation
The protein contents were measured according to Bradford Assay reagent method against
Bovine Serum Albumin as standards (Bradford 1976) Dye stock was made to dissolved
50mg comassie blue in 25 ml methanol The solution is added to 50ml of 85 phosphoric
acid and diluted to 100 ml with distilled water 02g fresh leaf samples were mills in 5 ml
phosphate buffer pH7 5ml of assay reagent (diluting 1 volume of dye stock with 4 volume
distilled water) were added in 01 ml leaf extract used for enzyme assay Absorbance was
recorded at 590nm and was expressed in mgg-1 fresh weight Proteins were calculated
from the following best fit standard curve equation
Protein (microgml-1) = -329196 + 1142755 plusmn 53436
34
152 Observations and Results
1521 Vegetative and Reproductive growth
Effect of sea salt on vegetative growth including height fresh and dry weight of Cajanus
cajan is presented in (Figure 14 and 15 Appendix-VI) Comparative analysis showed
that plant growth (all three parameters) was significantly increased with time (plt 0001)
however it was linearly decreased (plt 0001) with increasing salinity (Figure 16
Appendix-VI) shows the water content succulence relative growth rate (RGR) and
specific shoot length (SSL) of Cajanus cajan Under saline conditions all parameters were
significantly reduced in comparison to control however SSL showed decline after ECe38
dSm-1 Salt induced growth reduction was more pronounced at ECe 38 and 43 dSm-1 in
which plants died before reaching the reproductive maturity after 12 and 14 weeks at sea
salt treatments respectively Therefore further analysis was carried out in plant grown up
to ECe= 35 dSm-1 sea salt concentrations
Salinity significantly reduced (plt 0001) reproductive parameters including
number of flowers pods seeds and seed weight (Figure 17 Appendix-VII) Among all
treatments highest reduction was observed in 315 dSm-1 in which number of flowers and
pods reduced up to 7187 and 70 respectively Similar trend was observed in total
number and weight of seeds which showed 80 and 8793 reduction respectively
1522 Study on some biochemical parameters
i Photosynthetic pigments
Figure 18 Appendix-VII shows the effect of salinity on pigments (chlorophyll a b ab
ratio and carotenoids) of C cajan leaves A slight increase in total chlorophyll contents
(1828) and chlorophyll ab ratio (1215) was observed at low salinity (ECe= 16 dSm-
1) however they were significantly reduced (4125 and 3630 respectively) in high salt
treatment (plt 0001) Chlorophyll a was higher than chlorophyll b in all treatments
however chlorophyll b was un-affected by salinity whereas total chlorophyll content and
ab ratio was disturbed due to change in chlorophyll a This reduction was more
pronounced at high salinity (ECe= 35 dSm-1) in which chlorophyll a total chlorophylls
and ab ratio was decreased by 505 412 and 3630 respectively Carotenoid content
was maintained at ECe= 16 dSm-1 and decreased with further increase in salinity
35
ii Total soluble sugars
Total soluble sugars in leaves of C cajan is presented in Figure 19 Appendix-VII Total
leaf sugars in C cajan were remained un-affected at 16 dSm-1 and subsequently decreased
with further increase in medium salinity Although total sugars were decreased at ECe 28
and 35 dSm-1 a significant increase (~25) of soluble sugars was observed at higher
salinities However this increment was accounted for decrease (504 ) in insoluble sugar
content at that salinity levels
iii Protein
Total protein in leaves of C cajan is presented in Figure 19 Appendix-VII An increase
in leaf protein content in C cajan was found at lower salinity regime (ECe= 16 dSm-1)
which was followed by significant reduction with further increase in salinity This decline
was 2040 at 28 which was more pronounced (5646 ) at high salinity level (ECe=
35dSm-1)
36
Months (2009)
Jun Jul Aug Sep Oct Nov Dec
Valu
es
0
10
20
30
40
50
60
70
80
90
Rainfall (cm)Low Temp (
oC)
High Temp (oC)
Humidity at noon () Wind (kmph)
Humidity at midnight ()
Figure 13 Environmental data of study area during experimental period (July-November 2009)
Time (Weeks)
2 4 6 8 10 12 14 16 18
Pla
nt heig
ht (c
m)
0
30
60
90
120
150
180
210
43 38 35 28 16 Control
Figure 14 Effect of salinity using irrigation water of different sea salt concentrations on height of C cajan
during 18 weeks treatment (Lines represent means plusmn standard error of each treatment represents
significant differences at p lt 005)
37
Sea salt (ECe= dSm
-1)
Cont 16 28 35 38 43
Sea salt (ECe= dSm
-1)
Cont 16 28 35 38 43
Fre
sh w
eig
ht (g
)
0
5
10
15
20
25
30
35Initial Final
a
b b
c c cab b
c c cC 16 28 35 38 43
Fre
sh w
eig
ht
(g)
012345 a
bb
bc ca a ab b c c
Dry weightMoisture
Figure 15 Effect of salinity using irrigation water of different sea salt concentrations on initial and final
biomass (fresh and dry) of C cajan (Bars represent means plusmn standard error of each treatment Different
letters represent significant differences at p lt 005)
Mo
istu
re (
)
0
20
40
60
80
100
Succu
lance
(
)
0
20
40
60
80
100
Sea salt (ECe= dSm
-1)
Co
nt
16
28
35
38
43
RG
R (
)
0
20
40
60
80
100
Co
nt
16
28
35
38
43
SS
L (
)
0
20
40
60
80
100
Sea salt (ECe= dSm
-1)
ab
b b
c c
a
b bc c c
a
b b
c c c
a a a ab
c
Figure 16 Percent change (to control) in moisture succulence relative growth rate (RGR) and specific
shoot length (SSL) of C cajan under increasing salinity using irrigating water of different sea
salt concentrations (Bars represent means plusmn standard error of each treatment Different letters
represent significant differences at p lt 005)
38
Sea salt (ECe= dSm-1)
Control 16 28 35
Tota
l seeds (
Pla
nt-1
)
0
20
40
60
80
100
120
140 Seed w
eig
ht (g
pla
nt -1
)
0
5
10
15
20
25
Num
ber
10
20
30
40
50
60
70 a
b
cc
a
a
b
b
b c
c
a
b
a
c c
Flowers
Pods
Seed weightTotal seeds
Figure 17 Effect of irrigating water of different sea salt solutions on reproductive growth parameters
including number of flowers pod seeds and seed weight of C cajan (Values represent means
plusmn standard error of each treatment Different letters represent significant differences at p lt
005)
39
Sea salt (ECe=dSm-1
)
Control 16 28 35
Caro
tinoid
s (
mg g
-1 F
W)
000
005
010
015
020
025
030
Chlo
rophyll
(mg g
-1 F
W)
00
02
04
06
08
ab
ratio
00
05
10
15
20
25
ab
ab
b
a
cd
b
a
c
d
a
b
c
d
a
a
ab
b
Figure 18 Effect of irrigating water of different sea salt solutions on leaf pigments including chlorophyll a
chlorophyll b total chlorophyll and carotenoids of C cajan (Bars represent means plusmn standard
error of each treatment Different letters represent significant differences at p lt 005)
40
Figure 19 Effect of irrigating water of different sea salt solutions on total proteins soluble insoluble and
total sugars in leaves of C cajan (Bars represent means plusmn standard error of each treatment
Different letters represent significant differences at p lt 005)
Sea salt (ECe= dSm
-1)
C 16 28 35
Pro
tein
(m
g g
-1 F
W)
00
01
02
03
04
05
06
Su
gar
s (m
g g
-1 F
W)
00
02
04
06
08
a ab b
a a
b b
a ab b
a
b
ab
c
SoluableInsoluable
41
16 Experiment No 5
Range of salt tolerance of nitrogen fixing symbiotic bacteria associated
with root of Cajanus cajan
161 Materials and methods
1611 Isolation Identification and purification of bacteria
Nodules of C cajan grow in large clay pots and irrigated with running tap water at
biosaline agriculture research field were collected from the lateral roots (about 15 cm soil
depth) Nodules were surface sterilized with sodium hypochloride (2) for 5 min and
vigorously washed with sterilized distilled water Each nodule was crushed with sterilized
rod in 5 ml distilled water The bacterial suspension was streaked on yeast extract mannitol
agar (YEM) (K2HPO4 05 g MgSO 4 025g Na Cl 01 g Manitol 10g Yeast Extract 1g
Agar 20 g in 1000 ml of Distilled water) with the help of sterilized wire lope Colonies
were identified by studying different phenotypic characters as Rhizobium fredii
(Cappuccino and Sherman 1992 Sawada et al 2003) Pure culture of Rhizobium species
was stored at -20oC temperature
1612 Preparation of bacterial cell suspension
Bacteria were multiplied by growing in YEM broth for 48 hrs on shaking incubator (140
rpm) at 37oC in dark The culture in broth was centrifuged at 4000 rpm for 10 min to
obtained bacterial cell pellet Pellet was washed and centrifuged twice with sterilized
distilled water Pellet then re-suspended in sterilized distilled water before use
1613 Study of salt tolerance of Rhizobium isolated from root nodules of
C cajan
Assessment for salinity tolerance of Rhizobium species was assessed on YEM agar
Salinity levels of 0 05 10 15 20 25 and 30 having electrical conductivity 06 90
188 242 306 366 and 423 dSm-1 respectively were maintained with NaCl Bacterial
cell suspension of 01 ml (5times 103 colony forming unitsml) was poured in each sterilized
Petri dish 10 ml of molten YEM agar was poured immediately and shake well before
solidification of agar Petri plates were incubated at 37deg C in dark Colonies were observed
and counted in colony counter after 48 h and photographed (Dubey et al 2012 Singh and
42
Lal 2015) There were three replicates of each treatment and data were transformed to
log10 before analysis
162 Observations and Results
Colonies of Rhizobium on YEM agar at different salinity levels is presented in Figure 110
and 111 Appendix-VIII A significant decrease (plt0001) in rhizobial colonies was
observed with increasing salinity However the difference between non saline control and
90 dSm-1 and as that of 242 dSm-1 and 302 dSm-1 salt (NaCl) concentration showed
nonsignificant difference in rizobial colonies Whereas drastic decreased was observed on
further salinity levels Rhizobial colonies were not found at 423 dSm-1salt concentration
NaCl (ECw= dSm
-1)
06 9 188 242 306 366 423
Rh
izo
bia
l co
lonie
s (l
og
10)
0
1
2
3
4 a a
b
c c
d
e
Figure 110 Growth of nitrogen fixing bacteria associated with root of C cajan under different NaCl
concentrations (Bars represent means plusmn standard error of each treatment among the treatments
is recorded at p lt 005)
43
Figure 111 Photographs showing growth of Rhizobium isolated from the nodules of C cajan invitro on
YEM agar supplemented with different concentrations of NaCl (ECw)
188
423 90
Control
366
306 242
44
17 Experiment No 6
Growth and development of Ziziphus mauritiana in large size clay pot
being irrigated with water of two different sea salt concentrations
171 Materials and methods
1711 Experimental design
The grafted plants obtained from the local nursery of Mirpurkhas Sindh were transported
to the Biosaline Agriculture Research field Department of Botany University of Karachi
and were transplanted carefully in large earthen pots containing 20 Kg sandy loam soil
mixed with cow dung manure at 91 ratio having about 5 liters of water holding capacity
with a basal hole for drainage of excess salts to avoid accumulation in the rhizosphere
Over irrigation with about 15 liters of non-saline saline water was kept weekly in summer
and biweekly in winter to avoid accumulation of salts in rhizosphere Plants were irrigated
to start with non-saline tap water for about two weeks for establishment All the older
leaves were fallen and new leaves were developed during establishment period Following
irrigation schedule of non-saline (control) and saline water was selected in view of Z
mauritiana being moderately salt tolerant plant which includes both low and as well as
higher concentrations of the salt in irrigation
Sea salt () ECiw (dSm-1)
of irrigation water
Average resultant ECe (dSm-1) of soil
with some fluctuation often over
irrigation
Non saline (Control) 06 12
04 63 72
06 101 111
ECiw = Electrical conductivity of irrigation water ECe = Electrical conductivity of saturated soil
Healthy and well established plants were selected of nearly equal height and
divided into three sets each contain three replicates (total nine pots) Salinity was provided
through irrigation water of different sea salt concentrations All pots except non-saline
control were initially irrigated with 01 sea salt solution and then sea salt concentration
45
in irrigation medium was increased gradually upto the required salinity level The salinity
level of soil was monitored by taken the electrical conductivity of saturated soil paste the
end of experiment The electrical conductivity of soil (ECe) maintained at the level of 12
72 and 111 dSm-1 respectively as described by Mass and Hoffman (1977)
1712 Vegetative and reproductive growth
Vegetative growth in terms of shoot height fresh and dry weight of shoot and number of
branches were noted at destructive harvesting at initial (establishment) 60 and 120 days
of growth For dry weight shoots were dried in oven at 70˚C for three days Shoot
succulence specific shoot length (SSL) moisture percentage and relative growth rate
(RGR) was calculated at final harvest by using formulas given in Experiment No 4
Whereas number of flowers in reproductive data were recorded at onset of reproductive
period
As regard of fruit formation the duration of experiment was not sufficient for fruit
setting and furthermore the amount of sol in pots was not sufficient for healthy growth of
this plant Secondly flowering and fruiting is reported to be poor at the time of 1st initiation
of reproductive period (Azam-Ali 2006) Furthermore statistical significance of flower
and fruit count also become far less due to their excess dropping at early stage Hence it
was decided to proceed with study of fruit formation in forthcoming field trials of their
intercropping culture
1713 Analysis on some biochemical parameters
Biochemical analyses were performed at the grand period (at the time of flower initiation)
in fully expended fresh leaves Chlorophyll contents soluble sugar contents and soluble
proteins were analyzed Leaves samples taken from 3rd 4th node below the apex according
to the procedures given in Experiment No 4
46
172 Observations and Results
1721 Vegetative and Reproductive growth
Effect of sea salt on vegetative growth of Z mauritiana including height fresh and dry
weight is presented in (Figure 112 Appendix-IX) Comparative analysis showed that
plant growth (all three parameters) was significantly increased with time (plt 0001)
however number of branches was decreased (plt 0001) with increasing salinity
Figure 113 shows the moisture content succulence relative growth rate (RGR)
and specific shoot length (SSL) of Z mauritiana A non-significant difference in shoot
succulence SSL and moisture content was observed with time salinity and interaction of
both factors However RGR showed decline Salt induced growth reduction was more
pronounced at higher salinities
In Z mauritiana plants number of flowers showed significant decrease (plt0001)
with increasing salinity treatment Flower initiation seems non-significant at early growth
(60 days) period in controls and salinity treatments However drastic decrease was
observed with increasing salinity in 120 days of observation (Figure 114 Appendix-IX)
1722 Study on some biochemical parameters
i Photosynthetic pigments
The effect of Z mauritiana leaves pigments (chlorophyll a b ab ratio) on salinity shower
a slight difference in chlorophyll lsquoarsquo over control However chlorophyll lsquobrsquo contents
showed increase over control in both salinity treatments due to which the total chlorophylls
were also enhanced compared to controls Chlorophyll ab ratio was significantly
(plt0001) decreased in both salinities as compared to control (Figure 115 Appendix-IX)
ii Sugars and protein
In Z mauritiana plant soluble sugars were significantly decreased (plt0001) over controls
whereas proteins showed little decrease under salinity treatments compared to controls
(Figure 116 Appendix-IX)
47
Control 72 111
Fre
sh w
eig
ht (g
)
0
150
300
450
600
750
900
Sea salt (ECe= dSm
-1)
Control 72 111
Dry
weig
ht (g
)
0
150
300
450
600
750
900
Num
ber
of bra
nches
3
6
9
12
15
18
Heig
ht (c
m)
20
40
60
80
100
120
140
160
Initial 60 days 120 days
AcBb
Ba
AcBb Ba
AcBb Ba
Ac
BbBa
Figure 112 Effect of salinity using irrigation water of different sea salt concentrations on height number of
branches fresh weight and dry weight of shoot of Zmauritiana after 60 and 120 days of
treatment (Bars represent means plusmn standard error of each treatment Different letters represent
significant differences at p lt 005)
48
120 days 60 days InitialS
uccula
nce (
g g
-1 D
W)
00
03
06
09
12
Sea salt (ECe= dSm
-1)
SS
L (
cm
g-1
)
00
01
02
03
04
05
Control 72 111
Mois
ture
(
)
0
10
20
30
40
50
60
Control 72 111
RG
R (
mg g
-1 d
ay
-1)
0
5
10
15
20
a a aa a a a a a a
a aa a a a a a
a a aa a a a a a a a
b
b b
c
Figure 113 Effect of salinity using irrigation water of different sea salt concentrations on succulence
specific shoot length (SSL) moisture and relative growth rate (RGR) of Z maritiana (Bars
represent means plusmn standard error of each treatment Different letters represent significant
differences at p lt 005)
49
Sea salt (ECe= dSm
-1)
Control 72 111
Num
ber
of flow
ers
0
20
40
60
80
100
120
140 60 days120 days
Ac
BbBa
Figure 114 Effect of salinity using irrigation water of different sea salt concentrations on number of flowers
of Z mauritiana (Bars represent means plusmn standard error of each treatment Different letters
represent significant differences at p lt 005)
Sea salt (ECe= dSm
-1)
Control 72 111
Ch
loro
ph
yll
(mg g
-1)
00
03
06
09
12
15
18
bba
bba
bb
a
chl b chl a ab
ab
ra
tio
00
05
10
15
20
Figure 115 Effect of salinity using irrigation water of different sea salt concentrations on leaf pigments
including chlorophyll a chlorophyll b total chlorophyll and chlorophyll ab ratio of Z mauritiana (Values
represent means plusmn standard error of each treatment Different letters represent significant differences at p lt
005)
50
Figure 116 Effect of salinity using irrigation water of different sea salt concentrations on total sugars and
protein in leaves of Z mauritiana (Bars represent means plusmn standard error of each treatment
Different letters represent significant differences at p lt 005)
Sea salt (ECe= dSm
-1)
C 04 06
Pro
tein
s (m
g g
-1)
0
10
20
30
40
50
60
70
80
Solu
ble
sugar
s (m
g g
-1)
0
3
6
9
12
15
18a
a
bb
b b
Control 72 111
51
18 Discussion
Seed germination is the protrusion of radicle from the seed which is adversely affected by
salinity stress (Kaymakanova 2009) Salinity imposes the osmotic stress by accumulation
of Na+ and Cl- which decrease soil water potential that ultimately inhibits the imbibition
process (Othman 2005) Effect of seed germination against salinity is reported in linear
threshold response model of Maas and Hoffman (1977) The germination of a salt tolerant
desert legume Indigofera oblongifolia and a desert graminoid Pennisetum divisum are
also reported to behave to salinity in similar manner (Khan and Ahmad 1998 2007) Many
workers used chemical (organic inorganic) salt temperature biological and soil matrix
priming techniques to enhance seed germination percentage and especially germination
rate in saline medium (Ashraf et al 2008 Ashraf and Foolad 2005)Encouraging results
in most of the species of glycophytes and hydrophytes were found by presoaking in pure
water prior to germinating under saline condition Our study supports this finding and
seeds soaked in distilled water prior to germination performed better than those which
were presoaked in sea salt solutions Salinity adversely affects at all germination
parameters (germination percentage germination rate coefficient of germination velocity
and germination index) directly proportional with increasing salinity (Tayyab et al 2015)
With increase in time a delayed germination at higher salinity was found Higher sea salt
(168 dSm-1 for pure water presoaking and 35 dSm-1 for presoaking in respective
salinities) showed 50 or more reduction in all germination indices as compared to control
(Table 13-16 Figure 11)Our results are parallel with the finding of other workers such
as Kafi and Goldani (2001) who found the same trend in chickpea at higher salinities Pujol
et al (2000) reported that increased salinity inhibit the seed germination as well as delays
germination initiation in various halophyte species as well Similar response was also
found in some other crops such as pepper (Khan et al 2009) sunflower (Vashisth and
Nagarjan 2010) and eggplant (Saeed et al 2014) Salt tolerance within species may vary
at germination and other growth phases (Khan and Ahmad 1998)
According to our results C cajan appeared to be a salt sensitive in initial growth
phase specially when presoaked in saline medium (Figure 12) however at later growth
stages it proved relatively salt tolerant Salt stress delays or either seize the metabolic
activities during seed germination in salt sensitive and even in salt tolerant plants (Khan
and Ahmad 1998 Ali et al 2013b) Salinity also imposes the oxidative stress due to
52
overproduction of reactive oxygen species which may alter metabolic activities during
germination growth and developmental stages (Zhu 2001 Munns 2005
Lauchli and Grattan 2007)
In our study seeds of pigeon pea were unable to emerge beyond ECe39 dSm-1 sea
salt concentration Height of seedling was significantly affected by increasing salinity
(Figure 12) Similar results are also reported in Indian mustered (B juncea Almansouri
et al 2001) some Brassica species (Sharma et al 2013) and tomato cultivars (Jamil et
al 2005) Growth retardation with increasing salinity may be due to reduced
photosynthetic efficiency and inhibition of enzymatic and non-enzymatic proteins
(Tavakkoli et al 2011) Furthermore salt stress also limit the DNA and RNA synthesis
leads to reduced cell division and elongation during germination growth and
developmental stage
Khan and Sahito (2014) found variation in salt tolerance within species subspecies
and provenance level Furthermore the salt tolerance of a species may also vary at
germination and growth phases (Khan and Ahmad 1998 Ali et al 2013a) Srivastava et
al (2006) suggested that the genetic variability influences salinity tolerance eg wild
species like Cajanus platycarpus C scaraboides and C sericea showed better salt
tolerance than C cajan In this connection Wardill et al (2006) has also reported genetic
diversity in Acacia nilotica C cajan in this study appeared to be a salt sensitive at
germination in compression with later stages of growth Seedling establishment at saline
solution faces adverse effects when emerging radicle and plumule come in contact with
salt effected soil particle or saline water hence percent seedling establishment remains
less than germination percentage observed at petri plate Ashraf (1994) found that salinity
tolerance of different varieties of C cajan do not much differ at germination and early
growth stages whereas at adult growth stage show improvement in salt tolerance
Soil salinity is a major limiting factor for plant growth and yield production
particularly in leguminous plants (Guasch-Vidal et al 2013 Tayyab et al 2016) In
present study Plant height RGR fresh and dry biomass were severely reduced with
increasing salinity and plant was unable to grow after ECe= 43 dSm-1(Figure 14-16)
This growth inhibition of C cajan may be accounted for individual and synergistic effect
of water stress nutrient imbalances and specific ions toxicities (Hasegawa et al 2000
Silvera et al 2001) Salt induced ion imbalance results in lower osmotic potential which
53
alter physiological biochemical and other metabolic processes leading to overall growth
reduction (Del-Amor et al 2001) Excessive amount of salt in cytoplasm challenge the
compartmentalization capacity of vacuole and disrupts cell division cell elongation and
other cellular processes (Munns 2005 Munns et al 2006) Our results are parallel with
some other studies in which significant growth inhibition of peas chickpea and faba beans
have been reported against salt stress (El-Sheikh and Wood 1990 Delgado et al 1994)
Singla and Garg (2005) also observed a similar salt sensitive growth response in Cicer
arietinum In our study the fresh and dry biomass of C cajan also showed inhibitory
behavior to salt stress (Figure 15) Hernandez et al (1999) also found significant reduction
in dry biomass of pea plant and common bean (40 and 84 respectively) when grown
in saline medium Mehmood et al (2008) also found similar results in Susbania sasban
Salinity also has imposed deleterious effects on reproductive growth of C cajan
Production of flowers and pods are significantly decreased in response to salinity (Figure
19) Increase in flower shedding leads to decreased number of pods indicating salt
sensitivity of plant at reproductive phase which was more pronounced at high salinity
(Vadez et al 2007) Furthermore seed production and weight of seed per plant was also
linearly decreased Salt induced reduction of reproductive growth has also been found in
mung bean in which 60 and 12 less pods and seeds were produced respectively at 06
saline solution (Qados 2010) Similar results are reported in faba bean (De-Pascale and
Barbieri 1997) tomato (Scholberg and Locascio 1999) maiz sunflower (Katerji et al
1996) and watermelon (Colla et al 2006) Salinity reduces reproductive growth by
inhibiting growth of flowers pollen grains and embryo which leads to inappropriate ovule
fertilization and less number of seeds and fruits (Torabi et al 2013)
On biochemical parameters total chlorophyll and chlorophyll ab ratio has
increased in low salinity in contrast the adverse effect at higher salinity could be due to
high Na+ dependent breakdown of these pigments (Li et al 2010 Yang et al 2011)
Chlorophyll a is usually more prone to Na+ concentration and decrease in total chlorophyll
is mainly attributed to the destruction of chlorophyll a (Fang et al 1998 Eckardt 2009)
This diminution could be due to the destruction of enzymes responsible for green pigments
synthesis (Strogonov et al 1973) and increased chlorophyllase activity (Sudhakar et al
1997) Thus insipid of leaf was a visible indicator of salt induced chlorophyll damage
which was well correlated with quantified values as reported in other legume species
54
(Soussi et al 1998 Al-Khanjari et al 2002) In this study chlorophyll a was found to be
more sensitive than chlorophyll b (Figure 18) Garg (2004) also found similar reduction
in chlorophyll pigments (a b and total chlorophyll) in chickpea cultivars under salinity
stress
At low salinity (16 dSm-1) total carotenoids remained unaffected along with
increased total chlorophyll (Figure 18) which may suggest a role of carotenoids in
protection of photosynthetic machinery (Sharma et al 2012) Similar response was found
in Cajanus indicus and Sesamum indicum (Rao and Rao 1981) however
Sivasankaramoorthy (2013) and Ramanjulu et al (1993) reported slight increase of leaf
carotenoids in Zea maiz and mulberry when exposed to NaCl High salinity was destructive
for both leaf pigments (chlorophyll and carotenoids) of C cajan which was in accordance
with Reddy and Vora (1985) who found similar decrease in some other salt sensitive crops
Salinity led to the conversion of beta-carotene to Zeaxanthin which protect plants against
photo-inhibition (Sharma and Hall 1991)
In present study with increasing salinity water content and succulence of C cajan
were significantly reduced which indicated loss of turgor (Figure 16) Our data suggest
that decreased succulence by lowering water content may help in lowering leaf osmotic
potential when exposed to increasing salinity which is in agreement with findings of Parida
and Das (2005) and Abideen et al (2014) In addition increased production and
accumulation of organic substances is also necessary to sustain osmotic pressure which
provide osmotic gradient to absorb water from saline medium (Hasegawa et al 2000
Cha-um et al 2004) Compatible solutes including carbohydrates amino acids proteins
and ammonium compounds play important roles in water relations and cell stabilization
(Ashraf and Harris 2004) In this study C cajan produce more soluble sugars (Figure 18)
which is considered as a typical plant response under saline conditions (Murakeozy et al
2003) Sugars serve as organic osmotica and their available concentration is related to the
degree of salt stress and plantrsquos tolerance (Ashraf 1994 Murakeozy et al 2003) Sugars
are involved in osmoprotection osmoregulations carbon storage and radical scavenging
activities (Pervaiz and Satyawati 2008) On the other hand insoluble and total sugars were
reduced in higher salinity which is also supported by Parida et al (2002) and Gadallah
(1999) who found similar results in Bruguiera parviflora and Vicia faba
55
Total soluble proteins of C cajan were reduced due to deleterious effects of salinity
(Figure 18) The accumulation of Na+ in cytosol disrupts the protein and nucleic acid
synthesis (Bewley and Black 1985) Gill and Sharma (1993) and Muthukumarasamy and
Panneerselvam (1997) also reported decreased protein content with increasing salinity in
Cajanus cajan seedlings Similar results were found when tomato (Azeem and Ahmad
2011) Zingiber officinale (Ahmad et al 2009) and Sorghum bicolor (Ali et al 2013a)
were grown under variable salt concentrations (Figure 19)
Nodule formation of Rhizobium in Legume depends upon interaction between soil
chemistry of salt composition and osmotic regimes of salt and water (Velagaleti et al
1990 Zahran 1991 Zahran and Sprent 1986) Salinity reduces plant growth directly
through ion and osmotic effects and indirectly by inhibiting Legume-Rhizobium
association (El-Shinnawi et al 1989) Studies demonstrated a more sensitive response of
rhizobial N-fixing mechanism than growth of plant to abiotic stresses including salinity
(Mhadhbi et al 2004) In nodules metabolic disturbance initiated with the production of
ROS leading to tissues injury and loss of nodule function (Becana et al 2000) In general
it slow down the nitrogenase activity and decrease nodule protein and leghemoglobin
content which decreased becteroid development (Mhadhbi et al 2008) In consequence
plant suffer directly by salt induced ion toxicity low water uptake and photosynthetic
damage and indirectly through weak association of symbionts due to high energy demand
for nodule function (Pimratch et al 2008) In our study the isolated rhizobial strain from
nodules of C cajan was found to be tolerant to salinity even up to 2 (ECw= 306 dSm-1)
NaCl (Figure 110 and 111) Some of the other species of Rhizobium such as Brady
Rhizobium have been shown salt tolerant even at higher concentration than their
leguminous hosts (Zahran 1999) For instance a number of rhizobial species can tolerate
up to 06 NaCl (Yelton et al 1983) while Rhizobium meliloti can tolerate 175 to
40 NaCl and R leguminosarum can tolerate can tolerate upto 2 NaCl (Abdel-Wahab
and Zahran 1979 Sauvage et al 1983 Breedveld et al 1991 Helemish 1991
Mohammad et al 1991 Embalomatis et al 1994 Mhadhbi et al 2011) Rhizobia
isolated from soybean and chickpea can tolerate up to 2 NaCl with a difference of fast-
growing and slow growing strains (El-Sheikh and Wood 1990 Ghittoni and Bueno 1996)
Similarly Rhizobium from Vigna unguiculata can survive up to up to 55 NaCl
(Mpepereki et al 1997)
56
Present study shows an increase in vegetative growth in terms of plant height and
fresh and dry weight of shoot with increasing time under non-saline and saline conditions
but the increase was rapid at early period of growth (Figure 112) All the vegetative
growth parameters determined were reduced under salinity stress compared to non-saline
control Measurements of shoot moisture succulence specific shoot length and RGR
(Figure 113) indicate that Z mauritiana adjusted in its water relation over coming
negative water and osmotic potential with increase in salinity levels increased There is
evidence that water and osmotic potentials of salt tolerant plants become more negative in
higher salinities (Khan et al 2000) These altered water relations and other physiological
mechanisms help plants to get by adverse abiotic stress like that of drought and salinity
(Harb et al 2010) However the results clearly showed that salinity had an inhibitory
effect on growth but the decline was less at early sixty days and more during later 60-120
days in compression to controls Growth inhibition in shoot has been observed in number
of plants including different species of halophytes (Keiffer and Ungar 1997) chickpea
(Cicer arietinum Kaya et al 2008) and different wheat cultivars (Triticum aestivum
Moud and Maghsoudo 2008)
Salinity also caused reduction in the number of branches and the number of flowers
in Z mauritiana however reduction in the number of flowers is non-significant in ECe=
72 dSm-1 salinity treatment in comparison with non-saline control (Figure 114) The main
reason for this reduction could be attributed to suppression of growth under salinity stress
during the early developmental stages (shooting stage) of the plants These results are
similar to those reported by Ahmad et al (1991) and Khan et al (1998) As affirmed by
Munns and Tester (2008) suppression of plant growth under saline conditions may either
be due to osmotic effect of saline solution which decreases the availability of water for
plants or the ionic effect due to the toxicity of sodium chloride High salt concentration in
rooting medium also reduced the uptake of soil nutrients a phenomenon which affected
the plant growth thus resulting in less number of branches per plant Various abiotic
stresses such as temperature drought salinity light and heavy metals altered plant
metabolism which ultimately affects plant growth and productivity Amongst these
salinity stress is a major problem in arid and semiarid regions of the world (Kumar et al
2010) Salinity has an adverse effect on several plant processes including seed
germination seedling establishment flowering and fruit formation and ripening (Sairam
and Tyagi 2004) Salinity stress also imposes additional energy requirements on plant
57
cells and less carbon is available for growth and flower primordial initiation (Cheesman
1988) The lesser decrease in number of flowers at lower salinity (ECe= 72 dSm-1) has
been attributed to the fact that the cells of apex are un-vacuolated and the incoming salts
accumulated in the cytoplasm Munns (2002) further suggested a well-controlled phloem
transport of toxic ions from these cells prevented any change in reproductive development
Our findings showed an increase in total chlorophyll contents particularly
chlorophyll b contents were enhanced more than chlorophyll a contents under salinity
stress (Figure 115) In general the total chlorophyll contents decreased under high salinity
stress and this may be due to accumulation of toxic ions in photosynthetic tissues and
functional disorder of stomatal opening and closing (Khan et al 2009) The increase in
total chlorophylls appearing at salinity levels is considered as an important indicator of
salinity tolerance in plants (Katsuhara et al 1990 Demiroglu et al 2001) In another
study on Z mauritiana (cv Banara sikarka) the chlorophyll contents has shown decrease
with increasing salinity and sodicity but the seedlings treated with low salinity (ECe of 5
mmhoscm-1) shows slightly higher values than controls (Pandey et al 1991) Our study
also suggests that increase in total chlorophylls adapted this plant increased its tolerance
to salt stress
Slight decrease in protein has been shown under salinity treatments compared to
controls (Figure 16) Proteins play diverse roles in plants including involvement in
metabolic pathways as enzyme catalyst source of reserve energy and regulation of osmotic
potential under salt stress (Pessarakli and Huber 1991 Mansour 2000) Salts may
accumulate in cell cytoplasm and alter their viscosity depending on the response of plant
to salinity stress (Hasegawa et al 2000 Paravaiz and Satyawati 2008) The decrease in
protein contents under increasing salinity has also been documented in several plants
including Lentil lines (Ashraf and Waheed 1993) sorghum (Ali et al 2013a) and sugar
beet (Jamil et al 2014)
Soluble sugars were also decreased with increasing salinity treatments in our study
(Figure 16) Decrease in soluble sugars due to salinity has also been reported in Viciafaba
(Gadallah 1999) some rice genotypes (Alamgir and Ali 1999) Bruguiera parviflora
(Parida et al 2002) and Lentil (Sidari et al 2008) However the accumulation of soluble
sugars under salinity stress is considered as strategy to tolerate stress condition due to their
58
involvement in osmoprotection osmotic adjustment and carbon storage (Parida et al
2002 Parvaiz and Satyawati 2008)
From these experiments it is evident that C cajan is a salt sensitive plant at every
level of its life cycle starting from germination to growth phases Germination capacity
and salt tolerance ability of this species can be enhanced by water presoaking treatment
Growth reduction with increasing salinity could be attributed to physiological and
biochemical disturbances which ultimately affect vegetative and plant reproductive
growth Its roots are well associated with nitrogen fixing rhizobia and these
microorganisms were salt tolerant in in-vitro cultures Another fruit baring species of
marginal lands Z mauritiana showed growth improvement in lower salinity and its growth
was not much affected in high saline mediums owing to its controlled biochemical
responses
59
2 Chapter 2
Intercropping of Z mauritiana with C cajan
21 Introduction
Increasing soil salinity fresh water scarcity and agricultural malpractice creating shortage
of food crops for human and animal consumption (Bhandari et al 2014) and making
prices high Traditional agriculture which has been practiced since centuries using multi
species at a time in a given space could be a potential solution to narrow down the growing
edges of this supply demand scenario Plant species with innate resilience to abiotic
stresses like salinity and drought could be considered suitable to serve this purpose
especially for arid regions where marginal lands can be utilized to generate economy
Presence of such type of local systems in the region highlight their potential advantage in
crop production income generation as well as sustainability (Somashekar et al 2015)
For instance reports are available on successful intercropping of multipurpose trees
shrubs and grasses like millets pulses and some oil seed and fodder crops Green part of
these species usually mixed and used for cattle feed especially during the lean period The
utilization of the inter-row spaces of fruit trees like Ziziphus mauritiana for growing edible
legumes can generate further income by similar input (Dayal et al 2015) As an option
to this Cajanus cajan could serve as better intercropped as it provides protein rich food
nutritious fodder and wood for fuel which helped to uplift the socio-economic condition
of poor farmers Integrated agricultural practices improve the productivity of each crop by
keeping cost of production under sustainable limits (Arabhanvi and Pujar 2015)
Keeping in mind the above mentioned scenario in present study the possibility to
increase production of a non-conventional salt tolerant fruit tree (Z mauritiana) by
intercropping with a leguminous plant (C cajan) was investigated to produce edible fruits
and fodder simultaneously from salt effected waste lands
60
22 Experiment No 7
Physiological investigations on Growth of Ziziphus mauritiana and
Cajanus cajan intercropped in drum pot (Lysimeter) culture being
irrigated with water of sea salt concentration at two irrigation intervals
221 Materials and Methods
2211 Growth and Development
Experiment was designed to investigate the effect of intercropping on growth and
development of Z mauritiana (a fruit tree) and C cajan (a leguminous fodder) in drum
pot culture irrigated with water of 03 sea salt concentrations at two irrigation intervals
2212 Drum pot culture
Drum pot culture as recommended by Boyko (1966) and modified by Ahmed and
Abdullah (1982) was used for the present investigation as described in chapter 1
2213 Experimental Design
Three sets of 18 plastic drums (lysimeter) were used in this experiment One plant of Z
mauritiana were grown in each lysimeter Three replicates were kept for each treatment
comprising of 06 drums in each set which was further divided in two sub-sets First sub-
set was irrigated at every 4th and second subset at every 8th day
Set ldquoArdquo =Ziziphus mauritiana (Sole crop)
Set ldquoBrdquo = Cajanus cajan (Sole crop)
Set ldquoCrdquo = Ziziphus mauritiana + Cajanus cajan (intercropped)
The effect of salinity on sole crops of C cajan and Z mauritiana on salinity created
by various dilutions of sea salt has been investigated in chapter 1 Concentration of 03
sea salt considered equal level to its 50 reduction has been selected in present
experiment In addition irrigation was given in sub-sets in two intervals to investigate to
have some idea of its water conservation
61
2214 Irrigation Intervals
Sub-set 1 Irrigation was given every 4th day
Sub-set 2 Irrigation was given every 8th day
In set lsquoArsquo and lsquoCrsquo six month old saplings of Ziziphus mauritiana (vern grafted
ber) plants of nearly equal height and good health were transplanted in drum pots Plants
were irrigated to start with non-saline tape water for about two weeks for purpose of
establishment All the older leaves fell down and new leaves immerged during
establishment period
In set lsquoBrsquo and lsquoCrsquo Ten healthy sterilized seeds of Cajanus cajan imbibed in distill
water were sown in each drum pot and irrigated to start with tap water and after
establishment of seedlings only six seedlings of equal size with eqal distance (about one
feet) between C cajan and that of Z mauritiana were kept for further study The sowing
time of cajanus cajan seeds in both sets (B and C) was the same In drum pot lsquoCrsquo it was
sown when sapling of Z mauritiana have undergone two weeks of their establishment
period in tap water
When seedlings of C cajan reached at two leaves stage irrigation in all the sets
(ABC ) was started with gradual increase sea salt concentration till it reached to the
salinity level of treatment (03) in which they were kept up to end of experiment Each
drum was irrigated with enough water sea salt solution which retains 15 liters in soil at
field capacity Rest of water drain down with leaching of accumulated salt in root
rhizosphere
Vegetative growth of Z mauritiana plant was noted monthly in terms of height
volume of canopy while in C cajan height and number of branches was noted Shoot
length root length number of leaves fresh and dry weight of leaf stem and root leaf
weight ratio root weight ratio stem weight ratio specific shoot and root length plant
moisture leaves succulence and relative growth rate was observed and calculated at final
harvest in both the plant species growing individually (sole) or as intercropping at two
irrigation intervals
Investigations were undertaken on nitrate content relative water content and
electrolyte leakage at grand period of growth Amount of photosynthetic pigments soluble
62
carbohydrates proline content soluble phenols and Protein contents were also investigated
in fully expended leaves
Activity of catalase (CAT) ascorbate peroxidase (APX) guaiacol peroxidase
(GPX) superoxide dismutase (SOD) (Anti-oxidant enzymes) and nitrate reductase (NR)
activity was also observed in on both the Z mauritiana and C cajan leaves growing as
sole and as intercropped at two different irrigation intervals
The procedures of above mentioned analysis as follows
Leaf succulence (dry weight basis) Specific shoot length (SSL) and relative
growth rate (RGR) were measured according to the equations given in chapter 1
2215 Estimation of Nitrate content
NO3 was estimated through Cataldo et al (1975) 01g fresh leaf samples were boiled in
50 mL distilled water for 10 min 01mL of sample were added to mixed in 04 mL 50
salicylic acid (wv dissolved in 96 H2SO4 ) and allowed to stand for 20 min at room
temperature 95 mL of 2N NaOH was slowly mixed at last The samples were permissible
to cool NO3 concentration was observed at 410 nm and was calculated according to the
standard curve expressed in mg g-1 fresh weight
2216 Relative Water content (RWC)
Young and fully expended leaf was excise from each plant removing dust particles
preceding to Relative water content (RWC) Fresh weights (FW) were taken to all leaf
samples and were immersed in distilled water at 4 degC for 10 hours The soaked leaf samples
were taken out and surfeit water was removed by tissue paper Weighted again these leaf
samples for turgid weight (TW) and were oven dried at 70 degC Dry weight (DW) was
recorded after 24 hrs The RWC of leaf was calculated by the following formula
RWC () = [FW ndash DW] [TW ndash DW] x 100
2217 Electrolyte leakage percentage (EL)
EL was measured according to Sullivon and Ross (1979) Young and fully expended
leaves removing dust particles were taken 20 disc of 6mm diameter were made through
63
porer and were placed in the test tube containing 10ml de-ionized water First electrical
conductivity (EC lsquoarsquo) was record after shaken the tubes These test tubes now were placed
at 45-50oC warmed water bath for 30 min and observed second Electrical conductivity (EC
lsquobrsquo) Finally tubes were placed at 100oC water bath for ten min and obtained third and final
Electrical conductivity (EC lsquocrsquo) The electrolyte leakage was calculated in percentage by
using following formula
EL () = (EC b ndash EC a) EC b x 100
2218 Photosynthetic pigments
Photosynthetic pigments including chlorophyll a chlorophyll b total chlorophyll
chlorophyll ab ratio and carotinoids were estimated according to the procedure given in
chapter 1
2219 Total soluble sugars
Dry leaf samples (01g) were milled in 5mL of 80 ethanol and were centrifuged at 4000
g for 10 minutes and were estimated according to the procedure described in chapter 1
22110 Proline content
The proline contents were determined through Bates et al (1973) Each dried leaf powder
sample (01 g) was grinded and homogenized in 5 ml of 3 (wv) sulphosalicylic acid and
were centrifuged at 5000 g for 20 minutes 2ml supernatant was boiled by adding 2 ml
glacial acetic acid and 2 ml ninhydrin reagent (prepared by dissolving 125 g ninhydrin in
30 ml of glacial acetic acid and 20 ml 6 M phosphoric acid) in caped test tube The tubs
were kept in boiling water bath (100oC) for 1 hour After cooling 4 ml of toluene was
added to each tube and vortex Two layers were appeared the chromophore layer of
toluene was removed and their absorbance was recorded at 590nm against reference blank
of pure toluene The proline concentrations in leaves were determined from a standard
curve prepared from extra pure proline of (Sigma Aldrich) and were calculated from the
equation and were expressed in mgg-1 of leaf dry weight
Proline (microgmL-1) = -074092 + 1660767 (OD) plusmn054031
64
22111 Soluble phenols
The dried leaf powder (01g) was milled in 3ml of 80 methanol and was centrifuged at
10000g for 15 min (Abideen et al 2015) Final volume (5ml) were adjusted by adding
80 methanol Soluble phenols were determined by using Singleton and Rossi (1965) ie
5 ml of Folin-Ciocalteu reagent (19 ratio in distilled water) and 4 ml of 75 Na2CO3
were added to 01 ml supernatant The absorbance was recorded at 765 nm after incubation
of 30 minutes at room temperature The soluble phenols concentration in leaf tissues was
determined from a standard curved prepared from Gallic acid
22112 Total soluble proteins
The protein contents were measured according to Bradford Assay reagent method against
Bovine Serum Albumin as standards (Bradford 1976) Procedure was followed as given
in chapter 1
22113 Enzymes Assay
Enzyme extract prepared as given below was used for study of enzymes mentioned in text
The juvenile and expended leaf excised was frozen in liquid nitrogen and were stored at -
20 degC These leaf samples (100mg) was firmed in liquid nitrogen and were mills in 3 ml
of ice chilled potassium phosphate buffer (pH = 7 01 M) with 1mM EDTA and 1 PVP
(wv) The homogenate was filtered through a four layers of cheesecloth and were
centrifuged at 21000 g using refrigeration centrifuge (Micro 17 TR Hanil Science
Industrial Co Ltd South Korea) at 4 degC for 20 min The supernatant was separated and
stored at -20 degC and used for investigation on following enzymes
i Superoxide dismutase (SOD)
SOD (EC 11511) antioxidant enzymeactivity was measured through Beauchamp and
Fridovich (1971) derived on the inhibition of nitroblue tetrazolium (NBT) reduction by
produced O2minus using riboflavin photo-reduction 50 mM of pH 78 phosphate buffer (with
01mM EDTA 13 mM methionine) 75 microM nitroblue tetrazolium (NBT) 2 microM riboflavin
and 100 microl of enzyme extract was added to 3ml reaction mixture Riboflavin was added at
the last before the reaction was initiated under fluorescent lamps for 10 min Exposed and
un-exposed to florescence lamp without enzyme extract were used to serve as calibration
65
standards Activity was measured at 560nm Unit of SOD activity was defined as the
amount of enzyme required for 50 inhibition of NBT conversion
ii Catalase (CAT)
CAT (EC 11116) antioxidant enzyme activity was precise according to Aebi (1984)
derived on H2O2 reduction at 240nm for 30 s (ε = 36 M-1 cm-1)100mM potassium
phosphate buffer (pH=7) with 30mM H2O2 and 50 microl of diluted enzyme extract (adding in
last) was added to 3ml reaction mixture The decrease in absorbance due to H2O2 reduction
was measured at 240 nm and expressed in micromol of H2O2 reduced m-1g-1 fresh weight at 25
degC
iii Ascorbate peroxidase (APX)
Nakano and Asada (1981) method was used for APX (EC 111111) antioxidant
enzymeactivity by measuring the decrease in ascorbate oxidation by H2O2 The reaction
mixture (3ml) contained potassium phosphate buffer (50mM pH=7) 01mM H2O2 050
mM Ascorbate and 100 microl of enzyme extract and were observed 290 nm for 1 min 25 degC
(extinction coefficient 28 mM-1cm-1)
iv Guaiacol peroxidase (GPX)
GPX (EC 11117) antioxidant enzymeactivity was estimated through Anderson et al
(1995) 3ml of 50 mM potassium phosphate buffer (pH 7) guaiacol 75 mM H2O2 10 mM
reaction mixture with 20 microl of enzyme extract adding at last Increase in absorbance was
observed due to the formation of tetra-guaiacol at 470 nm for 2 min (extinction coefficient
266 mM-1cm-1)
v Nitrate reductase (NR)
The NR activity in leaves was observed through Long and Oaks 1990 Fresh leaf samples
(01g) were placed in 5ml of 100mM potassium phosphate pH 75 (added to 10
Isopropanol and 25mM KNO3) Tubes were vacuumed for 10 min to remove air from the
mixture and were placed in water bath shaker at 33oC for 60 min in dark The tubes were
placed in hot water (100oC) for 5 min 15 mL from the reaction mixture were added in 05
mL 20 sulphanilamide (wv dissolve in 5N HCl) and 025 mL 008 N-1-Napthylene-
66
diamine dihydrochloride Final volume up to 60 ml was made by adding distilled water
Color developed over the next 20 min Absorbance was measured at 540 nm using
spectrophotometer
67
222 Observations and Results
Sole and intercropped Ziziphus mauritiana
2221 Vegetative growth
Growth of Z mauritiana in terms of shoot root and plant length and number of leaves in
two different cropping system (sole and intercrop with C cajan) in two different irrigation
intervals has been presented in Figure 21 Appendix-XII A significant increase (plt0001)
in plant length was observed in 8th day irrigation in both the cropping systems in Z
mauritiana At 4th day of irrigation interval a non-significant increase in length was
observed in intercropped plants compared to sole crop Similarly at 8th day of irrigation
plants attain almost same heights in both the cropping systems
A significant increase (plt001) in root length was observed in sole Z mauritiana
at 8th day of irrigation compared to other treatments Smallest root length revealed in plants
that were irrigated at 4th day under sole crop system
The shoot length was significantly increase (plt0001) in plants which were
irrigated at 8th day under intercropped system However shoot length remains unaffected
when comparing the different cropping system at both the irrigation intervals
A significant increase (plt0001) in number of leaves was observed in intercropped
Z mauritiana plants compared to plants cultivated according to sole system However
more increase was observed in 4th day irrigated intercropped plant as compared to 8th day
The difference in number of leaves in sole crop at both irrigating intervals remains same
i Fresh weight
Figure 22 Appendix-XII showed fresh and dry weight of stem root and leaf of Z
mauritiana plant in two different cropping system (sole and intercrop with C cajan) in
two different irrigation intervals A significant increase (plt0001) in fresh weights of leaf
stem and root was observed in intercropping (with C cajan) 4th and 8th day of irrigation
interval compared to individual cropping of Z mauritiana In 4th day of irrigation the
increment was more pronounced in fresh weights of root (7848) leaves (4130) and
stem (4047) respectively with comparison to the crop growing alone Similarly
intercropping in 8th day of irrigation showed better growth of leaves (28) stem (12)
68
and root (31) against sole crop Whereas decrease in leaves 33 (plt005) stem 70
(plt0001) and root 60 (plt0001) fresh weights were observed in 8th day of irrigation
compared to 4th day intercropping However the difference was non-significant between
two sole crops irrigated at 4th and 8th day interval
ii Dry weight
Intercropping with comparison to the sole crop showed significant (plt0001) increase in
dry weights of leaves root and stem of Z mauritiana at 4th and 8th day of irrigation (Figure
22 Appendix-XII) At 4th day of irrigation intercropping showed an increment in dry
weights of Leaves (4366) stem (4109) and root (754) compared to the sole crop
Similar increase was observed in leaves (plt0001) stem (plt0001) and root (plt0001)
weights after 8th day of irrigation However intercropping at 8th day irrigation showed an
increment in root (19) stem (11) whereas a slight decrease (1) in leaves dry weight
When comparing irrigation time an increase in stem dry weight at 4th day whereas decline
in leaves dry weight was observed Root dry weights were more or less similar at both
irrigation intervals
iii Leaf weight ratio (LWR) root weight ratio (RWR) stem weight
ratio (SWR)
Leaf weight ratio (LWR) root weight ratio (RWR) stem weight ratio (SWR) of Z
mauritiana plant grown in two different cropping system (sole and intercrop with C cajan)
in two different irrigation intervals has been presented in Figure 23 Appendix-XII An
increased in LWR and SWR was recorded at 8th day of irrigation compared to 4th day of
irrigation in both cropping systems whereas decrease in RWR was observed LWR and
SWR remained un-change in sole and inter crop system However RWR increased in
intercrop system compared to sole crop system
iv Specific shoot length (SSL) specific root length (SRL)
Specific shoot length (SSL) specific root length (SRL) of Z mauritiana plant grown in
two different cropping system (sole and intercrop with C cajan) in two different irrigation
intervals has been presented in Figure 23 Appendix-XII SSL was observed higher in 8th
day of irrigation compared to 4th day in both the cropping systems However the increase
69
in SSL was lesser in sole crop compared to intercropping Similarly SRL was recorded
lesser in 4th day of irrigation compared to 8th day of irrigation in both cropping systems
Intercropped plants showed decline in SRL compared to sole crop plants Greatest SRL
revealed in plants that were irrigated after 8th day and planted according to sole crop
system
v Plant moisture
The moisture content of Z mauritiana plant grown in two different cropping system (sole
and intercrop with C cajan) in two different irrigation intervals has been presented in
Figure 23 Appendix-XII The moisture content of plants was significantly decreased
(plt005) in sole crop while increased (plt005) in intercropping at 8th day of irrigation
compared to 4th day At 4th day moisture remained same in both cropping system
However significant increase in moisture contents was observed in inter-crop system
compared to sole crop system after 8th day of irrigation
vi Plant Succulence
Succulence of Z mauritiana plant grown in two different cropping system (sole and
intercrop with C cajan) in two different irrigation intervals has been presented in Figure
23 Appendix-XII Plant succulence in 8th day was significantly reduced in sole crop
whereas increased in intercropping system In 4th day irrigated plants decrease in
succulence was noticed compared to plants that were irrigated at 8th day under sole crop
system However significant increase (plt0001) was observed in intercropped plants
irrigated at 4th day compared to 8th day
vii Relative growth rate (RGR)
Relative growth rate (RGR) of Z mauritiana plant grown in two different cropping system
(sole and intercrop with C cajan) in two different irrigation intervals has been presented
in Figure 23 Appendix-XII Relative growth rate remains unchanged at both irrigation
times under sole crop system However decline in 8th day was observed compared to 4th
day of irrigation under intercrop system Greatest RGR was recorded in plants that were
irrigated at 4th day under intercrop system
70
2222 Photosynthetic pigments
Photosynthetic pigments including Chlorophyll a chlorophyll b total chlorophyll
Chlorophyll ab ratio and carotinoids of Z mauritiana plant grown in two different
cropping system (sole and intercrop with C cajan) in two different irrigation intervals has
been presented in Figure 24 Appendix-XII
i Chlorophyll contents
A significant increase (plt0001) in chlorophyll a b and total chlorophyll was observed in
plants growing as sole crop compared to intercropped system at both the irrigation
intervals Higher chlorophyll contents were also recorded in plants that were irrigated at
8th day compared to 4th day of irrigation The chlorophyll ab ratio increased in 4th day
while decline in 8th day in intercropped system compared to sole crop However overall
results showed non-significant changes
ii Carotinoids
A significant increase (p lt 0001) in leaf carotinoids was observed in sole crop compare
to intercropped system at both irrigation times in Z mauritiana Least carotene content
was estimated in plants that were irrigated at 4th day under intercrop system
2223 Electrolyte leakage percentage (EL)
Electrolyte leakage percentage (EL) of Z mauritiana plant grown in two different
cropping system (sole and intercrop with C cajan) in two different irrigation intervals has
been presented in Figure 25 Appendix-XII A non-significant result was observed in
electrolyte leakage in plant growing at varying cropping system and irrigating intervals
2224 Phenols
Total phenolic contents in leaves of Z mauritiana plant grown in two different cropping
system (sole and intercrop with C cajan) in two different irrigation intervals has been
presented in Figure II25 Appendix-XII A significant increase (plt001) in total phenolic
contents was observed in intercropped growing at both irrigation interval compared to sole
crop However the increase was more pronounced at 8th day of irrigation Maximum
phenolic contents were measured in plants irrigated at 8th day under intercropped plants
71
2225 Proline
Total proline contents in leaves of Z mauritiana plant grown in two different cropping
system (sole and intercrop with C cajan) in two different irrigation intervals has been
presented in Figure 25 Appendix-XII A significant decreased (plt0001) was observed
in Z mauritiana cultivated according to intercropped system in both irrigation intervals
Maximum decrease was observed in intercropped plants irrigated at 8th day whereas
highest phenolic contents were observed in plants irrigated at 4th day under sole crop
system
2226 Protein and sugars
Protein and sugar contents in leaves of Z mauritiana plant grown in two different cropping
system (sole and intercrop with C cajan) in two different irrigation intervals has been
presented in Figure 26 Appendix-XII A nonsignificant difference in total protein and
sugar contents in Z mauritiana plants was observed in two different (4th and 8th day)
irrigation intervals However the interaction with time and irrigation interval also showed
nonsignificant result
2227 Enzyme essays
Antioxidant enzymes like Catalase (CAT) Ascorbate peroxidase (APX) Guaiacol
peroxidase (GPX) Superoxide dismutase (SOD) and Nitrate reductase activity in leaf of
Z mauritiana plant grown in two different cropping system (sole and intercrop with C
cajan) in two different irrigation intervals has been presented in Figure 27 and 28
Appendix-XII
i Catalase (CAT)
A significant decreased (plt0001) in catalase activities was observed in Z mauritiana
leaves in intercropped system in both time interval with compare to sole crop at 4th day
irrigated plant However maximum decline was in sole plants irrigated at 8th day interval
However their interaction with time was nonsignificant
72
ii Ascorbate peroxidase (APX)
A significant increase (plt0001) in APX activity was observed in 8th day irrigation in both
sole and intercropped plants with compare to sole and intercropped at 4th day irrigation
interval More increase (plt0001) was observed in intercropped Z mauritiana at 8th day
Whereas nonsignificant decrease was observed in two different cropping system in 4th day
irrigation interval However interaction between time and the treatments shows significant
values
iii Guaiacol peroxidase (GPX)
A significant (plt0001) increase in GPX was observed in 8th day intercropped Z
mauritiana plant with compare to irrigation intervals as well as cropping system However
at 4th day both cropping system showed nonsignificant difference Whereas more decline
was observed in 8th day sole crop The ANOVA reflects significant (plt005) interaction
between time and the cropped system
iv Superoxide dismutase (SOD)
A nonsignificant increase in SOD was observed in intercropped at 8th day irrigation
interval Whereas there was nonsignificant differences in 4th day intercropped and at both
time intervals of sole crop However interaction between time interval and the two
cropping system shows nonsignificant result
v Nitrate and Nitrate reductase
A significant increase (plt0001) in nitrate content and activity of nitrate reductase was
observed in intercropped plants of both irrigation intervals Increase in activity was
observed (plt0001) in intercropped Z mauritiana at 4th day
73
Sole and intercropped Cajanus cajan
2228 Vegetative growth
Growth of C cajan in terms of shoot root and plant length and number of leaves was
observed in two different cropping system (sole and intercrop with Z mauritiana) in two
different irrigation intervals has been presented in Figure 21 Appendix-XIII XIV A
significant increase (plt001) in plant length was observed in intercropped C cajan
compared to sole crop at both irrigation interval Whereas sole crop at 8th day interval
showed better results as compare to sole of 4th day Similarly root length remains
unaffected and showed non-significant change in both cropping systems and even at two
different irrigation intervals While shoot length was significantly (Plt001) decreased in
sole crop compared to intercropped at 4th day irrigation Whereas non-significant
difference be observed in rest of cropping systems growing at different irrigation interval
A significant increase (plt001) in leaves number was observed in intercropped
plants compared to sole crop at 4th and 8th day irrigation interval However most
significant decrease (plt0001) was observed in sole crop at 4th day
i Fresh weight
Figure 22 Appendix-XIV showed fresh and dry weight of stem root and leaf of C cajan
plant in two different cropping system (sole and intercrop with C cajan) in two different
irrigation intervals A significant increase (plt001) in fresh weight of leaf was observed in
intercropping (with Z mauritiana) at 4th and 8th day of irrigation interval compared to
individual cropping of C cajan The increase in intercropped system compared to sole
crop was more pronounced at 4th day (42) of irrigation than the 8th day (1701) Plants
showed higher leaves fresh weights in 8th day of irrigation compared to 4th day Similarly
the interaction between cropping system and the irrigation interval was significant
(Plt005)
An insignificant difference was observed in stem at 4th (15) and 8th (12) days
fresh weights in both intercropping system at two different irrigation intervals The
interaction between cropping system and the irrigation interval also showed non-
significant result
74
A non-significant difference in root fresh weight was observed in two different
cropping systems (sole and intercropped) in 4th and 8th day of irrigation intervals However
fresh weight of crop at 8th day irrigation interval was significantly increase (plt0001) over
4th day irrigation interval Similar pattern was observed in 4th day irrigated sole and
intercropped C cajan
ii Dry weight
A significant increase in leaves (42) stem (24) and root (18) dry weights were
observed in 4th day irrigation under intercropped system compared to sole However in 8th
day of irrigation this increase of dry weights was not much prominent Under sole crop
system dry weights of leaves stem and root was increased markedly in 8th day compared
to 4th day However in intercrop system the difference in dry weights was insignificant
between 8th and 4th day of irrigation
iii Leaf weight ratio (LWR) root weight ratio (RWR) stem weight
ratio (SWR)
Leaf weight ratio (LWR) root weight ratio (RWR) stem weight ratio (SWR) of C cajan
grown in two different cropping system (sole and intercrop with Z mauritiana) in two
different irrigation intervals has been presented in Figure 23 Appendix-XIV A
significant increase (plt0001) in LWR was observed at 8th day of irrigation compared to
4th day intercropped Similar pattern was noticed in RWR however SWR showed
insignificant difference between 4th and 8th day of irrigation A slight increase in LWR was
noticed in intercropped plants compared to sole Whereas RWR declined in intercrop
compared to sole and SWR remains un-changed
iv Specific shoot (SSL) root length (SRL)
Specific shoot length (SSL) specific root length (SRL) of C cajan grown in two different
cropping system (sole and intercrop with Z mauritiana) in two different irrigation
intervals has been presented in Figure 23 Appendix-XIV SSL and SRL were observed
to increase in sole crop compared to intercrop at 4th day of irrigation However increase
SSL and SRL was recorded in intercropped compared to sole at 8th day of irrigation A
general decline in SSL and SRL was noticed in 8th day of irrigation compared to 4th day
75
v Plant moisture
The moisture content of C cajan plant grown in two different cropping system (sole and
intercrop with Z mauritiana) in two different irrigation intervals has been presented in
Figure 23 Appendix-XIV The moisture content of plants was decreased significantly
(plt005) at 8th day irrigation interval compared to 4th day in sole crop Whereas non-
significant increase was observe in intercrop plants at 8th day of water irrigation
vi Plant succulence
Succulence of C cajan plant grown in two different cropping system (sole and intercrop
with Z mauritiana) in two different irrigation intervals has been presented in Figure 23
Appendix-XIV A significant increase (plt001) was observed in intercropped plants of C
cajan compared to sole crop at both irrigation interval However succulence increased in
sole crop and decreased in intercrop plants at 8th day of irrigation compared to 4th day
vii Relative growth rate (RGR)
Relative growth rate (RGR) of C cajan plant grown in two different cropping system (sole
and intercrop with Z mauritiana) in two different irrigation intervals has been presented
in Figure 23 Appendix-XIV A significant increase in RGR was observed in 8th day
compared to 4th day in both the cropping systems Highest increase was observed in
intercropped at 8th day irrigation At 4th day irrigation intervals intercropped plants
showed better RGR compared to Sole crop
2229 Photosynthetic pigments
Photosynthetic pigments including Chlorophyll a chlorophyll b total chlorophyll
Chlorophyll ab ratio and carotinoids of C cajan plant grown in two different cropping
system (sole and intercrop with Z mauritiana) in two different irrigation intervals has been
presented in Figure 24 Appendix-XIV
i Chlorophyll contents
A significant increase (plt005) in Chlorophyll a b and total chlorophyll was observed in
intercrop plants at 8th day irrigation interval Whereas at 4th day irrigation interval Sole
76
plants showed better results as compare to intercrop plants Plants at 8th day significantly
increase chlorophyll a b and total chlorophyll compared to 4th day of irrigation
Interactions between cropping systems and irrigation intervals were found significant
(chlorophyll a (plt001) chlorophyll b (plt001) and total chlorophyll (plt0001)
respectively) However the ratio of chlorophyll ab showed non-significant values in
cropping irrigation interval and their interaction
ii Carotenoids
A significant increase (plt001) in carotinoids was observed in intercropped C cajan at 8th
day of irrigation Whereas non-significant increase was observed in sole crop at 4th day
irrigation interval with compare to intercrop However the irrigation intervals showed
significant (plt0001) difference Whereas interaction of cropping system with irrigation
time also showed significant correlation (plt0001)
22210 Electrolyte leakage percentage (EL)
Electrolyte leakage percentage (EL) of C cajan plant grown in two different cropping
system (sole and intercrop with Z mauritiana) in two different irrigation intervals has been
presented in Figure 25 Appendix-XIV A non-significant increase in EL percentage was
observed in sole crop compared to intercrop plants growing at 4th and 8th day of irrigation
No significant change was noticed between the irrigation times to C cajan The interaction
between cropping system (sole and intercropped) and irrigation interval (4th and 8th day)
also showed non-significant
22211 Phenols
Total phenolic contents in leaves of C cajan plant grown in two different cropping system
(sole and intercrop with Z mauritiana) in two different irrigation intervals has been
presented in Figure 25 Appendix-XIV A nonsignificant result was observed in total
phenolic contents of C cajan growing as sole and intercropped system at two different
irrigation intervals However the interaction between irrigation intervals with crop system
showed significant (p lt 005) results
77
22212 Proline
Total proline contents in leaves of C cajan plant grown in two different cropping system
(sole and intercrop with Z mauritiana) in two different irrigation intervals has been
presented in Figure 25 Appendix-XIV Proline contents in leaves of C cajan showed
nonsignificant increase at 4th day of irrigation interval in both sole and intercropped
system Whereas the interaction between irrigation intervals showed significant (Plt001)
results
22213 Protein and Sugars
Protein and sugar contents in leaves of C cajan plant grown in two different cropping
system (sole and intercrop with Z mauritiana) in two different irrigation intervals has been
presented in Figure 26 Appendix-XIV A less significant difference (plt005) was
observed in two different (4th and 8th day) irrigation intervals However there was
nonsignificant difference in two cropped system More decrease was observed at 4th day
intercropped plants Whereas nonsignificant increase in 8th day intercropped and 4th day
sole plants were observed However interaction between crop and time of irrigation
showed significant results (plt0001)
22214 Enzyme assay
Antioxidant enzymes like Catalase (CAT) Ascorbate peroxidase (APX) Guaiacol
peroxidase (GPX) Superoxide dismutase (SOD) and Nitrate reductase activity in leaf of
C Cajan plant grown in two different cropping system (sole and intercrop with Z
mauritiana) in two different irrigation intervals has been presented in Figure II27
Appendix-XIV
i Catalase (CAT)
A significant increase (plt001) in catalase activity was observed in intercropped C cajan
at 8th day of irrigation with compare to other irrigation time and cropped system Whereas
increase was observed in sole crop at 4th day irrigation interval with compare to 8th day
However the irrigation intervals and the interaction between cropping system with
irrigation interval also showed nonsignificant correlation
78
ii Ascorbate peroxidase (APX)
A non-significant increase in APX was observed in intercropped plant in 4th and 8th day
irrigation interval with compare to sole crops Sole crop at 8th day showed maximum
decline However the difference between cropping system and their interaction with
irrigation interval also showed nonsignificant results
iii Guaiacol peroxidase (GPX)
A significant increase (plt005) in GPX activity was observed in 8th day sole crop
However there was nonsignificant difference among intercropped at two time interval and
sole crop at 4th day irrigation Whereas interaction with time to irrigation interval also
showed less significant results
iv Superoxide dismutase (SOD)
A significant decrease (plt0001) in SOD activity was observed in intercropped at 8th day
irrigation interval with compare to 4th day Maximum decrease was observed in 8th day
intercropped Whereas sole crop at 8th day also showed better result to 4th day sole crop
However ANOVA showed significant correlation among crop system at two time interval
and 4th day irrigation
v Nitrate and Nitrate reductase
Nitrate content and activity of nitrate reductase was nonsignificant in both cropping
system using both irrigation intervals However nonsignificant increase was observed in
nitrate content and activity of nitrate reductase in intercropped Z mauritiana at 8th day
79
Sole IntercropSole Intercrop
No o
f le
aves
0
20
40
60
Len
gth
(cm
)
0
40
80
120
160
200
2404
th day
Cajanus cajan
a
RootShoot
ab
a
a
b
a
a
8th
day
Figure 21 Vegetative parameters of Z mauritiana and C cajan at grand period of growth under sole and
intercropping system at 4th and 8th day irrigation intervals (Bars represent means plusmn standard error
of each treatment and significance among the treatments was recorded at p lt 005)
Sole IntercropSole Intercrop
No of
leav
es
0
200
400
600
Len
gth
(cm
)
0
40
80
120
160
200
240
Ziziphus mauritiana
RootShoot
4th
day 8th
days
b b
a a
a
b
cc
80
Sole Intercrop
Dry
wei
ght
(g)
50
100
150
200
250
300
Fre
sh w
eight
(g)
100
200
300
400
500
Sole Intercrop
4th
day 8th
day
a
b
c
a
b b aa
b
b
c c
a
bc
a
c
ba
b
c
a
b
c
Leaf Stem Root
Ziziphus mauritiana
Sole Intercrop
Dry
wei
ght
(g)
2
4
6
8
10
12
Fre
ah w
eight
(g)
5
10
15
20
25
30
35
40
Sole Intercrop
4th
day 8th
day
aa
b
a
a
b
a
b
c
a
b
c
a
c
b
a a
b
a
b
c
a
b
c
Leaf Stem Root
Cajanus cajan
Figure 22 Fresh and dry weight of Z mauritiana and C cajan plants under sole and intercropping system
at 4th and 8th day irrigation intervals (Bars represent means plusmn standard error of each treatment
and significance among the treatments was recorded at p lt 005)
81
Figure 23 Leaf weight ratio (LWR) root weight ratio(RWR) shoot weight ratio(SWR)specific shoot
length (SSL) specific root length (SRL) plant moisture Succulence and relative growth rate (RGR) of
Zmauritiana and C cajan grow plants under sole and intercropping system at 4th and 8th
day irrigation
intervals (Bars represent means plusmn standard error of each treatment and significance among the treatments
was recorded at p lt 005)
Sole Intercrop
Mo
istu
re (
)
0
20
40
60
80
SS
L (
cm g
-1)
01
02
03
04
05
06
RW
R (
g g
-1 D
W)
005
010
015
020
LW
R (
g g
-1 D
W)
01
02
03
04
05
06
07
Sole Intercrop
Su
ccu
lan
ce
(g H
2O
g-1
DW
)00
05
10
15
20
25
RG
R
(g g
-1 d
ay-1
)
001
002
003
004
005
SR
L (
cm g
-1)
05
10
15
20
25
SW
R (
g g
-1 D
W)
02
04
06
08
10
Ziziphus mauritiana
a a
bb
b
a
bb
a
b
aa
a aa
b
a
bb
c
b
a
bb
b
aa a
ba
bc
4th day
8th day
82
(Figure 23 continuedhellip)
Sole Intercrop
Mo
istu
re (
)
0
20
40
60
80
SS
L (
cm g
-1)
2
4
6
8
10
12
RW
R (
g g
-1 D
W)
002
004
006
008
010
012
014
LW
R (
g g
-1 D
W)
01
02
03
04
05
06
07
08
Sole Intercrop
Su
ccu
lan
ce
(g H
2O
g-1
DW
)
00
05
10
15
20
25
RG
R
(g g
-1 d
ay-1
)
001
002
003
004
005
SR
L (
cm g
-1)
5
10
15
20
25
SW
R (
g g
-1 D
W)
02
04
06
08
10
Cajanus cajan
a aab
a aaa
a
bba
a
b b
c
a aab
a
bbb
abbb
aa
bc
8th day
4th day
83
Sole Intercrop
Car
oti
noid
s (m
g g
-1 F
W)
00
01
02
03
04
05
Ch
loro
phyll
(m
g g
-1 F
W)
00
03
06
09
12
15
Sole Intercrop
4th
day 8th
day
Ch
loro
phyll
ab
rat
io
00
05
10
15
20
25Chl ab
Ziziphus mauritiana
a a
bb
a
b
a
b
a ab
b
Chl aChl b
Figure 24 Leaf pigments of Zmauritiana and C cajan grow plants under sole and intercropping system at
4th and 8th
day irrigation intervals (Bars represent means plusmn standard error of each treatment and
significance among the treatments was recorded at p lt 005)
Sole Intercrop
Car
oti
noid
s (m
g g
-1 F
W)
00
01
02
03
04
05
Ch
loro
phyll
(m
g g
-1 F
W)
00
03
06
09
12
15
18
Sole Intercrop
4th
day 8th
day
ab r
atio
00
05
10
15ab
ab
Cajanus cajan
bb b
a
a
b
cc
bb b
a
84
Ele
ctro
lyte
lea
kag
e(
)
0
5
10
15
4th
day 8th
dayP
hen
ols
(m
g g
-1)
0
5
10
15
20
25
30
Sole Intercrop
Pro
line
( g g
-1)
0
10
20
30
40
Sole Intercrop
Ziziphus mauritiana
a a a
a
b b ba
a
b
c
d
Figure 25 Electrolyte leakage phenols and prolein of Z mauritiana and C cajan at grand period of growth
plants under sole and intercropping system at 4th and 8
th day irrigation intervals (Bars represent
means plusmn standard error of each treatment and significance among the treatments was recorded at
p lt 005)
85
(Figure 25 continuedhellip)
E
lect
roly
te l
eakag
e(
)
0
20
40
60
80
4th
day 8th
day
Phen
ols
(m
g g
-1)
0
2
4
6
8
10
12
Sole Intercrop
Pro
line
( g g
-1)
000
003
006
009
012
015
018
Sole Intercrop
Cajanus cajan
a aa
a
a a aa
aa a
a
86
Sole Intercrop
Sugar
s (m
g g
-1)
0
20
40
60
Sole Intercrop
Pro
tein
(m
g g
-1)
00
02
04
06
4th
day 8th
day
Ziziphus mauritiana
a aa a
a
a a a
Sole Intercrop
Sugar
s (m
g g
-1)
0
10
20
30
Sole Intercrop
Pro
tein
(m
g g
-1)
00
02
04
06
08
10
4th
day 8th
dayCajanus cajan
ab
a
c
a
b
cc
Figure 26 Total protein and sugars in leaves of Z mauritiana and C cajan plants under sole and
intercropping system at 4th and 8th
day irrigation intervals (Bars represent means plusmn standard
error of each treatment and significance among the treatments was recorded at p lt 005)
87
Sole Intercrop
SO
D (
Unit
s m
g-1
)
0
2
4
6
8
10
12
14
Sole Intercrop
Cat
alas
e (U
nit
s m
g-1
)
0
5
10
15
20
25
AP
X (
Unit
s m
g-1
)
0
20
40
60
80
GP
X (
Unit
s m
g-1
)
00
01
02
03
04
05
4th
day 8th
day
Ziziphus mauritiana
a
bc
c
a
b
cc
a
c
b
b
b bb
a
Figure 27 Enzymes activities in leaves of Z mauritiana and C cajan plants under sole and intercropping
system at 4th and 8th
day irrigation intervals (Bars represent means plusmn standard error of each
treatment and significance among the treatments was recorded at p lt 005)
88
(Figure 27 continuedhellip)
Sole Intercrop
SO
D (
Unit
s m
g-1
)
0
1
2
3
4
5
Sole Intercrop
Cat
alas
e (U
nit
s m
g-1
)
0
2
4
6
8
4th
day 8th
dayG
PX
(U
nit
s m
g-1
)
00
05
10
15
20
25
Cajanus cajan
aA
PX
(U
nit
s m
g-1
)
0
20
40
60
80
100
bb
b
aaa
b
a
bbb
a
c
a
b
89
Sole Intercrop
NO
3 (
mM
ol
g-1
)
00
02
04
06
08
10
12
14
8th
day
Sole Intercrop
Nit
rate
Red
uct
ase
(mM
ol
g-1
)
0
1
2
3
4
4th
day
Nitrate reductaseNO
3
Ziziphus mauritiana
a
b
c
cb
b
b
a
Sole Intercrop
NO
3 (
mM
ol
g-1
)
00
02
04
06
08
10
12
8th
day
Sole Intercrop
Nit
rate
Red
uct
ase
(mM
ol
g-1
)
0
2
4
6
8
10
12
4th
dayCajanas cajan
a
bb
b
aa
aa
Nitrate reductase NO3
Figure 28 Nitrate reductase activity and nitrate concentration in leaves of Z mauritiana and C cajan plants
under sole and intercropping system at 4th and 8th
dayirrigation intervals (Values represent means
plusmn standard error of each treatment and significance among the treatments was recorded at p lt
005)
90
23 Experiment No 8
Investigations of intercropping Ziziphus mauritiana with Cajanus cajan
on marginal land under field conditions
231 Materials and Methods
2311 Selection of plants
Ziziphus mautitiana and Cajanus cajan were selected for this study as described in chapter
1
2312 Experimental field
Field of Fiesta Water Park was selected to investigate intercropping of Z mauritiana with
Ccajan It is situated about 50 km from University of Karachi at super highway toward
HyderabadThe area of study has subtropical desert climate with average annual rain fall
is ~20 cmmost of which is received during the monsoon or summer seasonSince summer
temperature (April to October) are approx 30-35 degC and the winter months (November to
March) are ~20 degC Wind velocity is generally high all the year Topography of the area
was uneven with clay- loam soil having gravels Xerophytic plants are pre-dominantly
present in the area including Prosopis spp Acacia spp Euphorbia spp Caparus
deciduas etc
2313 Soil analysis
Before conducting experiment soil of Fiesta Water Park field was randomly sampled at
three locationsatone feet of depthusing soil augerThese soil samples were analyzed in
Biosaline Research Laboratory Department of Botany University of Karachi to
determine its physical and chemical properties
i Bulk density
Bulk density was determinedin accordance with Blake and Hartge (1986) by using the
following formula
Bulk density = Oven dried soil (g) volume of soil (cm3)
91
ii Soil porosity
Soil porosity was calculated in accordance with Brady and Weil (1996) by using the
following formula
Soil porosity = 1- (bulk density Particle density) times 100
Where particle density = 265 gcm3
iii Soil texture and particle size
Soil particle size was determined by Bouyoucos hydrometric method in accordance with
Gee and Or (1986)On the basis of clay silt and sand percentages soil texture was
determined by using soil texture triangle presented in Figure 31
iv Water holding capacity
Water holding capacity in percentages was calculatedaccording to George et al (2013)
v pH and Electrical conductivity of soil (ECe)
Soil saturated paste was made with de-ionized water and leave for 24 hours Soil solution
was extracted through Buckner funnel and suction pump (Rocker 300) pH of soil
solution was taken on Adwa AD1000 pHMV meter and ECe was taken on electrical
conductivity meter (4510 Jenway)
2314 Experimental design
Six months old grafted Ziziphus mauritiana saplings were carefully transported in field of
Fiesta Water Park
Three equal size plots of 100times10 sq ft were prepared for this experiment
Plot ldquoArdquo = Ziziphus mauritiana (Sole crop)
Plot ldquoBrdquo = Cajanus cajan (Sole crop)
Plot ldquoCrdquo = Ziziphus mauritiana + Cajanus cajan (intercropped)
In plot lsquoArsquo and lsquoCrsquo pits of two cubic feet depth were prepared in two parallel rows
at a distance of 10 feet (Yaragattikar amp Itnal 2003)so that the distance of pits within the
row and the distance of pits between the rows were same Each row bears nine pits
Eighteen healthy saplings of nearly equal height and vigor of Z mauritiana were
92
transplanted in the pits and were fertilized with cow-dong manure Plants were irrigated
with underground (pumped) water initially on alternate day for two weeks older leaves
fall down completely and new leaves appeared in this establishment period Later the
irrigation interval was kept fortnightly Electrical conductivity of irrigated water (ECiw)
was 24 plusmn 05 dSm-1
After establishment of Z mauritiana water soaked seeds of intercropping plant (C
cajan) were sown in plot lsquoCrsquo Three vertical lines (strips design) of equal distance were
made between the rows of Z mauritiana The distance between the line was one feet
Eleven C cajan were maintained in each line at a distance of one feet which constitute a
total of 33 C cajan in 3 lines There were 264 plants of C cajan arranged in strip pattern
as intercrop for eighteen Z mauritiana A sole crop of C cajan in plot lsquoBrsquo was arranged
with the same manner to serve as control Similarly plot lsquoArsquo was served as control of Z
mauritianaThe experiment was observed up to reproductive yield of each plant
Field diagram Theoritical model of intercropping system used in this study showing sole crop in Plot lsquoArsquo
(Z Mauritiana) and Plot lsquoBrsquo (C cajan) while Plot lsquoCrsquo represents intercropping of both
species at marginal land
Six Z mauritiana plants were randomly selected from their two rows of block lsquoCrsquo
which were facing two rows of C cajan on either sides Similarly ten plants of C cajan
facing Z mauritiana were randomly selected for further study At the same manner six Z
mauritiana from block lsquoArsquo and ten C cajan from block lsquoBrsquo grown as sole crop were
selected as control for further study
93
2315 Vegetative and reproductive growth
Vegetative growth of Z mauritiana plant was noted in terms of height volume of canopy
while height and number of branches in Ccajan bimonthly after establishment Fresh and
dry weightsof leaves stem and root were observed at final harvest in both plant species
growing as sole or intercropping
Reproductive growth of Z mauritiana such as number length and diameter fruit
weight per ten plant and average fruit yield was measured at termination of the experiment
Whereas reproductive growth in C cajan was monitored in terms of number of pods
number of seeds weight of pods and weight of seed
2316 Analyses on some biochemical parameters
Following biochemical analysis was conducted in Fully expended leavesof Z mauritiana
and C cajan growing as sole and as intercropped at grand period of growth Additionally
fruits of Z mauritiana were also analyzed for their protein soluble and insoluble sugars
and total phenolic contents
i Photosynthetic pigments
Photosynthetic pigments including chlorophyll a chlorophyll b and total chlorophyll were
estimated in leaves of Z mauritiana and C cajan according to procedure described in
chapter 1
ii Protein in leaves
Protein contents were estimated in leaves of Z mauritiana and C cajan according to
procedure described in chapter 1
iii Total soluble sugars in leaves
Total soluble sugars were estimated in leaves of Z mauritiana and C cajanaccording to
procedure described in chapter 1
94
iv Phenolic contents in leaves
Phenolic content were estimated in leaves of Z mauritiana and C cajan according to
procedure described in chapter 1
2317 Fruit analysis
i Protein in fruit
Protein content in fruit of Z mauritiana was estimated according to procedure described
in chapter 1
ii Total soluble sugars in fruits
Total soluble sugars in ripe fruits of Z mauritiana were estimated according to procedure
described in chapter 1
iii Phenolic contents in fruits
Phenolic contents in fruits of Z mauritiana were estimated according to procedure
described in chapter 1
2318 Nitrogen estimation
Nitrogen was also estimated in root zone soil as well as in fully expended leaves of Z
mauritiana and C cajan plants
Total nitrogen in leaves and soil was estimated through AOAC method 95504
(2005) One g of dried powdered sample in round bottle flask was digested in presence of
20 mL H2SO4 15 mL K2SO4 and 07g CuSO4 at 400oC heating mental After digestion 80
ml distilled water was added in digest Then distillation was done at 100oC by adding 100
mL of 45 NaOH (drop wise) in digested solution Steam was collected in 35 mL of 01M
HCl in a flask Three samples of 10 mL each steam collected solution were taken and 2-3
drops of methyl orange was added as indicator Titration was made with 01M NaOH
Changeappearance of color indicates the completion of reactionPercent nitrogen was
calculated through following equation
N = (mL of acid times molarity) ndash (mL of base times molarity) times 14007
95
2319 Land equivalent ratio and Land equivalent coefficient
The LER defined the total land area needed for sole crop system to give yield obtained
mixed crop It is mainly used to evaluate the performance of intercropping (Willey 1979)
Land equivalent ratio (LER) of two crops was estimated according to (Willey 1979) by
using formula
Whereas partial LER of Z mauritiana calculated according to
Similarly Partial LER of Ccajan were calculated as
Land equivalent coefficient (LEC) an assess of dealings the effectiveness of relationship
of two crops (Alhassan et al 2012) was calculated by using (Adetiloye et al 1983)
equation as
Yield was calculated in gram fresh weight LER and LEC of height and total chlorophyll
were also calculated by using above formula by substituting their values with yield (fruits
of Z mauritiana and seeds of C cajan) to height fruits and chlorophyll respectively
23110 Statistical analysis
Data were analyzed by using (ANOVA) and the significant differences between treatment
means wereexamined by least significant difference (Zar 2010) All statistical analysis
was performed using SPSS for windows version 14 and graphs were plotted using Sigma
plot 2000
LER= Yield of Z mauritiana + Yield of C cajan (in intercropped) + Yield of C cajan + Yield of Z mauritiana (in intercropped)
Yield of Z mauritiana (sole) Yield of C cajan (sole)
Partial LER = Yield of Z mauritiana + Yield of C cajan (in intercropped)
Yield of Z mauritiana (sole)
Partial LER = Yield of C cajan + Yield of Z mauritiana (in intercropped)
Yield of C cajan (sole)
LEC = Partial LER of Z mauritiana times Partial LER of C cajan
96
232 Observations and Results
2321 Vegetative parameters
Vegetative growth parameters of Z mauritiana include plant height volume of canopy
grown individually as well as intercropped with C cajan is presented in Figure 29
Appendix-XV A significant increase in height and canopy volume of Z mauritiana with
time (p lt 0001) and cropping system (p lt 005) was observed However the interaction
between time and cropping system showed non-significant results In general the
intercropped plants were showed higher values in all vegetative parameters than sole crop
and this increase was more pronounced after 60 days
Figure 29 Appendix-XVII showed the vegetative growth parameters of C cajan
including height and number of branches Height of C cajan was significantly increased
(plt0001) with increasing time in plants growing sole and as intercropped with Z
mauritiana The interaction with time to crop height also showed significant (plt0001)
results in both cropping systems However slight decline in height of intercropped C
cajan was noticed at 120 days compared to sole crop Number of branches was significant
increased (plt0001) in both crops with increasing time The interaction of time with
branches also showed significant (plt0001) results in both cropping systems However
number of branches was slightly increased in intercropped plants at 120 days compared to
sole crop
2322 Reproductive parameters
i Fruit number and weight (fresh and dry)
Reproductive parameters of Z mauritiana and C cajan at grand period of growth under
sole and intercropping system has been presented in Figure 210 Appendix-XVI XVIII
Individual and interactive effect of time (p lt0001) and treatment (plt001) on number and
fresh weight of fruits of Z mauritiana was showed significant results Similarly plants
grown with C cajan showed significant increase (p lt0001) in fresh weight of fruits (p
lt005) whereas fruit dry weight and circumference was non-significant in comparison to
sole crop
97
In C cajan flowers were appeared only at blooming phase (during 60 days of treatment)
and no difference in number of flowers was observed in both cropping systems (sole and
with Z mauritiana (Figure 210 XVII)
Leguminous pods were initiated soon after flowering period (during 60 days) and
last till end of the experiment (120 days) A significant increase (plt0001) in pod numbers
was observed with increasing time in both sole and intercropped system But non-
significant differences in number of pods of both cropping system and their interaction
with time were observed
Similarly number and weight of C cajan seeds were showed non-significant difference
in both cropping systems
2323 Study on some biochemical parameters
i Photosynthetic pigments
Leaf pigments of Zmauritiana and C cajan grow plants under sole and intercropping has
been presented in Figure 211 Appendix-XVI XVIII In Z muritiana leaves A significant
increase (plt005) in chlorophyll a chlorophyll b total chlorophyll and carotinoids was
observed when grown as intercrop whereas the effect on chlorophyll ab ratio was non-
significant as that of sole one
In C cajan a slight decrease (plt005) in chlorophyll lsquobrsquo and total chlorophyll
(plt001) was observed in intercropped plants compare to sole one Whereas chlorophyll
lsquoarsquo chlorophyll ab ratio and carotinoids showed nonsignificant difference between sole
and intercropped C cajan
ii Total proteins sugar phenols
Sugars protein and phenols in leaves of Z mauritianaand C cajan at grand period of
growth under sole and intercropping system is presented in Figure 212 Appendix-XVI
XVIII Total proteins and soluble and insoluble sugar content of Z mauritiana leaves was
unaffected throughout the experiment However an increase in total phenolic content
(plt001) was observed in intercropped Z mauritiana plants than grown individually
98
In C cajan total soluble sugars protein and phenols in leaves showed non-
significant differences between sole to intercropped plants
Sugars protein and phenols in fruits of Z mauritiana grown under sole and
intercropping system is presented in Figure 213 Appendix-XVI A non-significant
increase was observed in phenolic as well as in soluble insoluble and total sugar contents
in fruits of Z mauritiana plants grown with C cajan (intercrop) as compare to the fruits
of sole crop
2324 Nitrogen Contents
Nitrogen in leaves and in soil of Z mauritiana and C cajan growing under sole and
intercrop system is presented in Figure 214 Appendix-XVI XVIII ANOVA showed a
non significant effect on nitrogen content of leaf as well as root zone soil of Z mauritiana
and C cajan grown individually or as intercropping system
2225 Land equivalent ratio (LER) and land equivalent coefficient
(LEC)
Land equivalent ratio (LER) Land equivalent coefficient (LEC) of height chlorophyll and
yield of of Z 98auritiana and C cajan growing as sole and intercropping system in has
been presented in Table 22 The LER using height of both species was nearly 2 in which
PLER of Z mutitania was 48 and PLER of C cajan was 519 Whereas the calculated
values of the land equivalent coefficient (LEC) of Z mauritiana and C cajan remained
9994
The LER using yield of both species was above 2 in which PLER of Z mauritiana
was 46 Whereas PLER of C cajan was 543 However the calculated values of LEC
of both species were 100
The LER using total chlorophylls of both species were more than 25 in which
PLER of Z mauritiana was 344 and as that of PLER of C cajan was 655 Whereas
the calculated values of LEC was 999 of both the species
99
Table 21 Soil analysis data of Fiesta Water Park experimental field
Serial number Parameters Values
1 ECe (dSm-1) 4266plusmn0536
2 pH 8666plusmn0136
3 Bulk density (gcm3) 123plusmn0035
4 Porosity () 53666plusmn1333
5 Water holding capacity () 398plusmn2811
6 Soil texture Clay loam
7 Sand () 385plusmn426
8 Silt () 3096plusmn415
9 Clay () 305plusmn1
Ece is the electrical conductivity of saturated paste of soil sample
Figure 29 Soil texture triangle (Source USDA soil classification)
100
Ziziphus mauritiana
Days
0 60 120
Volu
me
(m3)
0
10
20
30
Days
0 60 120
Hei
ght
(cm
)
0
50
100
150
200
250
Sole Intercrop
a
a
bb
c c
aa
bb
c c
Cajanus cajan
Days
0 60 120
Bra
nch
es (
)
0
10
20
30
Days
0 60 120
Hei
ght
(cm
)
0
50
100
150
200
250
300
Sole Intercrop
aa
bb
c c
aa
bb
c c
Figure 210 Vegetative growth of Z mauritiana and C cajan growing under sole and intercropping
system (Bars represent means plusmn standard error of each treatment and significance among the
treatments was recorded at p lt 005)
101
Ziziphus mauritiana
Fresh Dry
Fru
it w
eig
ht
(g)
0
50
100
150
200
Days
0 60 120 180
Nu
mb
er o
f F
ruit
s
0
100
200
300
Sole Intercrop
a
b
a
b
c
c
dd
Cajanus cajan
0 60 120
Num
ber
of
Pods
0
50
100
150
200
Days
0 60 120
Num
ber
of
Flo
wer
s
0
50
100
150
Sole Intercrop
Days
aa
bb
c c
Sole Intercrop
Num
ber
of
See
ds
0
100
200
300
400
500
See
d W
eight
(g)
0
10
20
30
40
50
60Number of seedsSeed weight
Figure 211 Reproductive growth of Z mauritiana and C cajan growing under sole and intercropping
system (Bars represent means plusmn standard error of each treatment and significance among the
treatments was recorded at p lt 005)
102
Ziziphus mauritiana
Cajanus cajan
Figure 212 Leaf pigments of Zmauritiana and C cajan growing under sole and intercropping (Bars
represent means plusmn standard error of each treatment and significance among the treatments was
recorded at p lt 005)
Sole Intercrop
Car
ote
noid
s (m
g g
-1)
00
01
02
03C
hlo
rophyl
l (m
g g
-1)
00
02
04
06
08
ab r
atio
00
05
10
15
20
25
ab
ab
Sole Intercrop
Car
ote
no
ids
(mg
g-1
)
00
01
02
03
Ch
loro
ph
yll
(m
g g
-1)
00
02
04
06
08
10
ab
rat
io
0
1
2
3
4ab
ab
103
Ziziphus mauritiana
Sole Intercrop
Lea
f P
hen
ols
(m
g g
-1)
0
2
4
6
8
10
12
Lea
f P
rote
ins
(mg
g-1
)
0
2
4
6
8
Lea
f S
ug
ars
(mg
g-1
)
0
5
10
15
20
25
30
35SoluableInsoluable
Figure 213 Sugars protein and phenols in leaves of Z mauritiana and C cajan at grand period of growth under
sole and intercropping system (Bars represent means plusmn standard error of each treatment and
significance among the treatments was recorded at p lt 005)
104
(Figure 212 continuedhellip)
Cajanus cajan
Sole Intercrop
Lea
f P
hen
ols
(m
g g
-1)
0
2
4
6
8
Lea
f P
rote
ins
(mg g
-1)
00
05
10
15
20
Lea
f S
ugar
s (m
g g
-1)
0
2
4
6
8
105
Ziziphus mauritiana
Sole Intercrop
Fru
it P
hen
ols
(m
g g
-1)
0
2
4
6
8
10
12
14
Fru
it P
rote
ins
(mg g
-1)
00
02
04
06
08
10
Fru
it S
ugar
s (m
g g
-1)
0
5
10
15
20
25
30
35 SoluableInsoluable
Figure 214 Sugars protein and phenols in fruits of Z mauritiana grown under sole and intercropping
system (Bars represent means plusmn standard error of each treatment and significance among the
treatments was recorded at p lt 005)
106
Z mauritiana
Sole Intercrop
Nit
rogen
(
)
0
1
2
3
4
5
6
7 LeafSoil
Cajanus cajan
Sole Intercrop
Nit
rogen
(
)
0
1
2
3
4
5
6
7 LeafSoil
Figure 215 Nitrogen in leaves and in soil of Z mauritiana and C cajan growing under sole and intercrop
system (Bars represent means plusmn standard error of each treatment and significance among the
treatments was recorded at p lt 005)
107
Table 22 Land equivalent ratio (LER) and Land equivalent coefficient (LEC) with reference to height chlorophyll and yield of of Z mauritiana and C cajan growing
under sole and intercropping system
Plant species Parameters Formulated with
reference to Height
Formulated with
reference to Total
Chlorophyll
Formulated with reference to Yield
(fresh weight of Z mauritiana fruit
and seed of C cajan)
Z mauritiana Partial LER 1027 1666 1159
C cajan Partial LER 0950 0877 0993
Intercropped
Total LER 1977 2543 2152
Z mauritiana amp C cajan
(Sole and intercropped) LEC 0975 1461 1151
107
108
24 Discussion
Intercropping is a common practice used to obtain better yield on a limited area through
efficient utilization of given resources which may not be achieved by growing each crop
independently (Mucheru-Muna et al 2010) In this system selection of appropriate crops
planting rates and their spatial arrangement can reduce competition for light water and
nutrients (Olowe and Adeyemo 2009) In general increased growth (biomass height
volume circumference biomass succulence SSL SRL SSR LWR SWR RWR and
RGR) of each species is a good indicator of successful intercropping The SRL and SSL
measure the ratio between the lengths of root or shoot per unit dry weight of respective
tissues (Wright and Westoby 1999) The weight ratio of leaf stem and root to total plant
weight (LWR SWR and RWR) describes the allocation of biomass towards each organ to
maximize overall relative growth rate (RGR) which explains how plant responds to certain
type of condition (Reynolds and Antonio 1996) In this study height and canopy volume
of Z mauritiana and height and branches of C cajan were increased when grown together
in comparison to sole crop in field experiment (Figure 29) Whereas in drum pot culture
biomass generally the length of plant canopy volume number of leaves RGR LWR
SWR RWR SSL and SRL were either higher or unaffected in both species growing in
intercropping at 4th and 8th days intervals (Figure 21-23) Similar beneficial effects on
growth of other intercrops have also been reported under different conditions (Yamoah
1986 Atta-Krah 1990 Kass et al 1992 Singh et al 1997) Dhyani and Tripathi (1998)
observed increased height stem diameter crown width and timber volume of three
intercropped species than sole crop Bhat et al (2013) also revealed significant
improvement in annual extension height and spread in apple plants intercropped with
leguminous plants
The increased growth of both intercropped plants of this study was well reflected
by their biochemical parameters Leaf pigments like chlorophyll a chlorophyll b and total
chlorophyll were either higher or remained unaffected (Figure 211) in both intercropped
plants than sole crops of field experiments Whereas in drum pot culture chlorophyll
content (Figure 24) was higher only in intercropped C cajan (specially in 8th days) Bhatt
et al(2008) and Massimo and Mucciarelli (2003) also reported the increased accumulation
of chlorophyll a b and total chlorophylls in leaves of soybean and peppermint when
109
grown with their respective intercrops Our results are also in agreement with Liu et al
(2014) and Otusanya et al (2008) reported similar results in Lycopersican esculentum and
later in Capsicum annum as well Some other reports are also available which shows non-
significant effect on leaf pigments in both cropping systems (Shi-dan 2012 Luiz-Neto-
Neto et al 2014)The synthesis and activity of chlorophyll depends on severity and type
of applied stress it generally increase in low saline mediums (Locy et al 1996) or
remained unaffected however sometimes stimulated (Kurban et al 1999 Parida et al
2004 Rajesh et al 1998)
Proteins and carbohydrates (sugars) perform vast array of functions which are
necessary for plant growth and reproduction (Copeland and McDonald 2012) Variation
in their contents helps to predict plant health which is usually decreased with applied stress
(Arbona et al 2013) Both are also the compulsory factors of animals diet since they
cannot manufacture sugars and some of the components of proteins which must be
obtained from food (Bailey 2012) In our experiment protein content was either remained
unchanged or increased which indicated a good coordination of both intercrops in field
and drum pot experiments (Figure 26 and 212) Liu et al (2014) also found that protein
and sugars were not affected in tomatogarlic intercrops In another experiment similar
results were found when corn was grown with and without intercropping (Borghi et al
2013)
Reactive oxygen species (ROS) are produced as a spinoff of regular metabolism
however under stress the overproduction of ROS may lead to oxidative damage (Baxter et
al 2014) In low concentrations ROS worked as messengers to regulate several plant
processes and also helps to improve tolerance to various biotic and abiotic stresses (Miller
et al 2009 Nishimura and Dangl 2010 Suzuki et al 2011) but when the concentration
goes beyond the critical limit ROS would become self-threatening at every level of
organization (Foreman et al 2003) To maintain a proper workable redox state an
efficient scavenging system of enzymatic (SOD CAT GPX and APX) andor non-
enzymatic (polyphenols sugars glutathione and ascorbic acid) antioxidants is required
which would be of critical importance when plant undergoes stress (Sharma et al 2012)
Among these enzymes SOD is a first line of defense which converts dangerous superoxide
radicals into less toxic product (H2O2) In further CAT APX and GPX worked in
association to get rid off from the excessive load of other oxygen radicals or ions (H2O2
110
OH- ROO etc) In this study antioxidant enzymes (SOD CAT GPX and APX) were
found to work in harmony which was not affected during 4th day treatment in both species
in comparison to sole crop (Fig 27) showing strong antioxidant defense which was not
compromised by cropping system When comparing in 8th day treatment a significant
general increase in all enzyme activities were observed in both species except for SOD
and GPX of C cajan (Fig 27) These results displayed relatively better performance and
tight control over the excessive generation of ROS which would be predicted in this case
due to less availability of water than in 4th day treatment (Karatas et al 2014 Doupis et
al 2013) Similarly by coping oxidative burst and maintaining cellular redox equilibrium
plants were able to improve growth performance especially in Z mauritiana (Fig 21)
Water deficit affect stomatal conductance which could bring about changes in
photosynthetic performance hence overproduction of ROS is usually found among
different crops (Moriana et al 2002 Miller et al 2010) As a response tolerant plants
overcome this situation by increased activity of antioxidant enzymes which was evident in
Wheat Rice olive etc (Zhang and Kirkham 1994 Sharma and Dubey 2005 Guo et al
2006 Sofo et al 2005)
Phenolic compounds despite their role in physiological plant processes are
involved in adsorbing and neutralizing reactive oxygen species (ROS Ashraf and Harris
2004) The overproduction of ROS may cause several plant disorders Plants produce
secondary compounds like polyphenols to maintain balance between ROS generation and
detoxification (Posmyk et al 2009) Increased synthesis and accumulation of phenolic
compounds is reported to safeguard cellular structures and molecules especially under
biotic abiotic constraints (Ksouri et al 2007 Oueslati et al 2010) In this study
intercropped Z mauritiana of field and both species in drum pot culture showed higher
phenolic content than individual crop (Figure 25 and 212) which may be attributed to
adaptive mechanism for scavenging free radicals to prevent cellular damage (Rice-Evans
1996)
In terms of fruit yield we observed that Z mauritiana is suitable for intercropping
as suggested by Yang et al (1992) Number of flowers fruits and fruit fresh weight of
both species either increased considerably or no-affected in intercropped plants compared
to individual ones (Figure 210) Moreover fruit quality of Z mauritiana includes proteins
phenols and soluble extractable and total sugars were also higher in intercropped plants
111
(Figure 213) Results of this study are better than other experiments reported by
Sharma (2004) Kumar and Chaubey (2008) and Kumar et al (2013) who did not find
influence of other understory forage crops (like Aonla) on the yield of Z mauritiana
However in other case the yield of intercropped ber was some time higher (Liu 2002)
Singh et al 2013 found no adverse effects on the yield of pigeonpea when intercropped
with mungbean however it improved the grain yield of associated species
A leguminous plant C cajan is used in this experiment as secondary crop which
can supplement Z mauritiana by improving soil fertility Results of both experiments
showed that the nitrogen was higheror un-affected (Figure 214) in soils of intercropped
plants which supports our hypothesis that leguminous intercrop increase N supply This
can be achieved by acquisition of limited resources to manage rootrhizosphere
interactions which can improve resource-use efficiency (Zhang et al 2010
Shen et al 2013 White et al 2013b Ehrmann and Ritz 2014 Li et al 2014) As a
consequence it impact on overall plant performance which starts from high photosynthetic
activity by increasing chlorophyll results in more availability of photoassimilate for
growth and reproductive allocation (Eghball and Power 1999) Use of C cajan in tree
intercropping proved beneficial for producing high yield crops and for the environment
(Gilbert 2012 Glover et al 2012)
Land equivalent ratio (LER) is commonly used to evaluate the effectiveness of
intercropping by using the resources of same environment compared with sole crop
(Vandermeer 1992 Rao et al 1990 1991 Cao et al 2012) It is the ratio of area for sole
crop to intercrop required to produce the equal amount of yield at the same management
level (Mead and Willey 1980 Dhima et al 2007) On the other hand land equivalent
coefficient (LEC) describe an association that concern with the strength of relationship It
is the proportion of biomassyield of one crop explained by the presence of the other crop
The LER 1 or more indicate a beneficial effect of both species on each other which increase
the yield of both crops as compare to single one (Zada et al 1988) In this experiment all
LER values were about 2 or more than 2 while LEC values were around 1 or more than
one in ZizyphusCajnus intercropping Both LER and LEC values were in descending
order of chlorophylls gt yield gt height (Table 22) However the partial LER was higher in
Zizyphus than Cajanus in all cases These results describe the superiority of intercropping
over sole cropping where LER values are even gt2 Some other studies reported LER from
112
09-14 (Bests 1976) 12-15 (Cunard 1976) and up to 2 (Andrews and Kassam 1976)
Similar results were reported in poplarsoybean system (Rivest et al 2010) black
locustMedicago sativa (Gruenewald et al 2007) wheatjujube (Zhang et al 2013)
Acacia salignasorghum (Droppelmann et al 2000 Raddad and Luukkanen 2007) The
high LER values in our system indicating a harmony in resource utilization in both species
which was also corroborated with their respective LEC values The greater LEC values (gt
025) suggesting an inbuilt tendency of studied crops to give yield advantage (Kheroar and
Patra 2013) Experiments based on traditional practices of growing legumes with cereals
demonstrated greater and continuous cash returns than individual-crops (Baker 1978) In
addition the same authors found further increase in cash returns by increasing the
proportion of cereal and incorporating maize with sorghum and millet In agreement with
our findings similar reports are also available from different intercropping systems
including sesamegreengram (Mandal and Pramanick 2014) maizeurdbean (Naveena et
al 2014) and pegionpeasorghum (Egbe and Bar-Anyam 2010)
After detailed investigations of both species using two different experiment designs
(drum pot and field) it is evident that intercropping had beneficial effects on growth
physiology biochemisty and yield of both species Furthermore by using this system
higher outcome interms of edible biomass and green fodder using marginal lands can be
obtained in a same time using same land and water resources which can help to eliminate
poverty and uplift socio-economic conditions
113
3 Chapter 3
Investigations on rang of salt tolerance in Carissa carandas
(varn karonda) for determining possibility of growing at waste
saline land
31 Introduction
Carissa carandas commonly known as Karonda or lsquoChrist thornrsquo belonging to family
Apocynaceae shows capability of growing under haloxeric conditions It is an important
plant which has established well at tropical and subtropical arid zone under high
temperatures It is large evergreen shrub and having short stem It has fork thorn and hence
used as hedges or fence around fields The leaves are oval or elliptic 25 to 75 cm long
dark green leathery and secrete white milk if detached The fruits are oblong broad- ovoid
or round 125- 25 cm long It has thin but tough epicarp Fruits are in clusters of 3-10
Young fruits are pinkish white and become red or dark purple on maturation
The plant is propagated through seed in August and September Budding and cutting
could also be undertaken Planting is started after first shower of monsoon Plants raised
from seeds are able to flower within two years Flowering starts in March and fruit ripen
from July to September (Kumar et al 2007) The fruit possess good amount of pectin and
acidity hence used in prickle jelly jam squash syrup and in chutney by the commercial
name lsquoNakal cherryrsquo (Mandal et al 1992) They are rich in vitamin C and good source
of Anthocyanin (Lindsey et al 2000) Its fruits also are one of the richest source of iron
(391 mg 100gm) (Tyagi et al 1999) Juice of its root is also used to treat various
microbial diseases such as diarrhea dysentery and skin disease (Taylor et al 1996)
Hence its range of salt and suitability for cultivation at waste saline land or with saline
water irrigation is being undertaken for commercial exploitation by preparing jams jellies
and prickles (Kumar 2014) Investigations on its growth and development at higher range
of salinities are being undertaken with an interest to cultivate it if profitable at highly saline
waste land
114
32 Experiment No 9
Investigation on the effect of higher range of salinities on growth of
Carissa carandas (varn karonda) created by irrigation of different
dilutions of sea salt
321 Materials and methods
3211 Drum Pot Culture
Drum pot culture as recommended by Boyko (1966) and modified by Ahmed and
Abdullah (1982) was used for the present investigation which was been already described
in Chapter 1 earlier
3212 Plant material
About six months old sapling of Carissa carandas (varn Karonda) having almost equal
height and volume poted in polythene bag in 3kg of soil fertilized with cow-dong manure
were purchased from the Noor nursery Gulshan-e-Iqbal Karachi Sindh and were
transported to the Biosaline research field department of Botany University of Karachi
3213 Experimental setup
Plants were transplanted in drum pot (Homemade lysimeter) filled with sandy loam mixed
with cow dung manure (91) Each drum pot was irrigated weekly during summer and
fortnightly during winter months with 20 liters tap water (Eciw= 0 6 dSm-1) or water of
sea salt concentrations of various ie 03 (Eciw = 42 dSm-1) 04 (Eciw =61 dSm-1)
06 (Eciw = 99 dSm-1) and 08 (Eciw = 129 dSm-1) The plants were established initially
by irrigation with tap water for two weeks and later salinity was gradually increased till
desired percentage is achieved for different treatments by dessolving of sea salt in
irrigation water Three replicates were maintained for each treatment Urea DAP and
KNO3 were the source of NPK provided in the ratio 312 50g granules Osmocot (Scotts-
Sierra Horticulture Products) and 50g Mericle-Gro (Scotts Miracle-Gro Products Inc)
were dissolved in irrigation water per drum after six months at six monthly intervals
Height and volume of canopy of these plants were recorded prior to the starting the
experiment and then after every six months interval
115
Since the vegetative growth performance in plants irrigated with 03 sea salt (Eciw = 42
dSm-1) was found comparatively better than control and only 26 decrease was noticed
in volume of canopy at plant irrigated with 04 sea salt (Eciw = 61 dSm-1) (Table III41)
the onward investigations were focused at higher salinity levels and plants were irrigated
with 06 (Eciw = 99 dSm-1) and 08 (Eciw = 129 dSm-1) sea salt in rest of experiment
3214 Vegetative parameters
Vegetative growth on the basis of plant height and volume were recorded while
reproductive growth was observed on the basis of number of flowers and number and
weight of fruits per plant Length and diameter of fruit were also recorded in ten randomly
selected fruits
3215 Analysis on some biochemical parameters
Following biochemical analysis of leaves was performed at grand period of growth (onset
of flowers)
i Photosynthetic pigments
Fresh fully expended leaves (01g) was crushed in 80 chilled acetone Further procedure
was followed described in chapter 1
ii Soluble sugars
Dry leaf samples (01g) were milled in 5mL of 80 ethanol and were centrifuged at 4000
g for 10 minutes Same procedure was followed as described in chapter 1
iii Protein content
The protein contents were measured according to Bradford Assay reagent method against
Bovine Serum Albumin which was taken for standard (Bradford 1976) as described in
chapter 1
iv Soluble phenols
The dried leaf powder (01g) was milled in 3ml of 80 methanol and was centrifuged at
10000g for 15 min Further procedure has been described in chapter 2
116
3216 Mineral Analysis
Estimation of Na+ and K+ were made according to Chapman and Pratt (1961) Oven dried
grinded Leaves (1g) furnace at 550ordmC for 6 hours and were digested in 5 ml of 2N HCl
Diluted and filtered solution was used to estimated Na+ and K+ in flame photometer
(Petracourt PFP I) The concentration of these ions was calculated against the following
standard curve equations
Na+ (ppm) = 0016135x1879824
K+ (ppm) = 0244346x1314603
117
322 Observations and Result
3221 Vegetative parameters
Vegetative growth in terms of height and volume of canopy of C carandas growing under
salinities created by irrigation of different dilutions of sea salt is presented in Table 32
Appendix-XIX A significant increase (plt0001) in plant height and volume of canopy
was observed with increasing time but the increase was rapid at early period of growth
However there was significant (plt0001) reduction under salinity stress The interaction
of time and salinity also showed significant (plt001) effect on plant parameters but the
increase in height and volume of canopy at Eciw= 42dSm-1of sea salt salinity was more
than control Plants irrigated with Eciw= 61 dSm-1 and Eciw= 99 dSm-1sea salt solution
showed decrease in height with respect to control but the difference between their
treatments was insignificantly higher decrease was observed in Eciw= 129 dSm-1 sea salt
irrigated plants
3222 Reproductive parameters
Reproductive growth in terms of flowers and fruits numbers flower shedding percentage
fresh and dry weight of ten fruit their length and diameter under salinities created by
irrigation of different dilutions of sea salt is presented in Table 33 Appendix-XX Number
of flowers and fruits significantly (plt0001) decreased with increasing salinity treatment
Difference in flower initiation seems non-significant at early growth period in controls and
salinity treatments However drastic decrease was observed in plants irrigated beyond
Eciw= 99 dSm-1 with increase in salinity
Flowers shedding percentage (Table 33 Appendix-XX) show an increase directly
proportional with increase in salinity however the difference in number of flowers
between the plants irrigated with Eciw= 99 dSm-1 and Eciw= 129 dSm-1 sea salt solution
is of little significance level (plt001)
Fresh and dry weight of average fruits (plt001) and their diameter (plt001) showed
decrease with increasing salinity whereas diameter and length of fruits showed non-
significant difference
118
3224 Study on some biochemical parameters
i Photosynthetic Pigments
Photosynthetic Pigments including Chlorophyll a chlorophyll b total chlorophyll
chlorophyll a b ratio and carotenoids of C carandas growing under salinities created by
irrigation of different dilutions of sea salt is presented in Figure 31 Appendix-XX The
chlorophyll contents of leaves significantly decreased (plt0001) over control with
increasing salinity however Chlorophyll rsquobrsquo at Eciw= 99 dSm-1salinity shows significant
increase (plt0001) over control Similarly Carotenoids at Eciw= 99 dSm-1 salinity show a
bit less significant increase (plt001) compare to control while at higher salinity (Eciw=
129 dSm-1) the decline is observed at all above mentioned parameters
iii Protein Sugars and phenols
Some biochemical parameters including Protein sugars and phenolic contents of C
carandas growing under salinities created by irrigation of different dilutions of sea salt is
presented in Figure 31 Appendix-XX Soluble proteins in leaves show non-significant
decrease at Eciw= 99 dSm-1salinity as compared with controls but a significant decrease
(plt005) was noted at Eciw= 129 dSm-1 salinity Sugars also showed non-significant
decrease at both the salinity whereas on contrary soluble phenols showed significant
increase (plt0001) with increasing salinity
3225 Mineral analysis
Mineral analysis including Na and K ions performed in leaves of C carandas growing
under salinities created by irrigation of different dilutions of sea salt is presented in Figure
32 Appendix-XX Sodium significantly increased (plt0001) all the way with increasing
salinity of growth medium Whereas significant decrease (plt0001) was observed in
Potassium with increasing salinity K+Na+ ratio show continuous increase with increasing
salinity
119
Table 31 Electrical conductivities of different sea salt concentration used for determining
their effect on growth of C carandas
Treatment
Sea salt ()
ECiw of irrigation water (dSm-1) ECe of soil saturated paste
(dSm-1)
Non-saline control 06 09
03 42 48
04 61 68
06 99 112
08 129 142
Whereas ECiw and ECe are the electrical conductivities of irrigation water and soil saturated past measured in deci semen per meter
120
Table 32Vegetative growth in terms of height and volume of canopy of C carandas growing under salinities created by irrigation of different dilutions of
sea salt
Treatment
Sea salt
(ECiw dSm-1)
Initial values prior to
starting saline water
irrigation
Growth at different salinities after 06 months
Height Volume Height Volume of canopy
cm m3 cm
increase
over initial
values
increase
decrease over
control
m3 increase over
initial values
increase
decrease
over control
Control 3734plusmn455 0029plusmn0001 8227plusmn4919 5363plusmn830 - 014plusmn0015 7952plusmn269 -
42 3674plusmn1415 0026plusmn0003 9930plusmn6142 6280plusmn205 +1710 019plusmn0017 8593plusmn098 +806
61 3752plusmn1243 0026plusmn0001 6490plusmn5799 4132plusmn485 -2305 012plusmn0010 7740plusmn117 -282
99 3819plusmn4499 0028plusmn0005 5793plusmn5821 3123plusmn1446 -4185 009plusmn0008 6759plusmn377 -1499
129 3676plusmn3114 0026plusmn0008 5250plusmn4849 2775plusmn1276 -4836 006plusmn0005 5690plusmn1110 -2844
LSD0 05
Salinity
Time Fisherrsquos least significant difference
91
172
002
0005
Values represent means plusmn standard error of each treatment and significance among the treatments was recorded at p lt 005
120
121
Table 33 Vegetative growth in terms of height and volume of canopy of C carandas growing under salinities
created by irrigation of different dilutions of sea salt
Treatment
Sea salt
(ECiw dSm-1)
Growth at different salinities after 12 months
Height Volume of canopy
cm
increase
over initial
values
increase
decrease over
control
m3
increase
over initial
values
increase
decrease over
control
Control 16214 plusmn633 7674plusmn307 - 077plusmn012 9689plusmn449 -
99 9736plusmn1048 6056plusmn561 -2109 034plusmn006 9367plusmn412 -333
129 6942plusmn565 4741plusmn480 -3822 022plusmn002 9064plusmn623 -645
Table 33 continuedhellip
Treatment
Sea salt
(ECiw= dSm-1)
Growth at different salinities after 18 months
Height Volume of canopy
Cm
increase
over initial
values
increase
decrease over
control
m3
increase
over initial
values
increase
decrease over
control
Control 1676plusmn1135 7776plusmn756 - 094plusmn011 9701plusmn578 -
99 10547plusmn842 6351plusmn666 -1833 045plusmn010 9445plusmn1024 -264
129 7581plusmn593 5154plusmn716 -3372 030plusmn003 9318plusmn580 -395
Table 33 continuedhellip
122
Table 33 continuedhellip
Treatment
Sea salt
(ECiw= dSm-1)
Growth at different salinities after 24 months
Height Volume of canopy
Cm
increase
over initial
values
increase
decrease over
control
m3
increase
over initial
values
increase
decrease over
control
Control 1911plusmn6
05 8055plusmn941 - 121plusmn015 9837plusmn522 -
99 1110plusmn5
31 6557plusmn543 -1859 053plusmn002 9509plusmn1032 -334
129 8754plusmn10
67 5990plusmn801 -2564 040plusmn008 9287plusmn745 -560
Table 33 continuedhellip
Treatment
Sea salt
(ECiw= dSm-1)
Growth at different salinities after 30 months
Height Volume of canopy
Cm
increase
over initial
values
increase
decrease over
control
m3
increase
over initial
values
increase
decrease over
control
Control 2052plusmn1126 8182plusmn676 - 146plusmn029 9873plusmn729 -
99 11700plusmn816 6743plusmn610 -1759 070plusmn011 9565plusmn850 -312
129 9628plusmn552 6189plusmn573 -2436 050plusmn004 9417plusmn1011 -462
LSD0 05 Salinity 77 007
Time 168 016
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and significance among the treatments was recorded at p lt 005
123
Table 34 Reproductive growth in terms of flowers and fruits numbers flower shedding percentage fresh and dry weight of ten fruit and their totals
perplant fruit length and diameter of C carandas growing under salinities created by irrigation of different dilutions of sea salt
Treatment
Sea salt
(ECiw= dSm-1)
Flower Fruits Flower
shedding
Weight of
Ten
fruit(fresh)
Weight of
Ten
fruit(dry)
Weight of
total fruitplant
(fresh)
Weight of
total fruitplant
(dry)
length
fruit
diameter
fruit
Numbers Numbers g g g g mm mm
Control 19467plusmn203 16600plusmn231 1468plusmn208 2282plusmn022 605plusmn009 37891plusmn891 10047plusmn283 1800plusmn003 1423plusmn006
99 12050plusmn202 7267plusmn491 3980plusmn307 1880plusmn035 530plusmn029 13695plusmn1174 3880plusmn469 1732plusmn037 1297plusmn011
129 12567plusmn549 6967plusmn203 4449plusmn082 1541plusmn023 435plusmn026 10742plusmn470 3041plusmn268 1711plusmn015 1233plusmn038
LSD0 05 Salinity 1514 1417 929 115 097 3785 1494 0971 097
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and significance among the treatments was recorded at p lt 005
123
124
Sea Salt (ECiw
= dSm-1
)
Cont 99 129
Car
ote
nio
ds
(mg
g-1
)
00
01
02
03
04
Ch
loro
ph
yll
(m
g g
-1)
00
01
02
03
04
05
06
ab
rat
io
00
05
10
15
20
25
30
35
ab
Chl a Chl b
a
a
a a
b
bcbc
a
b
c
a a
b
Figure 31 Chlorophyll a chlorophyll b total chlorophyll chlorophyll a b ratio carotenoids contents of C
carandas growing under salinities created by irrigation of different dilutions of sea salt (Bars
represent means plusmn standard error of each treatment and significance among the treatments was
recorded at p lt 005)
125
Sea Salt (ECiw
= dSm-1
)
Cont 99 129
Ph
eno
ls (
mg
g-1
)
0
5
10
15
20
Pro
tein
s (m
g g
-1)
0
1
2
3
4
Su
gar
s (m
g g
-1)
0
30
60
90
120
150Soluble Insoluble
a
a
a
a
a
a
b
b
b
c
ab
a
a
b
Figure 32 Total protein sugars and phenolic contents of C carandas growing under salinities created by
irrigation of different dilutions of sea salt (Bars represent means plusmn standard error of each treatment
and significance among the treatments was recorded at p lt 005)
126
Sea Salt (ECiw
= dSm-1
)
Cont 99 129
Ions
(mg
g-1
DW
)
0
20
40
60
80
100
120
KN
a ra
tio
00
01
02
03
04
05
06
07
Na K KNa
c
a
b
b
a
c
a
b
c
Figure 33 Mineral analysis including Na and K ions was done on leaves of C carandas growing under salinities
created by irrigation of different dilutions of sea salt (Bars represent means plusmn standard error of each
treatment and significance among the treatments was recorded at p lt 005)
127
33 Discussion
The volume and height of plants were increased per unit time under saline conditions This
increase was observed after six months in 03 sea salt (ECiw = 42 dSm-1) treated plants in
comparison to control (Table 32) Slight decrease was observed at 04 sea salt
(ECiw=61dSm-1) irrigation after which (Eciw= 99 dSm-1 and Eciw = 129 dSm-1sea salt) the
growth was significantly inhibited (Table 33) Noble and Rogers (1994) also noticed a general
decrease in growth of some of the glycophytes Humaira and Ahmad (2004) and Rivelli et al
(2004) also reported a proportional decrease in height of canola with increasing salinity
Cotton plants irrigated with saline water or those grown at saline soil are reported to increase
Na+ content in leaves accompanied by significant reduction in vegetative biomass (Meloni et
al 2001) Bayuelo-Jimenez et al (2003) observed salt induced growth inhibition of tomato
plant which was higher in shoot than root
Reproductive growth in terms of number of flowers number of fruits fruit length and
diameter were decreased and percent flower shedding increased with increasing salinity
(Table 34) These effects were higher at Eciw= 99 dSm-1and then maintained with further
salinity increment However weight of fruits (fresh and dry) and total fruits per plant were
linearly decreased with increasing medium salt concentrations A decrease in different phases
of reproductive growth like flowering fertilization fruit setting yield and quality of seeds etc
are reported to be seriously affected at different level of salinity by various workers (Lumis et
al 1973 Waisel 1991 Shannon et al 1994 Tayyab et al 2016) Cole and Mclead (1985)
and Howie and Lloyd (1989) reported severe effects of different salinity treatments on
flowering intensity fruit setting and number of fruits of Citrus senensis Walker et al (1979)
also reported reduction in the fruit weight during early ripening stage of Psidium guajava
Decrease in fruit diameter of strawberries (Fragaria times ananassa) has been reported with
salinity (Ehlig and Bernstein 1958)
In this study photosynthetic pigments of C carandas were decreased with salinity and
this decrease was more sever at Eciw = 129 dSm-1sea salt salinity (Figure 31) Such a decline
in amount of leaf pigments across different salinity regimes was also reported in cotton
(Ahmed and Abdullah 1979) Pea (Hernandez et al 1995 and Hernandez et al 1999) Vicia
128
faba (Gadallah 1999) Mulberry genotype (Agastian et al 2000) and B parviflora (Parida et
al 2004)
Leaf sugars and protein were decreased in both salinity levels (Figure 32) which could
be attributed to inhibition in transport of photosynthetic product (Levit 1980) Decrease
synthesis and mobilization of glucose fructose and sucrose has been demonstrated in number
of plants growing under salt stress (Kerepesi and Galiba 2000) Inhibition in the protein and
nucleic acid synthesis in Pisum sativum and Tamarix tetragyna plants were also reported by
Bar-Nun and Poljahoff-Mayber (1977) Melander and Harvath (1977) suggested that salt
induced reduction in protein is due to increase in protein hydrolysis
A significant increase in leaves phenol with increase in salinity (Figure 32) was
observed in present investigation was also demonstrated previously in Achilleacollina (Giorgi
et al 2009) Lactuca sativa (Kim et al 2008) and B parviflora (Parida et al 2004)
Inspite of over irrigation of saline water and maintaining leaching fraction of about
40 in drum pots accumulation of salts in rhizosphere soil was not completely avoided which
was evident in the differences between ECiw and ECe values (Table 31) Deposition of salts
in rhizosphere soil interferer absorption of minerals in plants For instance leaf Na+ content
of C carandas was significantly increased while K+ decreased with increasing soil salinity
(Figure 33) Over accumulation of toxic ions disturbed plant water status which directly
affects plant growth (Flowers et al 1977 Greenway and Munns 1980) A negative
relationship between Na+ and K+ concentration in roots and leaves of guava was also reported
by Ferreira et al (2001) Increase in Na+ content decreased K+ availability and K+Na+ ratio
in Vicia taba (Gadallah 1999) and also affect the uptake of other essential minerals in
Casurina equsetifolia (Dutt et al 1991)
Carissa carandas found to be a good tolerant to salinity and drought and it can produce
edible fruits from marginal lands of arid areas Fruits of this species can be consumed in a raw
form as well as in industrial products like pickles jams jellies and marmalades
129
4 Conclusions
In the light of above mentioned investigations it appears that pre-soaking treatment of Cajanus
cajan seeds has initiated metabolic processes at faster rate earlier which has helped seeds to
start germinative metabolism prior to be effected by toxic Na+ ions at higher salinities Cajanus
cajan and Ziziphus mauritiana were found to be the good companions for intercropping These
species synergistically enhanced the growth and biochemical performance of each other by
improving fertility of marginal land and maintaining harmony among different physiological
parameters which was missing in their sole crop Their intercropping could produce fodder
and delicious fruits even from under moderately saline substrate up to profitable extant
Carissa carandas also tolerated low and moderately salinities well by adjusting proper
regulation of physiological and biochemical parameters of growth It can provide protein rich
edible fruits jams jellies and pickles of commercial importance for benefit of poor farmer
from moderately saline barren land
130
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167
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168
6 THESIS APENDECES
Appendix-I One way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution of seed germination of pre-soaked seeds of C cajan in non-saline water prior to germination under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Mean
germination rate
(GR)
Salinity treatment 4422 20 221133 21015 0000
Error 441949 42 10522
Total 4864 62
Mean germination
velocity (GV)
Salinity treatment 418813 20 20941 51836 0000
Error 169671 42 40398
Total 588484 62
Mean
germination
time (GT)
Salinity treatment 0271 20 0013 8922 0000
Error 0064 42 0002
Total 0335 62
Mean germination
Index (GI)
Salinity treatment 4422 20 221133 21015 0000
Error 441949 42 10523
Total 4864607 62
Final
germination
(FG)
Salinity treatment 32107 20 1605397 25285 0000
Error 2666 42 63492
Total 34774 62
Appendix-II Two way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution of seed germination of pre-soaked seeds of C cajan in non-saline water prior to germination under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Germination percentage per
day
Salinity treatment 509583 20 25479 19187 0000
Time 53156 9 5906 4663 0002
Salinity treatment times time 251743 180 1398576 1053 ns
Error 531130 400 1327825
Total 1375283 629
Germination
rate per day
Salinity treatment
Time 761502 9 84611 83129 0000
Salinity treatment times time 442265 20 22113 24630 0000
Error 359117 400 0898
Total 2108622 629
Appendix-III One way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution of seed
germination of pre-soaked seeds of C cajan in respective saline water prior to germination under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Final mean germination
velocity (GV)
Salinity treatment 0538 6 0089 35585 0000
Error 0035 14 0003
Total 0573
Final mean
germination time (GT)
Salinity treatment 20862 6 3477 26256 0000
Error 1854 14 0132
Total 22716 20
Final mean germination
index (GI)
Salinity treatment 110514 6 18419 190215 0000
Error 1356 14 0097
Total 111869 20
Final
germination percentage (GP)
Salinity treatment 6857 6 1142857 40 0000
Error 400 14 28571
Total 7257 20
Appendix-IV Two way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution of seed
germination of pre-soaked seeds of C cajan in respective saline water prior to germination under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Germination percentage per
day
Salinity treatment 86644 6 14440816 505428 0000
Time 23378 6 3896 136373 0000
Salinity treatment times time 2717 36 75472 2641 0001
Error 2800 98 28571
Total 115540 146
Germination rate
per day
Salinity treatment 117386 6 19564 360762 0000
Time 128408 6 21401 394636 0000
Salinity treatment times time 58747 36 1632 30091 0000
Error 5314 98 0054
Total 309855 146
169
Appendix-V One way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution on seedling
emergence and height of germinating seeds of C cajan under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Seedling height of C cajan
Salinity treatment 200822 5 40056 169666 0000
Error 2833 12 0236
Total 203115 17
Seedling
emergence of C cajan
Salinity treatment 24805 6 4134 6381 000
Error 9070 14 647867
Total 33875 20
Appendix-VI Two way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution on growth and
development of C cajan in lysemeter (Drum pot) under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Plant height of
C cajan
Salinity treatment 261079 5 52215 720259 0000
Time 126015 8 15751 132488 0000
Salinity treatment times time 76778 40 1919 16144 0000
Error 11413 96 118893
Total 477028 161
Appendix-VII One way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution on growth
and development of C cajan in lysemeter (Drum pot) under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Number of
Flowers of C
cajan
Salinity treatment 3932 3 131075 39719 0000
Error 264 8 33
Total 419625 11
Number of pods
of C cajan
Salinity treatment 1473 3 491 23105 0000
Error 170 8 2125
Total 1643 11
Number of
seedspod of C cajan
Salinity treatment 3 3 1
Error 0 8 0
Total 3 11
Number of seeds plant of
C cajan
Salinity treatment 19332 3 6444 45621 0000
Error 1130 8 14125
Total 20462 11
Weight of
seeds plant of C cajan
Salinity treatment 592976 3 197658 85572 0000
Error 18478 8 2309
Total 611455 11
Chlorophyll a
of C cajan
Salinity treatment 0117 3 0039 81241 0000
Error 0004 8 0000
Total 0121 11
Chlorophyll b
of C cajan
Salinity treatment 0004 3 0001 15222 0001
Error 0001 8 0000
Total 0005 11
Total chlorophyll of
C cajan
Salinity treatment 0160 3 0053 164401 0000
Error 0002 8 0000
Total 0162 11
Chlorophyll a b
ratio of C cajan
Salinity treatment 242 3 0806 9327 0005
Error 0692 8 0086
Total 3112 11
Carotenoids of
C cajan
Salinity treatment 0015 3 0005 4510 0039
Error 0009 8 0001
Total 0025 11
Soluble sugars
of C cajan
Salinity treatment 0043 3 0014 6515 0015
Error 00178 8 0002
Total 0061 11
Insoluble
sugars of C
cajan
Salinity treatment 0118 3 0039 36262 0000
Error 0008 8 0001
Total 0127 11
Total sugars of
C cajan
Salinity treatment 0019 3 0006 4239 0045
Error 0012 8 0001
Total 0031 11
Protein of C cajan
Salinity treatment 0212 3 0070 15735 0001
Error 0036 8 0004
Total 0248 11
170
Appendix-VIII One way ANOVA for completely randomized design for range of salt tolerance of nitrogen fixing symbiotic bacteria
associated with root of C cajan
Variables Source Sum of Squares df Mean Square F-value P
Nodule
associated
Rhizobial colonies of C
cajan
Salinity treatment 35927 2 17963 229402 0000
Error 1409 18 0078
Total 37337 20
Appendix-IX Two way ANOVA for completely randomized design for growth and development of Z mauritiana in large size clay pot being irrigated with water of two different sea salt concentration
Variables Source Sum of Squares df Mean Square F-value P
Height of
Z mauritiana
Time 91030 2 45515 839 0000
Salinity treatment 3268 2 1634 10 0000
Time times Salinity treatment 1533 4 383 238 ns
Error 6751 42 161
Total 104554 71
Number of
branches of
Z mauritiana
Time 25525 2 127625 25333 0000
Salinity treatment 86333 2 43166 11038 0000
Time times Salinity treatment 27416 4 6854 1752 ns
Error 16425 42 3910
Total 6575 71
Number of
flowers of
Z mauritiana
Time 73506 2 36753 167777 0000
Salinity treatment 12133 2 6066 25061 0000
Time times Salinity treatment 27824 4 6956 28736 0000
Error 10166 42 242063
Total 127759 71
Fresh weight of
Shoot of
Z mauritiana
Time 3056862 2 1528431 340777 0000
Salinity treatment 107829 2 53914 12020 0000
Time times Salinity treatment 51303 4 12825 2859 0031
Error 251167 56 4485
Total 3515820 71
Dry weight of Shoot of
Z mauritiana
Time 784079 2 392039 338932 0000
Salinity treatment 26344 2 13172 11387 0000
Time times Salinity treatment 13042 4 3260 2818 0033
Error 64774 56 1156690
Total 913855 71
Succulence of
Z mauritiana
Time 0002 2 0001 0214 ns
Salinity treatment 0006 2 0003 0682 ns
Time times Salinity treatment 0007 4 0002 0406 ns
Error 0199 45 0004
Total 51705 54
Spacific shoot
length of Z mauritiana
Time 0000 2 914 0176 0000
Salinity treatment 0002 2 0001 2096 ns
Time times Salinity treatment 0003 4 0001 1445 ns
Error 0023 45 0001
Total 6413 54
Moisture
contents of Z mauritiana
Time 1264 2 0632 0243 ns
Salinity treatment 3603 2 1801 0691 ns
Time times Salinity treatment 4172 4 1043 0400 ns
Error 117146 45 2603
Total 131675 54
Relative growth
rate of Z mauritiana
Time 1584206 1 1584206 532968 ns
Salinity treatment 18921 2 9460 3183 ns
Time times Salinity treatment 61624 2 30812 10366 0000
Error 89172 30 2972
Total 4034 36
Appendix-X One way ANOVA for completely randomized design for growth and development of Z mauritiana in large size clay pot
being irrigated with water of two different sea salt concentration
Variables Source Sum of Squares df Mean Square F-value P
Chlorophyll a
of Z mauritiana
Salinity treatment 0004 2 0002 7546 0003
Error 0006 21 0000
Total 0010 23
Chlorophyll b of Z mauritiana
Salinity treatment 0037 2 0018 4892 0018
Error 0080 21 0003
Total 0117 23
171
Total
chlorophyll of
Z mauritiana
Salinity treatment 0144 2 0072 39317 0000
Error 0038 21 0002
Total 0182 23
Chlorophyll ab ratio of
Z mauritiana
Salinity treatment 1499 2 0749 33416 0000
Error 0471 21 0022
Total 1969 23
Total soluble
sugars of
Z mauritiana
Salinity treatment 378271 2 189135 36792 0000
Error 107952 21 5140
Total 486223 23
Total protein contents of
Z mauritiana
Salinity treatment 133006 2 66502 5861 0009
Error 238268 21 11346
Total 371274 23
Appendix-XI Three way ANOVA for split-split plot design for physiological investigations on growth of Z mauritiana and C cajan in
drum pot being irrigated with water of sea salt concentration at two irrigation intervals
Variables Source Sum of Squares df Mean Square F-value P
Height of
Z mauritiana
Time 4499 2 2249 28888 0004
Crop 448028 1 448028 2208 ns
Irrigation intervals 2523 1 2523 2774 ns
Time times Crop 928088 2 464044 2288 ns
Time times irrigation interval 1120400 2 560200 0615 ns
Crop times irrigation interval 2690151 1 2690 2957 ns
Time times Crop times irrigation interval 171927 2 85963 0094 ns
Error 10916 12 909732
Total 35
Canopy volume of Z mauritiana
Time 7943 2 3971 6554 ns
Crop 0382 1 0382 0579 ns
Irrigation intervals 0068 1 0069 0103 ns
Time times Crop 0265 2 0133 0201 ns
Time times irrigation interval 1142 2 0571 0852 ns
Crop times irrigation interval 0722 1 0722 1077 ns
Time times Crop times irrigation interval 1998 2 0999 1491 ns
Error 8043 12 0670
Total 29439 35
Appendix-XII Two way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C
cajan in drum pot being irrigated with water of sea salt concentration at two irrigation intervals
Variables Source Sum of Squares df Mean Square F-value P
Plant length of
Z mauritiana
Crop 2986 1 2986 75322 0000
Irrigation interval 2986 1 2986 75322 0000
Crop times Irrigation interval 15336 1 153367 3868 ns
Error 317166 8 39645
Total 292428 12
Shoot length of
Z mauritiana
Crop 1069741 1 1069741 30890 0000
Irrigation interval 1069741 1 1069741 30890 0000
Crop times Irrigation interval 253001 1 253001 73058 0026
Error 27704 8 3463
Total 103376 12
Root length of
Z mauritiana
Crop 19763 1 19763 2671 ns
Irrigation interval 481333 1 481333 65059 0000
Crop times Irrigation interval 800333 1 800333 108177 0000
Error 59186 8 7398
Total 49165 12
Main branches
of Z mauritiana
Crop 33333 1 33333 5797 0042
Irrigation interval 48 1 48 8347 0020
Crop times Irrigation interval 0333 1 0333 0057 ns
Error 46 8 575
Total 2888 12
Lateral
branches of Z mauritiana
Crop 1344083 1 1344083 41356 0000
Irrigation interval 54675 1 54675 16823 0000
Crop times Irrigation interval 784083 1 784083 24125 0000
Error 26 8 325
Total 22465 12
Leaf numbers of
Z mauritiana
Crop 22465 12 98283 96482 0000
Irrigation interval 25025 1 25025 24566 0001
Crop times Irrigation interval 11907 1 11907 11688 0009
Error 8149 8 1018667
172
Total 2037850 12
Shootroot ratio
of Z mauritiana
Crop 0027 1 0027 1842 ns
Irrigation interval 0001 1 0001 0097 ns
Crop times Irrigation interval 0825 1 0825 54909 0000
Error 0120 8 0015
Total 27776 12
Plant fresh
weight of Z mauritiana
Crop 398107 1 398107 577818 0000
Irrigation interval 139514 1 139514 20249 0000
Crop times Irrigation interval 146898 1 146898 21321 0000
Error 5511 8 688982
Total 7248659 12
Plant dry weight of Z mauritiana
Crop 87808 1 87808 471436 0000
Irrigation interval 57893 1 57893 31082 0000
Crop times Irrigation interval 61132 1 61132 32821 0000
Error 14900 8 186257
Total 1875710 12
Stem fresh
weight of
Z mauritiana
Crop 46687 1 46687 227539 0000
Irrigation interval 17933 1 17933 87402 0000
Crop times Irrigation interval 20180 1 20180 98351 0000
Error 16414 8 205185
Total 1718530 12
Root fresh weight of
Z mauritiana
Crop 58450 1 58450 2295 0000
Irrigation interval 42186 1 42186 165641 0000
Crop times Irrigation interval 37307 1 37307 146487 0000
Error 203746 8 25468
Total 357145 12
Leaf fresh weight of
Z mauritiana
Crop 29970 1 29970 19089 0000
Irrigation interval 117018 1 1170187 7453 0025
Crop times Irrigation interval 2310 1 2310 14714 0004
Error 125596 8 15699
Total 699711 12
Stem dry weight
of Z mauritiana
Crop 13587 1 13587 216591 0000
Irrigation interval 11856 1 11856 18899 0000
Crop times Irrigation interval 6787763 1 6787 108197 0000
Error 50188 8 62735
Total 4689795 12
Root dry weight
of Z mauritiana
Crop 1358787 1 13587 216591 0000
Irrigation interval 1497427 1 14974 118615 0000
Crop times Irrigation interval 128773 1 12877 1020052 0000
Error 100993 8 12624
Total 124421 12
Leaf dry weight
of Z mauritiana
Crop 2374 1 2374 135380 0000
Irrigation interval 8748 1 8748 4987 ns
Crop times Irrigation interval 26403 1 2640 150539 0000
Error 140313 8 17539
Total 127170 12
Plant moisture of Z mauritiana
Crop 22082 1 22082 5608 0045
Irrigation interval 38702 1 38702 9830 0013
Crop times Irrigation interval 44406 1 44406 11279 0009
Error 31496 8 3937
Total 29872 12
Stem moisture of Z mauritiana
Crop 0005 1 0005 0000 ns
Irrigation interval 110663 1 110663 12023 0008
Crop times Irrigation interval 0897 1 0897 0097 ns
Error 73633 8 9204
Total 28532 12
Root moisture of Z mauritiana
Crop 235266 1 235266 16502 0003
Irrigation interval 3923 1 3923 0275 ns
Crop times Irrigation interval 0856 1 0856 0060 ns
Error 114051 8 14256
Total 17572 12
Leaf moisture
of Z mauritiana
Crop 130413 1 130413 47746 0000
Irrigation interval 22256 1 22256 8148 0021
Crop times Irrigation interval 210662 1 210662 77127 0000
Error 21850 8 2731
Total 38888 12
173
Relative growth
rate of Z mauritiana
Crop 0000 1 0000 287467 0000
Irrigation interval 0000 1 0000 164217 0000
Crop times Irrigation interval 0000 1 0000 179626 0000
Error 0000 8 0000
Total 0009 12
Relative water
contents of Z
mauritiana
Crop 37381 1 37381 1380 ns
Irrigation interval 49871 1 49871 1841 ns
Crop times Irrigation interval 13496 1 13496 0498 ns
Error 216649 8 27081
Total 50855 12
Chlorophyll a of Z mauritiana
Crop 0103 1 0103 32466 0000
Irrigation interval 0003 1 0003 1075 ns
Crop times Irrigation interval 0000 1 0000 0187 ns
Error 0025 8 0003
Total 1498 12
Chlorophyll b
of Z mauritiana
Crop 0027 1 0027 196164 0000
Irrigation interval 0002 1 0002 15656 0004
Crop times Irrigation interval 0006 1 0006 45063 0000
Error 0001 8 0000
Total 0456 12
Total chlorophyll
of Z mauritiana
Crop 0257 1 0257 53469 0000
Irrigation interval 0001 1 0001 0315 ns
Crop times Irrigation interval 0002 1 0002 0442 ns
Error 0038 8 0004
Total 3736 12
Chlorophyll a b ratio of
Z mauritiana
Crop 0002 1 0002 0028 ns
Irrigation interval 0169 1 0169 1696 ns
Crop times Irrigation interval 1064 1 1064 10643 0011
Error 0799 8 0099
Total 43067 12
Carotenoids of
Z mauritiana
Crop 0018 1 0018 42747 0000
Irrigation interval 0002 1 0002 5298 0050
Crop times Irrigation interval 0003 1 0003 8118 0021
Error 0003 8 0000
Total 0451 12
Phenol of
Z mauritiana
Crop 24641 1 24641 13168 000
Irrigation interval 5078 1 5078 2714 ns
Crop times Irrigation interval 10339 1 10339 5525 0046
Error 14969 8 1871
Total 6289 12
Proline of Z mauritiana
Crop 0001 1 0001 52288 0000
Irrigation interval 0000 1 0000 6972 0029
Crop times Irrigation interval 0000 1 0000 0358 ns
Error 0000 8 0000
Total 0005 12
Protein of Z mauritiana
Crop 200001 1 200001 296 ns
Irrigation interval 69264 1 69264 102 ns
Crop times Irrigation interval 4453 1 4453 006 ns
Error 540367 8 67545
Total 814086 11
CAT enzyme of
Z mauritiana
Crop 74171 1 74171 11404 0009
Irrigation interval 299930 1 299930 46117 0000
Crop times Irrigation interval 15336 1 15336 2358 ns
Error 52029 8 65036
Total 441467 11
APX enzyme of
Z mauritiana
Crop 191918 1 191918 6693 0032
Irrigation interval 4665 1 4665 162723 0000
Crop times Irrigation interval 336912 1 336912 11750 0009
Error 229383 8 28672
Total 5423 11
GPX enzyme of
Z mauritiana
Crop 0000 1 0000 0020 ns
Irrigation interval 0103 1 0103 5893 0041
Crop times Irrigation interval 0109 1 0109 6220 0037
Error 0140 8 0017
Total 0353 11
SOD enzyme Crop 8471 1 8471 1364 ns
174
of
Z mauritiana
Irrigation interval 6220 1 6220 1001 ns
Crop times Irrigation interval 21142 1 21142 3405 ns
Error 49664 8 6208
Total 85498 11
NR enzyme of
Z mauritiana
Crop 7520 1 75208333333 37253364154 0003
Irrigation interval 1360 1 1360 6737 0318
Crop times Irrigation interval 0016 1 0016 0079 ns
Error 1615 8 0201
Total 10512 11
Nitrate of
Z mauritiana
Crop 003 1 003 3028 ns
Irrigation interval 0018 1 0018 1831 ns
Crop times Irrigation interval 0003 1 0003 0336 ns
Error 0079 8 0009
Total 0130 11
Appendix-XIII Three way ANOVA for split-split design for physiological investigations on growth of Z mauritiana and C cajan in drum
pot being irrigated with water of sea salt concentration at two irrigation intervals
Variables Source Sum of Squares df Mean Square F-value P
Height of
C cajan
Time 14990 2 7495 235059 0000
Crop 7848 1 7848 42235 0000
Irrigation intervals 749056 1 749056 9676 0009
Time times Crop 2638 2 1319140 7098 00262
Time times irrigation interval 309932 2 154966 2001 ns
Crop times irrigation interval 9127 1 9127 0117 ns
Time times Crop times irrigation interval 31974 2 15987 0206 ns
Error 928935 12 77411
Total 29065 35
Apendix-XIV Two way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C
cajan in drum pot being irrigated with water of sea salt concentration at two irrigation intervals
Variables Source Sum of Squares df Mean Square F-value P
Plant length of C cajan
Crop 1056563 1 1056563 12331 0007
Irrigation interval 21675 1 21675 2529 ns
Crop times Irrigation interval 137363 1 137363 1603 ns
Error 68544 8 8568
Total 334030 12
Shoot length of C cajan
Crop 808520 1 808520 36580 0000
Irrigation interval 165020 1 165020 7466 0025
Crop times Irrigation interval 285187 1 285187 12902 0007
Error 17682 8 22102
Total 224013 12
Root length of C cajan
Crop 16567 1 16567 0674 ns
Irrigation interval 3520 1 3520 0143 ns
Crop times Irrigation interval 26700 1 26700 1087 ns
Error 196453 8 24556
Total 11133 12
Main branches
of C cajan
Crop 80083 1 80083 64066 0000
Irrigation interval 10083 1 10083 8066 0021
Crop times Irrigation interval 075 1 075 06 ns
Error 10 8 125
Total 335 12
Letral branches
of C cajan
Crop 0 1 0
Irrigation interval 0 1 0
Crop times Irrigation interval 0 1 0
Error 0 8 0
Total 0 12
Leaf numbers
of C cajan
Crop 1776333 1 1776333 16679 0003
Irrigation interval 972 1 972 9126 0016
Crop times Irrigation interval 176333 1 17633 1655 0234
Error 852 8 1065
Total 22342 12
Shootroot ratio of C cajan
Crop 0385 1 0385 0638 0447
Irrigation interval 0007 1 0007 0011 0916
Crop times Irrigation interval 2669 1 2669 4424 0068
Error 4825 8 0603
Total 264061 12
Crop 76816 1 76816 7494853 0025
175
Plant fresh
weight of
C cajan
Irrigation interval 730236 1 730236 7124832 0028
Crop times Irrigation interval 266869 1 266869 2603812 0145
Error 81993 8 102491
Total 25941 12
Plant dry weight of C cajan
Crop 38270 1 38270 1150145 0009
Irrigation interval 53046 1 53046 15942 0003
Crop times Irrigation interval 20202 1 20202 6071 0039
Error 26619 8 3327
Total 4150 12
Stem fresh weight of
C cajan
Crop 16100 1 16100 1462 ns
Irrigation interval 9900 1 9900 0899 ns
Crop times Irrigation interval 00675 1 0067 0006 ns
Error 8806 8 11007
Total 3318 12
Root fresh weight of
C cajan
Crop 0190 1 0190 0248 ns
Irrigation interval 27331 1 27331 35753 0000
Crop times Irrigation interval 2698 1 2698 3529 0097
Error 6115 8 0764
Total 432050 12
Leaf fresh
weight of C cajan
Crop 541363 1 541363 13825 0005
Irrigation interval 347763 1 347763 8881 0017
Crop times Irrigation interval 208333 1 208333 5320 0049
Error 313246 8 39155
Total 7236 12
Stem dry weight
of C cajan
Crop 10323 1 10323 11530 0009
Irrigation interval 0452 1 0452 0505 ns
Crop times Irrigation interval 0232 1 0232 0259 ns
Error 7162 8 0895
Total 125151 12
Root dry weight
of C cajan
Crop 0007 1 0007 012 ns
Irrigation interval 0607 1 0607 972 0014
Crop times Irrigation interval 0367 1 0367 588 0041
Error 05 8 0062
Total 3515 12
Leaf dry weight
of C cajan
Crop 9363 1 9363 15649 0004
Irrigation interval 34003 1 3400 5683 0000
Crop times Irrigation interval 11603 1 11603 19392 0002
Error 4786 8 0598
Total 95072 12
Plant moisture of C cajan
Crop 199182 1 19918 6011 0039
Irrigation interval 272215 1 27221 8215 0020
Crop times Irrigation interval 76654 1 76654 2313 0166755
Error 265079 8 33134
Total 38272 12
Stem moisture
of C cajan
Crop 100814 1 10081 3290 0107246
Irrigation interval 53460 1 53460 1744 0223065
Crop times Irrigation interval 19778 1 1977 0645 0444938
Error 245119 8 30639
Total 31036 12
Root moisture
of C cajan
Crop 26266 1 26266 1389 ns
Irrigation interval 223809 1 223809 11836 0008
Crop times Irrigation interval 0097 1 0097 0005 ns
Error 151272 8 18909
Total 58346 12
Leaf moisture
of C cajan
Crop 2623 1 2623 39350 0000
Irrigation interval 1765 1 1765 26477 0000
Crop times Irrigation interval 1425 1 1425452 21378 0001
Error 533411 8 66676
Total 36263 12
Relative growth
rate of C cajan
Crop 0000 1 0000 17924 0002
Irrigation interval 0000 1 0000 21296 0001
Crop times Irrigation interval 0000 1 0000 88141 0017
Error 0000 8 0000
Total
Crop 256935 1 256935 1560 ns
Irrigation interval 268827 1 26882 1633 ns
176
Electrolyte
leakage of C
cajan
Crop times Irrigation interval 30379 1 30379 0184 ns
Error 1316923 8 16461
Total 50381 12
Chlorophyll a
of C cajan
Crop 0101 1 0101 7957 0022
Irrigation interval 0062 1 0062 4893 ns
Crop times Irrigation interval 0199 1 0199 15600 0004
Error 0102 8 0012
Total 5060 12
Chlorophyll b
of C cajan
Crop 0017 1 0017 7758 0023
Irrigation interval 0027 1 0027 12389 0007
Crop times Irrigation interval 0056 1 0056 25313 0001
Error 0017 8 0002
Total 1727 12
Total
chlorophyll of C cajan
Crop 0178 1 0178 14819 0004
Irrigation interval 0198 1 0198 16520 0003
Crop times Irrigation interval 0509 1 0509 42379 0000
Error 0096 8 0012
Total 13217 12
Chlorophyll a b
ratio of C cajan
Crop 0065 1 0065 0691 ns
Irrigation interval 0033 1 0033 0357 ns
Crop times Irrigation interval 0016 1 0016 0173 ns
Error 0756 8 0094
Total 35143 12
Carotenoids of C cajan
Crop 0021 1 0021 19599 0002
Irrigation interval 0028 1 0028 26616 0000
Crop times Irrigation interval 0041 1 0041 38531 0000
Error 0008 8 0001
Total 1443 12
Phenol of C cajan
Crop 0799 1 0799 3171 ns
Irrigation interval 0040 1 0040 0159 ns
Crop times Irrigation interval 0911 1 0911 3617 ns
Error 2016 8 0252
Total 970313 12
Proline of C cajan
Crop 0008 1 0008 14867 0004
Irrigation interval 0019 1 0019 34536 0000
Crop times Irrigation interval 0008 1 0008 14969 0004
Error 0004 8 0000
Total 0155 12
Protein of C
cajan
Crop 116376 1 116376 3990 ns
Irrigation interval 434523 1 434524 14899 0048
Crop times Irrigation interval 33166 1 33166 1137 ns
Error 233303 8 29163
Total 817371 11
CAT enzyme
of C cajan
Crop 0249 1 0249 0121 ns
Irrigation interval 2803 1 2803 13702 ns
Crop times Irrigation interval 92392 1 9239 4517 ns
Error 16362 8 2045
Total 28654 11
APX enzyme
of C cajan
Crop 855939 1 855939 4073 ns
Irrigation interval 1078226 1 1078226 5130 ns
Crop times Irrigation interval 13522 1 13522 64349 000
Error 1681112 8 210139
Total 17137 11
GPX enzyme
of C cajan
Crop 0965 1 0965 9265 0160
Irrigation interval 1167 1 1167 11195 0101
Crop times Irrigation interval 0887 1 0887 8514 0194
Error 0833 8 0104
Total 3854 11
SOD enzyme
of C cajan
Crop 4125 1 4125 9731 0142
Irrigation interval 4865 1 4865 11477 0095
Crop times Irrigation interval 20421 1 20421 48172 0001
Error 3391 8 0423
Total 32804 11
Nitrate
reductase
enzyme
Crop 0053 1 0053 0034 ns
Irrigation interval 0001 1 0001 0000 ns
Crop times Irrigation interval 10329 1 10329 6650 0327
177
of C cajan Error 12424 8 1553
Total 22808 11
Nitrate of
C cajan
Crop 0039 1 0039 0576 ns
Irrigation interval 0083 1 0083 1222 ns
Crop times Irrigation interval 0003 1 0003 0005 ns
Error 0545 8 0068
Total 0668 11
Appendix-XV Two way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C
cajan intercropped on marginal land under field condition
Variables Source Sum of Squares df Mean Square F-value P
Height of Z mauritiana
Time 79704 3 26568 77303 0000
Treatment 979209 1 979209 4702 0455
Time times Treatment 756019 3 252006 1210 3381 ns
Error 3332 16 208259
Total 90366 39
Canopy volume of Z mauritiana
Time 1049 3 3498 115444 0000
Treatment 3509 1 3509 5966 0266
Time times Treatment 3374 3 1124 1911 1684 ns
Error 9413 16 5883
Total 1284 39
flowers numbers of Z
mauritiana
Time 1794893 3 598297 770043 0000
Treatment 19980 1 19980 10152 0057
Time times Treatment 21017 3 7005 3559 0381
Error 31488 16 1968
Total 1882468 39
Fruits numbers
of Z mauritiana
Time 324096 3 108032 297941 0000
Treatment 10824 1 10824 64081 0000
Time times Treatment 7141 3 2380 14093 0001
Error 2702 16 168913
Total 351833 39
Appendix-XVI One way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C cajan intercropped on marginal land under field condition
Variables Source Sum of Squares df Mean Square F-value P
Weight of ten
fruits (FW) of
Z mauritiana
Treatment 557113 1 557113 6663 0032
Error 668923 8 83615
Total 1226036 9
Weight of ten fruits (DW) of
Z mauritiana
Treatment 4356 1 4356 0321 ns
Error 10862 8 13577
Total 112976 9
diameter of fruit of Zmauritiana
Treatment 0534 1 0534 0946 ns
Error 4514 8 0564
Total 5048 9
Fruit weight per plant of
Z mauritiana
Treatment 0739 1 0739 4022 ns
Error 1471 8 0184
Total 2211 9
Fruit sugar
(soluble) of
Z mauritiana
Treatment 5041 1 5041 0081 ns
Error 497328 8 62166
Total 502369 9
Fruit sugar (extractable) of
Z mauritiana
Treatment 32041 1 32041 0424 ns
Error 604384 8 75548
Total 636425 9
Total fruit
sugars of Z mauritiana
Treatment 16 1 16 0780 ns
Error 164 8 205
Total 18 9
Chlorophyll a of
Z mauritiana
Treatment 0082 1 0082 1384 0020
Error 0024 4 0006
Total 0105 5
Chlorophyll b
of Z mauritiana
Treatment 0011 1 0011 8469 0043
Error 0005 4 0001
Total 0016 5
Total chlorophyll of
Z mauritiana
Treatment 0152 1 0152 11927 0025
Error 0051 4 0013
Total 0203 5
Treatment 0015 1 0015 0867 ns
Error 0067 4 0017
178
Chlorophyll a b
ratio of Z mauritiana
Total 0082 5
Carotinoids of Z mauritiana
Treatment 0011 1 0011 9719 0035
Error 0004 4 0001
Total 0015 5
Leaf protein of
Z mauritiana
Treatment 0106 1 0106 4 ns
Error 0106 4 0027
Total 0213 5
Leaf sugars
(soluble) of
Z mauritiana
Treatment 054 1 054 0025 ns
Error 848 4 212
Total 8534 5
Leaf sugars
(Extractable) of Z mauritiana
Treatment 486 1 486 8055 0046
Error 2413 4 0603
Total 7273 5
Total sugars in
leaf of Z
mauritiana
Treatment 216 1 216 0104 ns
Error 83333 4 20833
Total 85493 5
Leaf phenols of
Z mauritiana
Treatment 8166 1 8166 5665 ns
Error 5766 4 1442
Total 13933 5
Leaf nitrogen of Z mauritiana
Treatment 15 1 15 1939 ns
Error 3093 4 0773333
Total 4593 5
Soil nitrogen of
Z mauritiana
Treatment 0375 1 0375 21634 ns
Error 0693 4 0173
Total 1069 5
Appendix-XVII Two way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C
cajan intercropped on marginal land under field condition
Variables Source Sum of Squares df Mean Square F-value P
Height of Ccajan
Time 700196 2 350098 2716 0000
Treatment 594405 1 594405 16017 0000
Time times Treatment 488829 2 244415 6586 0004
Error 1001996 27 37111
Total 705495 59
Number of branches of
Ccajan
Time 8353 2 4176 1050050 0000
Treatment 24066 1 24066 18672 0000
Time times Treatment 24133 2 12066 9362 0000
Error 348 27 1288
Total 8572 59
Number of flowers of
Ccajan
Time 289297 2 144648 301277 0000
Treatment 365066 1 365066 0701 ns
Time times Treatment 730133 2 365066 0701 ns
Error 14059 27 520733
Total 317415 59
Number of pods
of Ccajan
Time 347682 2 173841 70559 0000
Treatment 159135 1 159135 1558 ns
Time times Treatment 8167 2 40835 0399 ns
Error 27574 27 1021276
Total 447407 59
Appendix-XVIII One way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C
cajan intercropped on marginal land under field condition
Variables Source Sum of Squares df Mean Square F-value P
Shoot weight
(FW) of
Ccajan
Treatment 0 1 0 0 ns
Error 87444 4 21861
Total 87444 5
Shoot weight
(RW) of Ccajan
Treatment 0 1 0 0 ns
Error 13808 4 3452
Total 13808 5
Number of
seeds of
Ccajan
Treatment 245 1 245 0005 ns
Error 940182 18 52232
Total 940427 19
Weight of seeds
of Ccajan
Treatment 02 1 02 0000 ns
Error 7585 18 421406
Total 7585 19
179
Chlorophyll a of
Ccajan
Treatment 0001 1 0001 5442 ns
Error 0001 4 0000
Total 0002 5
Chlorophyll b
of Ccajan
Treatment 0006 1 0006 9079 0039
Error 0002 4 0001
Total 0008 5
Total
chlorophyll of
Ccajan
Treatment 0017 1 0017 51558 0001
Error 0001 4 0000
Total 0019 5
Chlorophyll a b ratio of
Ccajan
Treatment 0183 1 0183 5532 ns
Error 0132 4 0033
Total 0316 5
Leaf protein of Ccajan
Treatment 0001 1 0001 0017 ns
Error 0228 4 0057
Total 0228 5
Leaf sugars of
Ccajan
Treatment 0015 1 0015 0003 ns
Error 1624 4 406
Total 16255 5
Leaf phenols of
Ccajan
Treatment 0201 1 0201 0140 ns
Error 5746 4 1436
Total 5948 5
Leaf nitrogen
of Ccajan
Treatment 1306 1 1306 3062 ns
Error 1706 4 04266
Total 3013 5
Appendix-XIX Two way ANOVA for completely randomized design for investigations on determining range of salt tolerance in Carissa
carandas
Variables Source Sum of Squares df Mean Square F-value P
Height of C carandas
Time 72042 5 14408 55957 0000
Salinity treatment 49345 2 24672 196775 0000
Time times Salinity treatment 16679 10 1667920 13302 000
Error 3009 24 125385
Total 143777 53
Volume of
canopy of
C carandas
Time 3329 4 0832 38126 000
Salinity treatment 1393 2 0696 67129 000
Time times Salinity treatment 0813 8 0102 9792 000
Error 0207 20 0010
Total 5969 44
Appendix-XX One way ANOVA for completely randomized design for investigations on determining range of salt tolerance in Carissa carandas
Variables Source Sum of Squares df Mean Square F-value P
Number of
flowers of C carandas
Salinity treatment 10288 2 5144194 1342937 0000
Error 229833 6 38305
Total 10518 8
Number of fruits of
C carandas
Salinity treatment 18000 2 9000 268215 0000
Error 201333 6 33555
Total 18201 8
Flower shedding
percentage of C carandas
Salinity treatment 1541647 2 770823 53455 0000
Error 86519 6 144199
Total 1628166 8
Weight of ten fruits (FW) of
C carandas
Salinity treatment 82632 2 41316 187678 0000
Error 1321 6 0220
Total 83953 8
Weight of ten
fruits (DW) of
C carandas
Salinity treatment 4355 2 2177 13753 0005
Error 095 6 0158
Total 5305 8
Fruits per plant
(FW) of
C carandas
Salinity treatment 133127 2 66563 278148 0000
Error 1435861 6 239310
Total 134563 8
Fruits per plant
(DW) of C carandas
Salinity treatment 8782 2 439117 117790 0000
Error 223677 6 37279
Total 9006 8
Size of fruits of C carandas
Salinity treatment 1301 2 0651 4125 ns
Error 0946 6 0158
Total 2248 8
Salinity treatment 5607 2 2804 17592 0003
180
Diameter of fruit
of C carandas
Error 0956 6 0159
Total 6563 8
Chlorophyll a of C carandas
Salinity treatment 0112 2 0056 119786 0000
Error 0003 6 0000
Total 0115 8
Chlorophyll b of
C carandas
Salinity treatment 0005 2 0002 434 0000
Error 0000 6 0000
Total 0005 8
Total chlorophyll of C carandas
Salinity treatment 0159 2 0079 104188 0000
Error 0005 6 0001
Total 0164 8
Chlorophyll a b
ratio of C carandas
Salinity treatment 9661 2 4831 324691 0000
Error 0089 6 0015
Total 9751 8
Carotenoids of C carandas
Salinity treatment 0029 2 0014 28822 0000
Error 0003 6 0001
Total 0032 8
Leaf Protein of
C carandas
Salinity treatment 2722 2 1361 98 0012
Error 0833 6 0138
Total 3555 8
Soluble sugar of
C carandas
Salinity treatment 234889 2 117444 12735 0006
Error 55333 6 9222
Total 290222 8
In soluble sugars
of C carandas
Salinity treatment 595395 2 297698 39094 0000
Error 45689 6 7615
Total 641085 8
Total sugar of
C carandas
Salinity treatment 1576898 2 788448 39201 0000
Error 120676 6 20113
Total 1697574 8
Phenols of C carandas
Salinity treatment 14675 2 7338 74202 0000
Error 0593 6 0099
Total 15268 8
Leaf Na+ of
C carandas
Salinity treatment 1346 2 673 673 0000
Error 6 6 1
Total 1352 8
Leaf K+ of C carandas
Salinity treatment 798 2 399 133 0000
Error 18 6 3
Total 816 8
Leaf K+ Na+
ratio of C carandas
Salinity treatment 0305 2 0153 654333 0000
Error 0001 6 0000
Total 0307 8
181
7 Publications
iii
Investigation on intercropping of Ziziphus mauritiana with Cajanus
cajan for fruit and fodder at marginal land and cultivation of Carissa
carandas for fruits through saline water irrigation
Thesis Approved
RESEARCH SUPERVISOR EXTERNAL EXAMINER
PROF DR RAFIQ AHMAD
FPAS FTWAS
Professor (Retd) Botany (Plant Physiology)
PI Biosaline Research Projects
Department of Botany
University of Karachi
iv
CERTIFICATE
It is hereby certified that this thesis is based on the results of the experimental work carried
out by Mr TAYYAB SO MUHAMMAD HANIF under my supervision on the topic
ldquoInvestigation on intercropping of Ziziphus mauritiana with Cajanus cajan for fruit
and fodder at marginal land and cultivation of Carissa carandas for fruits through
saline water irrigationrdquo
Mr TAYYAB had been enrolled under my guidance for the award of PhD in
Department of Botany University of Karachi I have personally checked all the research
work reported in the thesis and certify its accuracyvalidity It is further certified that the
materials included in this thesis have not been used partially or fully in a manuscript
already submitted or in the process of submission in partialcomplete fulfillment for award
of any other degree from any other university Mr TAYYAB has fulfilled requirements of
the University of Karachi for the submission of this dissertation and I endorse its
evaluation for the award of PhD Degree
RESEARCH SUPERVISOR
PROF DR RAFIQ AHMAD
FPAS FTWAS
Professor (Retd) Botany (Plant Physiology)
PI Biosaline Research Projects
Department of Botany
University of Karachi
Karachi-75270 Pakistan
v
DEDICATED TO MY FAMILY
MUHAMMAD HANIF (MY FATHER)
MRS ARIFA (LATE)
(MY BELOVED MOTHER)
SHAHEEN TAYYAB (MY WIFE)
vi
ACKNOWLEDGMENTS
All the praises for almighty Allah and all respects for Prophet Muhammad (Peace be Upon
Him) who has shown me the straight path
I am grateful to my supervisor Prof Dr Rafiq Ahmad for his keen interest
patronage and guidance during this research work which made successful submission of
this thesis
I also obliged to Prof Dr Ehtesham Ul Haque and Prof Dr Javed Zaki (Present
and Former Chairmen Department of Botany respectively) for providing me all the
necessary facilities and administrative support
Being employed as lecturer in Department of Botany Govt Islamia Science
College Karachi I am also thankful to Education and literacy Department Govt of Sindh
(Pakistan) for providing me facilities to perform this study
Thanks are due to Dr D Khan in assessing statistical data analysis and colleague
of Biosaline lab Dr M Azeem Dr Naeem Ahmed and M Wajahat Ali Khan for their
cooperation throughout the course of study
I am also gratefully acknowledged to Mr Noushad Raheem and Mr Noor Uddin
of Fiesta Water Park for providing field plot and facilities to perform this study I am also
thankful to Pakistan Metrological Department for providing environmental data
I am also obliged to Dr M Qasim and Dr M Waseem Abbasi for their suggestions
and support in writing this thesis
Assistance of Abbul Hassan (Lab attendant) Tajwar Khan (Biosaline field
Attendant) and Mr Wahid (Plant Physiology Lab Assistant) is also acknowledged
Thanks are also due to my friends Dr Rafat Saeed Dr Kabir Ahmad Dr Zia Ur
Rehman Farooqi Dr Noor Dr M Yousuf Adnan Asif Bashir Dr A Rauf A Hai Faiz
Ahmed MA Rasheed Jallal Uddin Saadi Ahsan Shaikh Saima Fehmi A Mubeen
Khan Dr Noor Ul Haq Saima Ahmad S Safder Raza SM Akber and my college
colleagues for giving me encouragement during this research work
vii
I can never forget the support and encouragement and good wishes of Mr M
Wilayat Ali Khan Mrs Shahnaz Rukhsana Mr Mansoor Mrs Rabia Mansoor Mrs
Chand Bibi and Mrs Saeeda Anwar
In the last I am highly grateful to my beloved father Muhammad Hanif my loving
mother Arifa (when she alive) my caring wife Shaheen and sweet childrenrsquos Sara and
Sarim my supportive brothers and sisters and all family members for their prayers love
sacrifices and encouragements provided during course of this research work
viii
TABLE OF CONTENTS
No Title Page no
Acknowledgement vi
Summary xix
Urdu translation of summary xxi
General introduction 1
Layout of thesis 11
1 Chapter 1 13
11 Introduction 13
12 Experiment No 1 15
121 Materials and methods 15
1211 Seed collection 15
1212 Experimental Design 15
122 Observations and Results 17
13 Experiment No 2 22
131 Materials and methods 22
1311 Seed germination 22
132 Observations and Results 23
14 Experiment No 3 28
141 Materials and methods 28
1411 Seedling establishment 28
142 Observations and Results 29
1421 Seedling establishment 29
1422 Shoot height 29
15 Experiment No 4 31
151 Materials and methods 31
1511 Drum pot culture 31
1512 Experimental design 31
1513 Vegetative and Reproductive growth 32
1514 Analysis on some biochemical parameters 32
152 Observations and Results 34
1521 Vegetative and Reproductive growth 34
ix
No Title Page no
1522 Study on some biochemical parameters 34
16 Experiment No 5 41
161 Materials and methods 41
1611 Isolation Identification and purification of bacteria 41
1612 Preparation of bacterial cell suspension 41
1613 Study of salt tolerance of Rhizobium isolated from root
nodules of C cajan
41
162 Observations and Results 42
17 Experiment No 6 44
171 Materials and methods 44
1711 Experimental design 44
1712 Vegetative and reproductive growth 45
1713 Analysis on some biochemical parameters 45
172 Observations and Results 46
1721 Vegetative and Reproductive growth 46
1722 Study on some biochemical parameters 46
18 Discussion (Chapter 1) 51
2 Chapter 2 59
21 Introduction 59
22 Experiment No 7 60
221 Materials and Methods 60
2211 Growth and Development 60
2212 Drum pot culture 60
2213 Experimental Design 60
2214 Irrigation Intervals 61
2215 Estimation of Nitrate content 62
2216 Relative Water content (RWC) 62
2217 Electrolyte leakage percentage (EL) 62
2218 Photosynthetic pigments 63
2219 Total soluble sugars 63
22110 Proline content 63
22111 Soluble phenols 64
x
No Title Page no
22112 Total soluble proteins 64
22113 Enzymes Assay 64
222 Observations and Results 67
2221 Vegetative growth 67
2222 Photosynthetic pigments 70
2223 Electrolyte leakage percentage (EL) 70
2224 Phenols 70
2225 Proline 71
2226 Protein and sugars 71
2227 Enzyme essays 71
2228 Vegetative growth 73
2229 Photosynthetic pigments 75
22210 Electrolyte leakage percentage (EL) 76
22211 Phenols 76
22212 Proline 77
22213 Protein and Sugars 77
22214 Enzyme assay 77
23 Experiment No8 90
231 Materials and Methods 90
2311 Selection of plants 90
2312 Experimental field 90
2313 Soil analysis 90
2314 Experimental design 91
2315 Vegetative and reproductive growth 93
2316 Analysis on some biochemical parameters 93
2317 Fruit analysis 94
2318 Nitrogen estimation 94
2319 Land equivalent ratio and Land equivalent coefficient 95
23110 Statistical analysis 95
232 Observations and Results 96
2321 Vegetative parameters 96
2322 Reproductive parameters 96
xi
No Title Page no
2323 Study on some biochemical parameters 97
2324 Nitrogen Contents 98
2325 Land equivalent ratio land equivalent coefficient 98
24 Discussion (Chapter 2) 108
3 Chapter 3 113
31 Introduction 113
32 Experiment No 9 114
321 Materials and methods 114
3211 Drum Pot Culture 114
3212 Plant material 114
3213 Experimental setup 114
3214 Vegetative parameters 115
3215 Analysis on some biochemical parameters 115
3216 Mineral Analysis 116
322 Observations and Result 117
3221 Vegetative parameters 117
3222 Reproductive parameters 117
3223 Study on some biochemical parameters 118
3224 Mineral analysis 118
33 Discussion (Chapter 3) 127
4 Conclusion 129
5 References 130
6 Appendices 168
7 Publications 181
xii
LIST OF FIGURES
Figure Title Page no
11 Effect of irrigation water of different sea salt solutions on seed
germination indices of C cajan
27
12 Effect of irrigating water of different sea salt solutions on
seedling emergence (A) and shoot length (B) of C cajan
30
13 Environmental data of study area during experimental period
(July-November 2009)
36
14 Effect of salinity using irrigation water of different sea salt
concentrations on height of C cajan during 18 weeks treatment
36
15 Effect of salinity using irrigation water of different sea salt
concentrations on initial and final biomass (fresh and dry) of C
cajan
37
16 Percent change in moisture succulence relative growth rate
(RGR) and specific shoot length (SSL) of C cajan under
increasing salinity using irrigating water of different sea salt
concentrations
37
17 Effect of irrigating water of different sea salt solutions on
reproductive growth parameters including number of flowers
pod seeds and seed weight of C cajan
38
18 Effect of irrigating water of different sea salt solutions on leaf
pigments including chlorophyll a chlorophyll b total
chlorophyll and carotenoids of C cajan
39
19 Effect of irrigating water of different sea salt solutions on total
proteins soluble insoluble and total sugars in leaves of C cajan
40
110 Growth of nitrogen fixing bacteria associated with root of C
cajan under different NaCl concentrations
42
111 Photographs showing growth of Rhizobium isolated from the
nodules of C cajan in vitro on YEM agar supplemented with
different concentrations of NaCl
43
xiii
Figure Title Page no
112 Effect of salinity using irrigation water of different sea salt
concentrations on height number of branches fresh weight and
dry weight of shoot of Z mauritiana after 60 and 120 days of
treatment
47
113 Effect of salinity using irrigation water of different sea salt
concentrations on succulence specific shoot length (SSL)
moisture and relative growth rate (RGR) of Z mauritiana
48
114 Effect of salinity using irrigation water of different sea salt
concentrations on number of flowers of Z mauritiana
49
115 Effect of salinity using irrigation water of different sea salt
concentrations on leaf pigments including chlorophyll a
chlorophyll b total chlorophyll and chlorophyll ab ratio of Z
mauritiana
49
116 Effect of salinity using irrigation water of different sea salt
concentrations on total sugars and protein in leaves of Z
mauritiana
50
21 Vegetative parameters of Z mauritiana and C cajan at grand
period of growth under sole and intercropping system at two
irrigation intervals
79
22 Fresh and dry weight of Z mauritiana and C cajan plants under
sole and intercropping system at 4th and 8th day irrigation
intervals
80
23 Leaf weight ratio (LWR) root weight ratio (RWR) shoot weight
ratio (SWR)specific shoot length (SSL) specific root length
(SRL) plant moisture Succulence and relative growth rate
(RGR) of Z mauritiana and C cajan grow plants under sole and
intercropping system at 4th and 8th day irrigation intervals
81
24 Leaf pigments of Z mauritiana and C cajan grow plants under
sole and intercropping system at 4th and 8th day irrigation
intervals
83
xiv
Figure Title Page no
25 Electrolyte leakage phenols and proline of Z mauritiana and C
cajan at grand period of growth plants under sole and
intercropping system at 4th and 8th day irrigation intervals
84
26 Total protein in leaves of Z mauritiana and C cajan plants
under sole and intercropping system at 4th and 8th day irrigation
intervals
86
27 Enzymes activities in leaves of Z mauritiana and C cajan plants
under sole and intercropping system at 4th and 8th day irrigation
intervals
87
28 Nitrate reductase activity and nitrate concentration in leaves of
Z mauritiana and C cajan plants under sole and intercropping
system at 4th and 8th day irrigation intervals
89
29 Soil texture triangle (Source USDA soil classification) 99
210 Vegetative growth of Z mauritiana and C cajan growing under
sole and intercropping system
100
211 Reproductive growth of Z mauritiana and C cajan growing
under sole and intercropping system
101
212 Leaf pigments of Z mauritiana and C cajan growing under sole
and intercropping
102
213 Sugars protein and phenols in leaves of Z mauritiana and C
cajan at grand period of growth under sole and intercropping
system
103
214 Sugars protein and phenols in fruits of Z mauritiana grown
under sole and intercropping system
105
215 Nitrogen in leaves and in soil of Z mauritiana and C cajan
growing under sole and intercrop system
106
31 Chlorophyll a chlorophyll b total chlorophyll chlorophyll a b
ratio carotenoids contents of C carandas growing under
salinities created by irrigation of different dilutions of sea salt
124
xv
Figure Title Page no
32 Total protein sugars and phenolic contents of C carandas
growing under salinities created by irrigation of different
dilutions of sea salt
125
33 Mineral analysis including Na and K ions was done on leaves of
C carandas growing under salinities created by irrigation of
different dilutions of sea salt
126
xvi
LIST OF TABLES
Table Title Page no
11 Electrical conductivities of different sea salt solutions
used in germination of C cajan
18
12 Effect of irrigation water of different sea salt solutions
on germination percentage (GP) per day of C cajan
seeds pre-soaked in non-saline water prior to
germination with duration of time under various salinity
regimes
19
13 Effect of irrigation water of different sea salt solutions
on germination rate (GR) per day of seeds C cajan pre-
soaked in non-saline water prior to germination with
duration of time under various salinity regimes
20
14 Effect of irrigation water of different sea salt solutions
on mean germination rate (GR) coefficient of
germination velocity (GV) mean germination time
(GT) mean germination index (GI) and final
germination (FG) of C cajan seeds pre-soaked in non-
saline water prior to germination under various salinity
regimes
21
15 Electrical conductivities of different sea salt solutions
used in germination of C cajan
24
16 Effect of irrigation water of different sea salt solutions
on germination percentage (GP) per day of C cajan
seeds pre-soaked in respective sea salt concentrations
with duration of time
25
17 Effect of irrigation water of different sea salt solutions
on germination rate (GR) per day of C cajan seeds pre-
soaked in respective sea salt concentrations with
duration of time
26
xvii
Table Title Page no
18 Electrical conductivities of different Sea salt
concentrations and ECe of soil saturated paste at the end
of experiment
30
21 Soil analysis data of Fiesta Water Park experimental
field
99
22 Land equivalent ratio (LER) and Land equivalent
coefficient (LEC) with reference to height chlorophyll
and yield of Z mauritiana and C cajan growing under
sole and intercropping system
107
31 Electrical conductivities of different sea salt
concentration used for determining their effect on
growth of C carandas
119
32 Vegetative growth in terms of height and volume of
canopy of C carandas growing under salinities created
by irrigation of different dilutions of sea salt
120
33 Vegetative growth in terms of height and volume of
canopy of C carandas growing under salinities created
by irrigation of different dilutions of sea salt
121
34 Reproductive growth in terms of flowers and fruits
numbers flower shedding percentage fresh and dry
weight of ten fruit and their totals per plant fruit length
and diameter of C carandas growing under salinities
created by irrigation of different dilutions of sea salt
123
xviii
LIST OF ABBREVIATIONS
APX Ascorbate peroxidase
CAT Catalase
DAP Diammonium Phosphate (fertilizer)
dSm-1 Deci Siemens per meter
ECe Electrical conductivity of the Soil saturated extract
ECiw Electrical conductivity of the irrigation water
GPX Guaiacol Peroxidase
GR Glutathione reductase
GSH Reduced glutathione
LEC Land equivalent coefficient
LER Land equivalent ratio
NPK Nitrogen Phosphate Potash (fertilizer)
NR Nitrate reductase
RGR Relative growth rate
ROS Reactive oxygen species
RWR Root weight ratio
SOD Superoxide dismutase
SRL Specific Root Length
SSL Specific Shoot Length
SWR Shoot weight ratio
xix
Summary
Salinity is a growing threat to crop production which affects sustainability of agriculture
in aridsemiarid areas Growth responses of plant to salinity vary considerably among
species Cajanus cajan Ziziphus mauritiana and Carissa carandas are sub-tropical crops
grown worldwide particularly in Asian subcontinent for edible and fodder purposes but
not much is known about their salinity tolerance and intercropping
Effect of salinity has been initially studied in present work at germination of C cajan
under different sea salt salinities using presoaked seeds with water and respective salt
solutions Seed germination decreased with increasing salinity and it was more sever in
presoaking under water of different salinities The 50 threshold reduction started at
ECiw= 35 dSm-1 sea salt in presoaking treatments However this threshold was decreased
up to ECiw= 168 dSm-1 sea salt at further seedling establishment stage Growth experiment
of C cajan in drum pot culture (Lysimeter) also showed a salt induced growth reduction
in which plant tolerate salinity up to 42 dSm-1 At this salinity leaf pigments (chlorophylls
and carotenoids) proteins and insoluble sugars decreased up to 50 whereas soluble
sugars were increased (~25) Reproductive growth was also affected at this salinity in
which at least 70 reduction in flowers pods and seeds were observed
Salt tolerance of symbiotic nitrogen fixing bacteria associated with root of C cajan
showed salinity tolerance up to ECw= 366 dSm-1 NaCl salinity invitro environment For
intercropping experiments Ziziphus mauritiana (grafted variety) was selected with C
cajan Preliminary investigations showed a growth promotion in Z mauritiana at low
salinity (ECe= 72 dSm-1) and growth was remained unaffected up to ECe= 111 dSm-1
Intercropping of C cajan with Z mauritiana was primarily done in drum pot (Lysimeter)
culture Result showed better growth responses of both species when growing together as
intercrops than sole in which encouraging results were found in 8th day irrigation interval
rather than of 4th day Biochemical parameters eg photosynthetic pigments protein
phenols electrolyte leakage and sugars of these species displayed increase or decrease
according to their growth responses Increased activity of antioxidant enzymes and that of
nitrate reductase and its substrate (NO3) also contributed in enhancement of growth
Field experiment of intercropping of above mentioned plants at marginal land
irrigated with underground water (Eciw= 28 dSm-1) showed better vegetative growth of
xx
both species than sole crop The overall reproductive growth remained unaffected
although the numbers size and weight of fruit were better in intercropping system
Photosynthetic pigments were mostly increased whereas leaf protein and sugars remained
unchanged In addition higher values of LER and LEC (gt 1) indicated the success of
intercropping system
Experiment on salinity tolerance of Carissa carandas (varn karonda) using drum
pots culture showed improvement at low salinity (up to ECiw= 42 dSm-1 sea salt) whereas
higher salinity (ECiw= 129 dSm-1 sea salt) adversely affected vegetative and reproductive
growth Plant managed to tolerate up to ECiw= 99 dSm-1 sea salt Salinity severely affected
biochemical parameters including photosynthetic pigments proteins and sugars whereas
leaf phenolics were increased Leaf accumulated high amount of Na+ whereas affect
absorption of essential minerals like K+ was decreased
In the light of above mentioned investigations it appears that C cajan can be
propagated in saline soils with good presoaking techniques in non-saline water which
would helped to grow at moderately saline conditions It could be a good option used as
intercrop species because of its ability to improve soil fertility even under water deficit
conditions The proposed Cajanus-Ziziphus intercropping system could help poor farmers
to generate income from unproductive soils by obtaining sufficient fodder from C cajan
for their cattle and producing delicious edible fruits from Z mauritiana for commercial
purposes Carissa carandas could also be introduced as new crop for producing fruits from
moderate saline waste lands and used for preparing prickle jam and jelly for industrial
purposes
xxi
لاصہ خ
کا عمل ے ں ب ڑھئ لف پ ودوں می ی ےمخ طرہ ہ
وا خ ا ہ ے ب ڑھی لئ داوار کے ی ں زرعی ب وں می
ر علاق ج
ن ی م ب
ر و ب ج ن کھاری پ ن کھاری پ ن ب
دا کروت ی ر اور ر ب ے ارہ ا ہ وت لف ہ ی ی مخ کاف ں ودگی می اص Subtropical کی موج ا اور خ ی و پ وری دب ں ج ی ں ہ صلی
کی ف طے
خ
وراک و ں ج می
ی ملکوں
ائ ی ش کھاکر ای کی ی ان پ ودوں کم لوگ ہ ہت کن ب ں لی ی ی ہ
وئ عمال ہ
ارے کے طور ب ر است ری پ ن سے خ
ں ی ے ہ ں علم رکھئ ارے می ے عمل کے ت گئ ے گائ
کر ا ھ ملا
ی سات ک ہ رواداری اور ات
وں ج ن ر کےب ے ارہ
ھگوئ ہلے سے ت ں ب کاز والے محلول می لف ارت ی
مک کے مخ
دری ں ں سمی ی مطالعہ می
دائ ی کھاری اب کا
کہ پ ن کے و ی ج وئ ع ہ
کمی واف ں ی ت می ب
کی طن وں ج ن
ھ ب ہ کے سات
اف ں اض کھاری پ ن می ا گی ا کی دہ اہ کا مش رات
iwEC =اب
1-35 dSm می خ ی کہ ت ی ج مک کے ب راب ررہ
دری ں زی سمی کا
ہ ارت ں ی ام می ی ت صدی dSm= iwEC 168-1پ ودوں کے ق
ق
ی ک رہ ں Lysemeterت ے والے پ ودوں می ڑھئ ں ب روان چ می 1-dSm 24 ں جوضلہ مک محلول می
دری ں زی سمی کا
ارت
ں کر می ر خل ب زب ر س ی
ات اور غ روز مادوں لمخی
گ اف الت ف کے رت ی ت
ائ ی ں ض کھاری پ ن می ی اس
گئ کھی
ت ت د زا ب رداش
ت صدی 05اف
ق
ی ش کم وب ں کر می ی کہ خل ب زب ر س ں 50کمی ج وں می ج ن
ھلی اورب ھول ت ں ت ن می ری ج دی ب ڑھوب ولی
ا پ ا رہ مات
ہ ں اف ت صدی اض
05ق
ی گئ کھی
ت ح طور د
کمی واض ت صدی
ق
ی وی شلک سہب ڑ سے می کی چ ر مک (Symbiotic)ارہ
کی ں ا رت ی
کٹ ی ے والے ب
کرئ مد خ
ن من روج ی
اب سے (NaCl)ت
ی ر کے سا dSmwEC 366 =-1رواداری ں ب ری ہ می ج ے عمل کے ت گئ ے
گائ
کر ا ھ ملا
ی سات ک ہ یات
گئ کھی
ت ک د ر ت ھ ارہ
ت
بی ق کے ب
حق ی ت دائ ی ا اب گی ا ی
کھاری پ ن کو ج کم ں ے می ج ں dSme (Ec 72 =-1(ن ی کہ می ری ج ں ب ڑھوب ی ر می e (Ec =ب
)1-111 dSm ہل ہلے ب ے عمل ب گئ ے
گائ
کر ا ھ ملا
ی سات ک ہ کو ات ر ی ر اور ب ی ارہ
ر رہ اب ر می ی
ک غ کی خد ت
Lysemeter ج ب رآم ت ا ی زا ب ی کے جوضلہ اف
اش ی ے سے آب
ف ف ھ دن کے و
سی ت آت
کی ی ار دن ی خ
گئ کی ں ں دمی ن می ے ج
وئ ہ
ے عمل گئ ے گائ
کر ا ھ ملا
ی سات ک ہ سی ت ات
کی ی ے پ ودوں
گائ
ن ہا ا کی پ ودوں ب شام
وں اق
ے دوپ ج گئ
ت ا ی زا ب ادہ جوضلہ اف ں زت می
ی ول ب ات ف روزمادوں لخمی
گ اف الت ف کے رت ی ت
ائ ی ضلاات می درخ ی می
ائ کی می ی
ائ ےجی
وئ Electrolyteب رآمد ہ
Leakage کی کر ں س ی وں می ب ی ان پ ودوںاور ب
ی ش کمی ب ں دار می ی دپ ں مق
ں دکھائ ر می
اظ ی ری کے ب
کے ب ڑھوب
xxii
Antioxidant ی ظرح سے ہ اور اس ہ اف ں اض کی سرگرمی وں می امروں
اور اس کے Nitrate Reeducatesخ
Substrate )3(NO ا ی کا سی ب ب ہ اف ں اض ما می وں
ش ھی ی
ت
ےdSmiw(Ec 28 =-1(معمولی گئ ے ئ کب راب ں سی ی می ائ ہ ت والے ت درج ں می ری ہ می ج
ی ت ئ ن ہا زمب کی ب الا پ ودوں
ے عمل گئ ے گائ
کر ا ھ ملا
ی سات ک ہ سی ت ات
کی ی ے پ ودوں
ادوں ب ر لگائ ی
ب ما ب وں
ش دی ی ولی
ے پ
وئ ج خاضل ہ
ت ا ی ر بہی ادہ ب ں زت می
ےض ر رہ ہی ں ب ام می ط ے ت گئ ے
گائ
کر ا ھ ملا
ی سات ک ہ شامت اور وزن ات عداد ج
کی ت ھلوں ی کہ ت ی ج ر رہ اب ر می ی
الت ف ی غ ی ت
ائ
ی وئ ں ہ ہی
ع ب ی دت لی واف ی ب
کوئ ں دار می کی مق کر
ات اور س ں لمخی ی وں می ب ی کہ ب ہ ج
اف ا اض مات
ں ں روزمادوں می
گ اف د کے رت LER مزت
ے LEC (gt1)اور ی ہ کرئ ارہ کی ظرف اس ی ائ کامی کی ام
ط ے ت گئ ے
گائ
کر ا ھ ملا
ی سات ری ات ک ہ
کی ب ڑھوب
ک دا کروت ں ری ہ می ج کھاری پ ن ) Lysemeterو کھاری پ ن روداری کے ت ا کم گی ا ں اگات iwEC = 142می
1-dSm ( کھاری پ ن ادہ ی کہ زت ی ج وئ ری ہ ہی ں ب مک( می
دری ں زی سمی کا
زی dSm= iwEC 129-1 ارت کا دری ارت سمی
ی وئ ر ہ
اب ری ب ری ظرح می
دی ب ڑھوب ولی
ی اور پ
ائ علی
ں ف مک( می
ی کہ ں ک dSm9= iw(Ec 9-1(ج مک ت
دری ں زی سمی کا
ارت
ت کب رداش ات اور س روز مادوں لخمی گ اف الت ف کے رت ی ت
ائ ی ضلاات می درخ ی می
ائ کی می ی
ائ ےجی اب رہ کامی ں ےمی
ر ب ری ظرح کرئ
ں ی وں می ب وا ب ہ ہ
اف ں اض ی ول می ب
ں ف ی وں می
ب ی کہ ب ں ج ی
وب ر ہ اب می
+Na ہ سے کی وج مع ی ج اف رلز کے K+اض روری می
ی سے ض ج
ی وئ ر ہ
اب کی ضلاجی ت می ے
کرئ زب چ
ا ت ق حق الا ت ہ ت درج ے ظر می
وئ ےہ
ھگوئ ں ت ی می
ائ ہلے سے ت کہ ب ی
ے آئ مئ ں ی ہ ت ات سا ی می
ئ کی روش ر ت ہ سے ارہ کی وج ے
ت ف
ھی مدد دے س ں ت ے می گئ ں ا ن می ن زمی مکی دل ں وکہ معی ے ج ا ہ اسکی ا خ ھی لگات
ں ت ن خالات می مکی کو ں وں ج ن
وزہ کے ب ے مج ا ہ کی
داواری ی ر ب ی ے عمل غ گئ ے
گائ
کر ا ھ ملا
ی سات ک ہ ی ر ات ر اور ب ی ضلاجی ت والی ارہ
اف ے اض لئ وروں کے
اپ کی صور ت خ ر ن ارہ زمی
ھی دا ت کروت ے ا ہ وسکی ت ہ اب کا ذرت عہ ت ے ی ب ڑھائ
کی آمدئ وں
کشاپ ی صورت
ارئ ح کی ت ل
ھ ی ت وردئ دار ج ی ر سے مزت ارہ اور ب ی خ
عئصت
صل کے طور ب ی ف ئے ب لئ ے کے
کرئ دا ی ھل ب ن سے ت کارآمد زمی ر ی
ن اور غ مکی
دل ں ے معی
لئ اضد کے ے رمق ا ہ اسکی ا خ کی ی ش ب
1
General Introduction
Intercropping is a major resource conservation technique for sustainable agriculture under
various climatic conditions (Zhang et al 2010 Li et al 2014) It can reduced operational
cost for the production of multiple crops with maintained or even higher level of
productivity (Vandermeer 2010 Perfecto and Vandermeer 2010) It can enhance the
water use efficiency by saving 20 to 40 irrigation water with improved fertilizer
management (Fahong et al 2004 Jat et al 2005 Jani et al 2008) Intercropping system
is more suitable in marginal areas with lower mechanization and cultivation input by
farmers on small tracts of farmlands (Ngwira et al 2012) It can enhance the cumulative
production per unit area and protect the small farmers against market fluctuations or crop
failure ensure the income improve soil fertility and food demands (Rusinamhodzi et al
2012) In this system dominating more compatible and productive species are selected or
replaced in which complementarity effects and beneficial interactions resulting enhanced
yield as compared to monoculture (Huston 1997 Loreau and Hector 2001) It was
estimated that in species diverse systems biomass production is 17 times higher as
compared to monoculture (Cardinale et al 2007)
It is suggested that intercropping is the best suitable cropping system which can
improve the resource-use efficiency by procurement of limiting resources enhanced
phyto-availability and effective plants interactions (Marschner 2012 White and
Greenwood 2013 Ehrmann and Ritz 2014) It is widespread in many areas of world
particularly in latin America it is estimated about 70-90 by small farmers which mainly
grow maiz potatoes beans and other crops under this system whereas intercropping of
maiz with different crops is estimated about 60 (Francis 1986) Additionally
agroforestry is more than 1 billion ha in this area (Zomer et al 2009) The land used for
intercropping system of various crops is greatly varied from 17 in India to 98 in Africa
(Vandermeer 1989 1992 Dupraz and Liagre 2011)
In intercropping system two or more crops or genotypes coexist and growing
together at a same time on a similar habitat (Li et al 2013) It may be divided into various
types such as in mixed intercropping system two or more crops simultaneously growing
without or with limited distinct arrangements whereas in relay intercropping system
second crop is planted when the first is matured while in strip intercropping both the crops
2
are simultaneously growing in strips which can facilitate the cultivation and crop
interactions (Ram et al 2005 Sayre and Hobbs 2004)
Several less-conventional fruit tress including Manilkara zapota (Chicko)
Ziziphus mauritiana (Jujubar) Carissa carndas (Karanda) Annona squamosa (Sugar
apple) and Grewia asiatica (Falsa) has been reported with high nutritional value with
capability to grow at marginal lands (Mass and hoffman 1997) Qureshi and Barrett-
Lennard (1998) suggested few grafted plants that can widely use to improve the quality
and productivity of fruits Grafting is also used to induce stress tolerance in plants against
various abiotic and biotic stresses including salinity stress (Rivero et al 2003) Both root
stocks and shoot stocks contribute to increase the tolerance level of plants Root stocks
represent the first part of defense to control the uptake and translocation of nutrients and
salts throughout the plant (Munns 2002 Santa-cruz et al 2002 Zrig et al 2011) while
shoot stocks develops physiological and biochemical changes to promote plant growth
under stress conditions (Moya et al 2002 Chen et al 2003)
Ziziphus mauritiana Lamk (varn grafted ber) belongs to the family Rhamnaceae
grows widely in most of the dry tropical and subtropical regions around the world Various
grafting methods are used for their propagation including wedge and whip or tongue
methods (Nerd and Mizrahi 1998) Intercropping of these grafted fruit trees with various
leguminous crops is also being successfully practiced in many countries thought the world
Leguminous crops are considered excellent symbiotic nitrogen fixing crops It can
effectively improve soil fertility and offset the critical problems of sub-tropical areas to
fight against desertification and soil degradation These plants are considered as an
excellent source of proteins for humans and animals They can fix the 90 of atmospheric
nitrogen and contribute 40 nitrogen to the soil thus increase the soil fertility (Peoples et
al 1995) However most of the leguminous plants are not salt tolerant while some
species are better drought tolerant and effectively contribute in marginal lands (Zahran
1999)
Among the leguminous plants Pigeon pea (Cajanus cajan (L) Millspaugh) of the
family Fabaceae is widely grown for food fodder and fuel production particularly in
semiarid areas The salinity tolerance of this specie is not well documented both at
germination and seedling stages This crop is still underexploited due to its edible and
3
economic importance While limited investigations has been made to uncover its
nutritional quality medicinal uses and drought tolerance
The identical physiological traits are important in both the mono and intercropping
systems to maximize the resource acquisition The exploitation of best possible
combination of traits of different plants in intercropping system is very important to
maximize the overall performance in intercropping system It depends on the above ground
beneficial plant interactions for light space and optimal temperatures (Wojtkowski 2006
Zhang et al 2010 Shen et al 2013 Ehrmann and Ritz 2014) as well as the
complementary below ground plant interactions with soil biotic factors (Bennett et al
2013 Li et al 2014)
Water is also a major limiting factor intercropping can enhanced the acquisition
of water by root architecture and distribution in the soil profile for effective utilization of
rainfall (Zegada-Lizarazu et al 2006 De Barros et al 2007) and enhanced the water use
efficiency for effective hydraulic redistribution by deep rooted crops and water stored in
the soil profile (Morris and Garrity 1993 Xu et al 2008) Mycorrhizal networks around
the roots of intercrop plants also enhanced the availability of water and available resources
and reduced the surface runoff (Caldwell et al 1998 Van-Duivenbooden et al 2000
Prieto et al 2012)
Intercropping with leguminous plants can enhanced the agricultural productivity in
less productive soils due to enhanced nitrogen availability and also improved the soil
fertility by effective nitrogen fixation (Seran and Brintha 2010 Altieri et al 2012) Due
to weaker soil nitrogen competition intercropping with legumes enhanced the nitrogen
availability to the non-leguminous intercrop which also absorbs the additional nitrogen
released in the soil or root nodules of the leguminous plant (Li et al 2013 White et al
2013a) The use of legumes in many intercropping systems is pivotal According to the
listing of Hauggaard-Nielsen and Jensen (2005) seven out of ten are the legumes among
the most frequently used intercrops around the world
The ecological range of adaptability of legumes reaches from the inner tropics to
arctic regions with individual species expressing tolerance to drought temperature
nutrient deficiency in soil water logging salinity and other environmental conditions
(Craig et al 1990 Hansen 1996) The woody perennial leguminous plants have a number
4
of purposes they can be used to reclaim degraded wastelands retard erosion and provide
shade fuel wood timber and green manure (Giller and Wilson 1991)
Trees with nitrogen fixing capability play an important role to offset the critical
problems of tropical and sub-tropical regions in their fight against desert encroachment
and soil impoverishment These plants are capable to live in N-poor soils through their
association with Rhizobium that fix atmospheric nitrogen Nitrogen fixing activity in the
field depends both on their N2-fixing potential and on their tolerance to existing
environmental stresses (Galiana et al 2002) Symbiotic N2 fixation in leguminous plants
can mainly be considered an excellent source of protein supply for human and animal
consumption They range from extensive pasture legumes to intensive grain legumes and
are estimated to contribution up to 40 of their nitrogen to the soil (Simpson 1987)
The traits in the monocropping system in the selected crop extensively exploit the
acquisition of limiting resources in the environment and continuously focused on the
availably of similar resources for the successful crop production (White et al 2013 ab)
whereas in intercropping with different crops cycling of resources can be optimized to
the complementarity or facilitation traits (Costanzo and Barberi 2014) to overcome
resource limitations during the growing season (Hill 1996 George et al 2014)
For the long term sustainable agriculture and food production in resource limiting
areas with lower input Intercropping systems have the potential to increase the
productivity With efficient mechanization cultural practices and optimized nutrient
management rapid improvements are also possible through this system In future
perspective intercrops with higher resource use efficiency through plant breeding and
genetics is likely to be the most effective option for sustainable agriculture and
development
Increase of world population and demand of additional food production
The demand and production gap of food fodder fuel wood and livestock products is
increasing day by day due to global population which will increase from about 7 billion
(FAO 2014) to 9 billion by 2050 (Haub 2013) The increasing urbanization further
intensifies the problem which will increase from 54 to 66 expected in 2050 (UN
2014) Majority of this rise in urbanization will occur in developing countries around the
5
globe The major problem is to meet the challenge of increasing food demand for this ever
growing population up to 70 more food crops to feed the additional 23 billion population
worldwide by 2050 (FAO 2010 2011) Hence there is great need to increase the re-
vegetation for fuel wood and fodder production (Thomson 1987) An increase in
production could be envisaged through increasing the yield of already productive land or
through more extensive use of unproductive land The high concentration of salts in soil
or water does not let the conventional crops grow and give feasible economic return
Hence it is necessary to search for unconventional crops for foods fodder and fuel which
could give profitable yield under saline conditions (Ahmad and Ismail 1993) Reclamation
of this land through chemical and engineering treatments is very expensive The most
appropriate use of saline wasteland is the production of high yielding salt tolerance fuel
wood timber and forage species (Qureshi et al 1993) Therefore the most attractive
option is to screen a range of species and identify those which have potential of being
commercially valuable for the degraded environments (Ismail et al 1993)
Pakistan is in semi-arid region and the 6th most populated county of the world
Population drastically increased in Pakistan which was 80 million in 1980 and annual
increase in population is about 4 million (UNDES 2011) This is continuously
overburdened and it is estimated that in 2025 it will reach to 250 million and 335 million
in 2050 which decrease the available water per capita to less than 600 m3 resulting 32
shortfall of water requirements causing an alarming condition particularly for Pakistan
Furthermore this shortfall in 2050 leading to severe food shortage upto 70 million tones
which indicates the further development and serious measures for the new resources
(ADB 2002) Subsequent severe food and fodder crises along with all the resource
limitations with continuous increase in urbanization from the current 35 to 52 in 2025
will further intensity the agriculture production and demand
Shortage of good quality irrigation water
On earth surface the major resources of available fresh water is deposited in the form of
ponds lakes rivers ice sheets and caps streams and glaciers whereas underground water
as underground streams and aquifers With the drastic increase in population the water
consumption rise as the twice of the speed of population growth The scarcity of water is
widespread to many countries of different regions Majority of population in developing
countries suffering from seasonal or year round water shortage which will increase with
6
expected climatic changes Currently almost 50 countries around the globe are facing
moderate to severe shortage of water
Due to the greenhouse effect it is estimated that since the start of 20th century 14
degF temperature is already risen which will likely rise at least another 2degF and over the next
100 years it is estimated about more than 11degF due to the consequences of biogenic gases
(El-Sharkawy 2014) This is mainly due to the product of human activities including
industrial malpractices excess fossil fuel consumption deforestation poor land use and
cultural practices
Rising in atmospheric CO2 concentration which probably reached 700 μmol (CO2)
molminus1 resulting severe climatic changes It will accelerate the melting of ice and glacier
resulting the rising rainfall and storms in tropics and high latitude consequently 06 to 1
meter rise in sea level on the expense of costal lowlands across the continents After this
initial high flows the decrease in inflow was very terrifying Due to these climatic changes
humans suffering from socioeconomic changes including degradation of lands with lower
agricultural output and degradation of natural resources will further enhanced the poverty
and hunger resulting dislocation and human migrations (Randalls 2010)
In the mean while scarcity of good quality water is increasing day by day with the
demands of water for domestic agricultural and industrial utilization which will further
increase up to 10 of the total available resources as estimated by 2025 which needs
serious water managements (Bhutta 1999) It is very challenging for the modern
agriculture to ensure the increasing demand of more arable and overburdened population
with the limiting resources including the unavailability of good quality water and
deterioration of even previously productive land (Du et al 2015)
In Pakistan Indus River basin is the back bone of agriculture and socioeconomic
development which contributes 65 of the total river flows and 90 for the food
production with a share of 25 to the GDP It is estimated that about 30-40 of its surface
storage capacity will reduce by 2025 due to siltation of reservoirs and climatic changes It
will impose serious threat to irrigated agriculture in near future consequently with
decreases in groundwater resources resulting shortage of fresh water and 15-20
reduction in grain yield in Pakistan (World Bank 2006)
7
Spread of saline soil and reduction in agricultural yield
Along with scarcity of water soil salinity is one of the major environmental stresses which
severely threaten the agriculture The damages of salinity is widespread around the world
which is so far effected the more than 800 million hectare (more than 6) of land
worldwide including 397 million ha by salinity associated with 434 million ha by sodicity
(FAO 2010) The out of total 230 million hactares of irrigated land more than 45 million
hactares (20) is so far effected by salinity which is about the 15 of total cultivated land
(Munns and Tester 2008)
In Pakistan out of 2036 million hectares of cultivated land more than 6 million
hectares is affected by salinity and water logging of various degrees (Qureshi et al 2004)
About 16 million hectares of tropical arid plains which have been put under crop
cultivation depend extensively on canal irrigation network This area (about 60) is now
seriously affected by water logging and salinity (Qureshi et al 2004) The rise of subsoil
water levels accompanied by its subsequent decline due to irrigation combined with
insufficient drainage has led to salinization of valuable agricultural land in arid zones all
over the world (Ahmad and Abdullah 1982) The dominated cation in salt-affected soil is
Na+ followed by Ca2+ and Mg2+ while the anions Cl and SO4 are almost equal in
occurrence (Qureshi et al 1993) Salt content varies in different regions of the salt-
affected areas but at certain sites could reach up to an ECe of 90-102 dSm-1 (Ahmad and
Ismail 1993)
Salinity is a chief anxiety to meet the ever growing demands of food crops Salinity
adversely affects the plant growth and productivity Plants differentially respond to salt
stress and categories into four classes Salt sensitive moderately salt sensitive moderately
salt tolerant and highly salt tolerant plants on the basis of their tolerance limits Whereas
mainly plants are divided into halophytes (salt tolerant) and glycophytes (salt sensitive) on
the basis of adaptive evolution (Flowers 2004 Munns and Tester 2008) Unfortunately
majority of cultivated crops are not able to withstand in higher salinity regimes and
eventually die under higher saline conditions which proposed serious attentions to manage
the dissemination of salinity (James et al 2011 Rozema and Flowers 2008)
Excessive accumulation of salts in rhizosphere initially reduced the water
absorption capacity of roots leading to hyperosmotic stress followed by specific ion
8
toxicity (Munns 2008 Rahnama et al 2010) Plants initially manage the overloaded salt
by various excluding and avoidance mechanisms depending on their tolerance levels The
management of salt inside the cytosol is depends on the compartmentalization capacity of
plants followed by osmotic adjustments and efficient antioxidant defense mechanisms
Whereas higher salt beyond the tolerance impose injurious effects on various
physiological mechanisms These are including disruption of membrane integrity
increased membrane injuries nutrient ion imbalances osmotic disturbance
overproduction of reactive oxygen species (ROS) compromised photosynthesis and
respiration due to stomatal closure and damages of enzymatic machinery (Munns and
Tester 2008) In specific ion toxicity Na+ and Cl- are the chief contributors in
physiological disorders Excessive Na+ in rhizosphere antagonize the uptake of K+
resulting lower growth and productivity (James et al 2011) Salt load in the cytosol trigger
the overproduction of ROS including H2O2 OH- super oxides and singlet oxygen They
are involved in sever oxidative damages to various vital cellular components including
DNA RNA lipids and proteins (Apel and Hirt 2004 Ahmad and Umar 2011)
Strategies to cope up the salinity problem
The development and cultivation of highly salt tolerant crop varieties for salt affected areas
is the major necessity to meet the future demands of food production whereas the majority
of available food crops are glycophytes Therefore it is an emergent need of crop
improvement methods which are more efficient cost effective and grow on limiting
resource The use of poor quality water for irrigation is also very important under the
proposed shortage of fresh water in near future For the development of salt tolerant
varieties more understanding of stress mechanisms are required at whole plant molecular
and cellular levels
The variability in stress tolerance of salt sensitive genotypes (glycophytes) and
highly salt tolerant plants (halophytes) showed genetic basis of salt tolerance It indicate
that salt tolerance is a multigenic trait which involves variety of gene expressions and
related mechanisms Salt stress induces both the qualitative and quantitative changes in
gene expression (Manchanda and Garg 2008) These multigenetic expressions play a key
role in upregulation of various proteins and metabolites responsible for the management
of anti-stress mechanisms (Bhatnagar-Mathur et al 2008) Plant breeding and transgenic
strategies are intensively used for decades to improve the crop performance under salinity
9
and aridity conditions Few stress tolerant varieties are so far released for commercial
production whereas in natural condition where plant exposed to variety of climatic
conditions the overall performance of plant have changed as compared to controlled in
invitro conditions (Schubert et al 2009 and Dodd and Perez-Alfocea 2012) The success
stories about transgenic approaches for crop improvement under stressful environments
are still very scanty because of the insufficient understanding about the sophisticated
mechanisms of stress tolerance (Joseph and Jini 2010) It indicates that there is less
correlation between the assessment of stress tolerance in invitro and invivo conditions
Although there have been some achievement in this connection in some model plants
including rice tobacco and Arabidopsis (Grover et al 2003) which proposed the
possibilities of success in other crops in future Variety of technicalities and associated
financial challenges are still associated with this strategy
In conventional cultivation practices continuous irrigation with poor quality water
can enhanced the salinization due to evapotranspiration leading to increased saline andor
sodic soils This problem can be cope up by intercropping system in which high salt
tolerant or salt accumulator plants are intercropped with salt sensitive crops which can
accumulate salt thus can reduce the risk of salt increment in soil Additionally better
cultivation practices including the micro-jet or drip irrigation and partial root zone drying
technique is also very fruitful to optimize the water requirements and avoid the risks
associated with conventional flooding irrigation system
In dry land agriculture plantation of deep rooted perennials during off season or
annuals can reduced the risk of salinization They continuously grown and utilize excess
amount of water create a balance between water utilization and rail fall Thus prevent the
chance of salt accumulation on soil surface due to increased water table and
evapotranspiration (Manchanda and Garg 2008) The efficient irrigation and
intercropping strategy is seemed quite attractive cost effective and very beneficial in less
mechanized poor marginal areas It can ameliorate the injurious effects of salinity and
increased production per unit area thus ensure the sustainable agriculture in semi-arid or
marginal lands (Venkateswarlu and Shanker 2009)
A number of plant species are available that are highly compatible with saline
sodic and marginal lands The cultivation of these species with proposed intercropping
system is economically feasible to grow in marginal soil Some plants including Carissa
10
carandus Ziziphus mauritiana and Cajanus cajan was selected to revealed their potential
for intercropping under saline marginal lands These are important plants which can
established well at tropical and subtropical arid zone under high temperatures Hence their
range of salt tolerance and suitability for cultivation at waste saline land or with saline
water irrigation is being undertaken for commercial exploitation
Objective of present investigation
The plan of present investigation has been worked out to look into possibility of increasing
production of an unconventional salt tolerant fruit tree (Z mauritiana) by intercropping
with a legume ( C cajan) which apart from increasing fertility of soil could be able to
provide fodder for grazing animals from salt effected waste land Possibility of making
use of saline water for irrigation has also been considered for growing leguminous plant
(C cajan) and salt tolerant unconventional fruit tree (Crissa carandas) under saline
condition
11
LAYOUT OF THESIS
Chapter 1 Monoculture of Cajanus cajan (Vern Arhar) and Ziziphus mauritiana
(Varn Ber) under different range of salinities created by irrigation of
various sea salt concentrations
A Experiments on Cajanus cajan
Following experiments were performed under A
Experiment No 1 Effect of Pre-soaked seeds of C cajan in distilled water for
germination in water of different sea salt concentrations
Experiment No 2 Effect of Pre-soaked seeds of C cajan in various dilutions of sea salt
for germination in water of respective sea salt concentrations
Experiment No 3 Seedling establishment experiment of C cajan on soil irrigated with
sea salt of different concentrations
Experiment No 4 Growth and development of C cajan in Lysimeter (Drum pot culture)
being irrigated with water of different sea salt concentrations
Experiment No 5 Range of salt tolerance of nitrogen fixing symbiotic bacteria
associated with root of C cajan
B Experiments on Ziziphus mauritiana
Experiment No 6 Growth and development of Z mauritiana in large size clay pot being
irrigated with water of two different sea salt concentrations
Discussion (Chapter 1)
Chapter 2 Intercropping of Ziziphus mauritiana with Cajanus cajan
Experiment No 7 Physiological investigations on Growth of Ziziphus mauritiana and
Cajanus cajan intercropped in drum pot (Lysimeter) culture being
irrigated with water of sea salt concentration at two irrigation
intervals
Experiment No 8 Investigations of intercropping Ziziphus mauritiana with Cajanus
cajan on marginal land under field conditions
12
Discussion (Chapter 2)
Chapter 3 Investigations on rang of salt tolerance in Carissa carandas (varn
karonda) for determining possibility of growing at waste saline land
Experiment No 9 Investigation on the effect of higher range of salinities on growth of
Carissa carandas (varn karonda) created by irrigation of different
dilutions of sea salt
Discussion (Chapter 3)
13
1 Chapter 1
Monoculture of Cajanus cajan (Vern Arhar) and Ziziphus mauritiana
(Varn Ber) under different range of salinity created by irrigation of
various sea salt concentrations
11 Introduction
Scarcity of good quality water enforced the growers to irrigate the crops with
lowmoderately saline water at marginal lands which ultimately enhance soil salinity due
to high evapo-transpiration (Azeem and Ahmad 2011) To overcome this situation people
are now focusing on less-conventional plants which can grow on resource limited areas
and can produce edible biomass for human and animal consumption
Ziziphus mauritiana (varn grafted ber) is salt and drought tolerant plant which can
grow on marginal and degraded land (Morton 1987) It has wide spread crown and a short
bole fast growing tree with average bearing life of 25 years The ripe fruit (drupe) is juicy
hard or soft sweet-tasting pulp has high sugar content vitamins A amp C carotene
phosphorus and calcium (Nyanga et al 2013 2008 Pareek 2013) The leaves contain 6
digestible crude protein and an excellent source of ascorbic acid and carotenoids The
leaves are used as forage for cattlesheepgoats and also palatable for human consumption
(Sharma et al 1982 Bal and Mann 1978 Agrawal et al 2013) The timber is very hard
can be worked to make boats charcoal and poles for house building Roots bark leaves
wood seeds and fruits are reputed to have medicinal properties The tree also used as a
source of tannins dyes silk (via silkworm fodder) shellac and nectar (Dahiru et al 2006
Chrovatia et al 1993 Gupta 1993)
Some atmospherics nitrogen fixing bacterial associated deep rooted drought
tolerent leguminious plants like Cajanus cajan can fix up to 200 Kg nitrogen ha-1 year-1
due to symbiotic association of Rhizobium with its deep penetrating roots (Bhattacharyya
et al 1995) Total cultivated area of Pigeon pea is about 622 million hectare and global
annual crop production is around 474 million tonnes whereas total seed production of
this crop is about 015 million tonnes (FAOSTAT 2013) Its seeds are an excellent source
of good quality protein (up to 24) and foliage is used as animal fodder with high
nutritional value (Pandey et al 2014) Besides being used as food and fodder this plant
14
also have therapeutic value and it is used against diabetes fever dysentery hepatitis and
measles (Grover et al 2002) It also use traditionally as a laxative and was identified as
an anti-malarial remedy beside other medicinal species (Ajaiyeoba et al 2013 Qasim et
al 2010 2011 2014)
Following experiments were conducted to evaluate the seed germination seedling
establishment and growth of C cajan as well as grafted sapling of Z mauritiana under
various salinity regimes Investigations were also undertaken to find-out of their
intercropping has any beneficial effect on growth at marginal saline land saline
environment
15
12 Experiment No 1
Effect of Pre-soaked seeds of Cajanus cajan in distilled water for
germination in water of different sea salt concentrations
121 Materials and methods
1211 Seed collection
Seeds of C cajan were purchased from local seed market Mirpurkhas Sindh and were
tested to determine the effect of salinity on germination at the biosaline laboratory Botany
department Karachi University Karachi The best lot of healthy seeds having 100
germination was selected for further experiments
1212 Experimental Design
Seeds of C cajan were surface sterilized with 01 sodium hypochlorite solution for 2-3
minutes washed in running tap water then soaked in sterilized distilled water for one hour
(Saeed et al 2014) Sterilized glass petri plates (9cm) lined with filter paper were moist
with 10 ml of distilled water at different saline water of different sea salt concentrations
and their germination percentage was observed Their electrical conductivities on these
sea salt dilutions are mentioned in Table 11 Three replicates were used for each treatment
Ten seed were placed in each petri plate which were kept in temperature controlled
incubator (EYELA LTI-1000 Japan) at 28 plusmn 1ordmC in dark Experiment was continued for 7
days Data were recorded on daily bases Analyses of varience by using repeated measures
and the significant differences between treatment means were examined by least
significant difference (Zar 2010) All statistical analysis was performed using SPSS for
windows version 14 and graphs were plotted using Sigma plot 2000
Germination percentage of C cajan was recorded every 24 hours per seedling
evaluation procedure up to 07 days The final percent germination related with salinity in
accordance with Maas and Hoffman (1977) The percent germination was calculated using
the following formula (Cokkizgin and Cokkizgin 2010)
16
Germination index for C cajan was recorded according to AOSA (1990) by using
following formula
Where Gt is the number of germinated seed on day t and Dt is the total number of
days (1 - 7)
Coefficient of germination velocity of C cajan was calculated described by Maguire
(1962)
Where G represents the number of germinated seeds counted per day till the end of
experiment
Mean germination time of C cajan was calculated by Ellis and Roberts (1981) by
using following formula
Where lsquonrsquo is the number of germinated seeds in day d whereas Σn is the total
germinated seeds during experimental period
Germination rate was of C cajan determined according to following formula
(Shipley and Parent 1991)
Where numbers of germinated seeds were recorded from 1 to 7
17
122 Observations and Results
Cajanus cajan (imbibed in distilled water) grown at different salinity regimes showed 50
reduction at 16 salt concentration corresponding ECiw 168 dSm-1 (Table 1 2 Appendix
I)
Rate of germination was inversely correlated with sea salt concentration It was
significantly (p lt 0001) decreased from first day to final (day 7) of observation Higher
germination rate was recorded in control and at lower concentrations of sea salt in early
days of seed incubation with contrast to higher concentrations of sea salt which was
reduced with increasing day of incubation (Table 13 Appendix I)
A significant decrease (p lt 0001) in coefficient of germination velocity was
observed with increasing salinity (Table 14 Appendix I)
A significantly increase (p lt 0001) in mean germination time of seeds was observed
with increasing sea salt concentrations However the difference was insignificant at lower
salinities (Table 14 Appendix I)
A significant decrease (p lt 0001) in mean germination index was observed with
increasing salt concentrations except lower salinities More reduction was observed
byhond 16 and onward sea salt concentration (Table 14 Appendix I)
18
Table 11 Electrical conductivities of different sea salt solutions used in germination of C cajan
Sea salt () ECiw (dSm-1)
Non saline control 06
01 09
02 16
03 35
04 42
05 58
06 62
07 79
08 88
09 99
10 101
11 112
12 128
13 131
14 145
15 159
16 168
ECiw is the electrical conductivity of irrigation water measured in deci semen per meter
19
Table 12 Effect of irrigation water of different sea salt solutions on germination percentage (GP) per day
of C cajan seeds pre-soaked in non-saline water prior to germination with duration of time under
various salinity regimes
Sea Salt
(ECiw= dSm-1)
GP
1st day
GP
2nd day
GP
3rd day
GP
4th day
GP
5th day
GP
6th day
GP
7th day
Control 8333plusmn667 90plusmn00 9333plusmn333 9333plusmn333 9333plusmn333 9333plusmn333 9333plusmn333
09 8667plusmn333 9333plusmn333 9667plusmn333 9667plusmn333 100plusmn00 100plusmn00 100plusmn00
16 7667plusmn667 80plusmn10 8333plusmn882 8333plusmn882 8333plusmn882 8333plusmn882 8667plusmn667
35 6667plusmn333 9333plusmn333 9333plusmn333 9333plusmn333 9333plusmn333 9333plusmn333 9333plusmn333
42 70plusmn00 8667plusmn333 90 plusmn00 90 plusmn00 90 plusmn00 90 plusmn00 90 plusmn00
58 6333plusmn667 7333plusmn333 8333plusmn333 90 plusmn00 90 plusmn00 90 plusmn00 90 plusmn00
62 5667plusmn667 80plusmn577 90 plusmn00 90plusmn00 90 plusmn00 90 plusmn00 90plusmn00
79 5333plusmn333 70plusmn00 8333plusmn333 8333plusmn333 8333plusmn333 8333plusmn333 8333plusmn333
88 4000plusmn00 6667plusmn667 8333plusmn333 8333plusmn333 8333plusmn333 8333plusmn333 8333plusmn333
99 2667plusmn333 60 plusmn00 90 plusmn00 90plusmn00 90 plusmn00 90 plusmn00 90 plusmn00
101 2333plusmn333 70plusmn577 7333plusmn333 7333plusmn333 7333plusmn333 7333plusmn333 7333plusmn333
112 70plusmn577 7667plusmn333 80plusmn00 8333plusmn333 8333plusmn333 8333plusmn333 8333plusmn333
128 6667plusmn333 6667plusmn333 6667plusmn333 6667plusmn333 6667plusmn333 6667plusmn333 6667plusmn333
131 3333plusmn882 50plusmn00 5333plusmn333 5333plusmn333 5333plusmn333 5333plusmn333 5667plusmn333
145 3333plusmn667 40 plusmn00 50 plusmn577 50plusmn577 50 plusmn577 5333plusmn333 5333plusmn333
156 3667plusmn667 40plusmn577 4667plusmn882 4667plusmn882 50plusmn577 50plusmn577 5333plusmn667
168 1667plusmn882 3333plusmn333 3333plusmn333 3333plusmn333 3667plusmn333 3667plusmn333 4333plusmn333
LSD 005 Salinity 18496
Time (days) 13322
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and LSD among the treatments is recorded at p lt 005
20
Table 13 Effect of irrigation water of different sea salt solutions on germination rate (GR) per day
of seeds C cajan pre-soaked in non-saline water prior to germination with duration of
time under various salinity regimes
Sea Salt
(ECiw= dSm-1)
GR
1st day
GR
2nd day
GR
3rd day
GR
4th day
GR
5th day
GR
6th day
GR
7th day
Control 833plusmn067 450plusmn00 311plusmn011 233plusmn008 187plusmn007 156plusmn006 133plusmn005
09 867plusmn033 467plusmn017 322plusmn011 242plusmn008 200plusmn00 167plusmn00 143plusmn00
16 767plusmn067 400plusmn050 278plusmn029 208plusmn022 167plusmn018 139plusmn015 124plusmn010
35 667plusmn033 467plusmn017 311plusmn011 233plusmn008 187plusmn007 156plusmn006 133plusmn005
42 700plusmn00 433plusmn017 300plusmn00 975plusmn750 180plusmn00 150plusmn00 129plusmn00
58 633plusmn067 367plusmn017 278plusmn011 225plusmn00 180plusmn00 150plusmn00 129plusmn00
62 567plusmn067 400plusmn029 300plusmn00 225plusmn00 180plusmn00 150plusmn00 129plusmn00
79 533plusmn033 350plusmn00 278plusmn011 208plusmn008 167plusmn007 139plusmn006 119plusmn005
88 400plusmn00 333plusmn033 278plusmn011 208plusmn008 167plusmn007 139plusmn006 119plusmn005
99 267plusmn033 300plusmn00 300plusmn00 225plusmn00 180plusmn00 150plusmn00 129plusmn00
101 233plusmn033 350plusmn029 244plusmn011 183plusmn008 147plusmn007 122plusmn006 105plusmn005
112 700plusmn058 383plusmn017 267plusmn00 208plusmn008 167plusmn007 139plusmn006 119plusmn005
128 667plusmn033 333plusmn017 222plusmn011 167plusmn008 133plusmn007 111plusmn006 095plusmn005
131 333plusmn088 250plusmn00 178plusmn011 133plusmn008 107plusmn007 089plusmn006 081plusmn005
145 333plusmn067 200plusmn00 167plusmn019 125plusmn014 100plusmn012 089plusmn006 076plusmn005
156 367plusmn067 200plusmn029 156plusmn029 117plusmn022 100plusmn012 083plusmn010 076plusmn010
168 167plusmn088 167plusmn017 111plusmn011 083plusmn008 073plusmn007 061plusmn006 062plusmn005
LSD 005 Salinity 0481
Time (days) 0378
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and LSD among the treatments is recorded at p lt 005
21
Table 14 Effect of irrigation water of different sea salt solutions on mean germination rate (GR)
coefficient of germination velocity (GV) mean germination time (GT) mean
germination index (GI) and final germination (FG) of C cajan seeds pre-soaked in non-
saline water prior to germination under various salinity regimes
Sea Salt
(ECiw= dSm-1) GR GV GT GI FG
Control 2624plusmn100 369plusmn005 027plusmn00 2624plusmn100 9667plusmn333
09 2743plusmn063 365plusmn009 027plusmn001 2743plusmn063 100plusmn00
16 2398plusmn218 423plusmn036 024plusmn002 2398plusmn218 8333plusmn882
35 2467plusmn086 378plusmn005 026plusmn00 2467plusmn086 9333plusmn333
42 3169plusmn733 311plusmn058 035plusmn008 3169plusmn733 9333plusmn333
58 2264plusmn081 399plusmn015 025plusmn001 2264plusmn081 90plusmn00
62 2253plusmn073 400plusmn013 025plusmn001 2253plusmn073 9333plusmn333
79 2074plusmn081 402plusmn00 025plusmn00 2074plusmn081 8333plusmn333
88 1927plusmn043 449plusmn008 022plusmn00 1927plusmn043 90plusmn577
99 1853plusmn033 486plusmn009 021plusmn00 1853plusmn033 90plusmn00
101 1635plusmn056 470plusmn022 021plusmn001 1635plusmn056 8667plusmn882
112 2263plusmn042 369plusmn020 027plusmn001 2263plusmn042 9667plusmn333
128 1953plusmn098 341plusmn00 029plusmn00 1953plusmn098 9667plusmn333
131 1368plusmn059 440plusmn018 023plusmn001 1368plusmn059 6667plusmn333
145 1276plusmn099 446plusmn019 023plusmn001 1276plusmn099 60plusmn577
156 1289plusmn153 447plusmn030 023plusmn002 1289plusmn153 8000plusmn100
168 876plusmn104 589plusmn078 018plusmn002 876plusmn104 8667plusmn333
LSD005 5344 3312 0064 5344 1313
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and LSD among the treatments is recorded at p lt 005
22
13 Experiment No 2
Effect of Pre-soaked seeds of Cajanus cajan in various dilutions of sea
salt for germination in water of respective sea salt concentrations
131 Materials and methods
1311 Seed germination
Procedure of seed germination has been mentioned in Experiment No 1 earlier The seeds
were pre-soaked in various sea salt concentrations instead of non-saline water and
germinated in respective sea salt concentrations Their electrical conductivities mentioned
in Table 15 Data were calculated and analysed according to formulas given in Experiment
No 1
Since these pre-soaked seeds in different sea salt concentration showed 50
germination at 03 equivalent to ECiw= 42dSm-1 sea salt solution any further work
beyond ECiw= 42dSm-1was not continued
132 Observations and Results
The final percent germination related with salinity in accordance with Maas and
Hoffman (1977) linear relative threshold response model as follows
Relative Final Germination = 100-200 (Ke ndash 005)
Where threshold salt concentration was 005 and Ke is the concentration of salts
at which relative final germination may be predicted This model indicated 50
declined in final germination at 030 salt concentration corresponding to ECiw= 42
dSm-1 (Table 16 Appendix II)
Rate of germination was significantly decreased (p lt 0001) from first day to final
(day 07) of observation and it was inversely correlated with sea salt concentration High
germination rate was recorded in control and low sea salt concentrations in early days of
seed incubation compared to higher sea salt concentrations but the difference in rate was
reduced (Table 17 Appendix II)
23
A progressive decline (p lt 0001) in coefficient of germination velocity was
observed with increasing salinity and fifty percent reduction was observed at 021 sea
salt concentration (ECiw = 319 dSm-1 Figure 11 Appendix II)
Final germination percentage was decreased significantly with increasing sea salt
concentrations However the difference was insignificant at lower (ECiw = 16 dSm-1)
salinity (Figure 11 Appendix II)
Mean germination time of seeds was increased significantly (p lt 0001) with
increasing sea salt concentrations However the difference was insignificant at lowest
(ECiw = 09 dSm-1) salinity (Figure 11 Appendix II)
Mean germination index was also significantly decreased (plt0001) with
increasing salt concentrations except for ECiw = 09 dSm-1 salinity Fifty percent reduction
in mean germination index was observed at 0188 sea salt concentration (ECiw = 289
dSm-1 Figure 11 Appendix II)
24
Table 15 Electrical conductivities of different sea salt solutions used in germination of C cajan
Sea salt () ECiw (dSm-1)
0 04
005 09
01 16
015 24
02 32
025 39
03 42
ECiw is the electrical conductivity of irrigation water measured in deci semen per meter
25
Table 16 Effect of irrigation water of different sea salt solutions on germination percentage (GP) per day of C cajan seeds pre-soaked in respective sea salt concentrations
with duration of time
Sea salt
ECiw (dSm-1)
GP
1st day
GP
2nd day
GP
3rd day
GP
4th day
GP
5th day
GP
6th day
GP
7th day
Control 6667plusmn333 8667plusmn333 10000plusmn000 10000plusmn000 10000plusmn000 10000plusmn000 10000plusmn000
09 7000plusmn000 7667plusmn333 9000plusmn000 10000plusmn000 10000plusmn000 10000plusmn000 10000plusmn000
16 4667plusmn333 6000plusmn000 7333plusmn333 8000plusmn000 8667plusmn333 8667plusmn333 9000plusmn577
24 4333plusmn333 5000plusmn000 6000plusmn577 6667plusmn333 7333plusmn333 7333plusmn333 8000plusmn000
32 3000plusmn000 3333plusmn333 3667plusmn333 4333plusmn333 5000plusmn577 6000plusmn577 7000plusmn577
39 1667plusmn333 2333plusmn333 2333plusmn333 4000plusmn577 4333plusmn333 5000plusmn000 6000plusmn000
42 667plusmn333 1333plusmn333 2333plusmn333 2333plusmn333 3333plusmn333 3667plusmn333 5000plusmn000
LSD 005 Salinity 327 Time 327
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and LSD among the treatments was recorded at p lt 005
25
26
Table 17 Effect of irrigation water of different sea salt solutions on germination rate (GR) per day of Ccajan
seeds pre-soaked in respective sea salt concentrations with duration of time
Sea salt
(ECiw= dSm-1)
GR
1st day
GR
2nd day
GR
3rd day
GR
4th day
GR
5th day
GR
6th day
GR
7th day
Control 667plusmn033 433plusmn017 333plusmn000 250plusmn000 200plusmn000 167plusmn000 143plusmn000
09 700plusmn000 383plusmn017 300plusmn000 250plusmn000 200plusmn000 167plusmn000 143plusmn000
16 467plusmn033 300plusmn000 244plusmn011 200plusmn000 173plusmn007 144plusmn006 129plusmn008
24 433plusmn033 250plusmn000 200plusmn019 167plusmn008 147plusmn007 122plusmn006 114plusmn000
32 300plusmn000 167plusmn017 122plusmn011 108plusmn008 100plusmn012 100plusmn010 100plusmn008
39 167plusmn033 117plusmn017 078plusmn011 100plusmn014 087plusmn007 083plusmn000 086plusmn000
42 067plusmn033 067plusmn017 078plusmn011 058plusmn008 067plusmn007 061plusmn006 071plusmn000
LSD 005 Salinity 014
Time 014 Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and LSD among the treatments is recorded at p lt 005)
27
Sea salt (ECiw = dSm-1
)
Contr
ol
09
16
24
32
39
42
Germ
ination Index(s
eedd
ays
-1)
0
2
4
6
8
Fin
al germ
ination (
)
0
20
40
60
80
100
Coeff
icie
nt of
germ
ination v
elo
city
(seedd
ays
-1)
00
01
02
03
04
05
06
07
Sea salt (ECiw = dSm-1
)
Contr
ol
09
16
24
32
39
42G
erm
ination tim
e (
Days
)
0
1
2
3
4
LSD005 = 0086
a = 0664 b = 1572
R2 = 0905 n =21
LSD005 = 062
a = 1239
b = 9836
R2 = 0894 n=21
LSD005 = 053
a = 8560b = -2272
R2 = 0969 n=21
RGF = 100-200 (Ke -005) Ke = 030
Figure 11 Effect of irrigation water of different sea salt solutions on seed germination indices of C cajan
(Bars represent means plusmn standard error of each treatment and significance among the treatments
was recorded at p lt 005)
28
14 Experiment No 3
Seedling establishment experiment of Cajanus cajan on soil irrigated with
sea salt of different concentrations
141 Materials and methods
1411 Seedling establishment
Seedling establishment experiment was carried out in Biosaline research field Department
of Botany University of Karachi Surface sterilized seeds pre-soaked were sown in small
plastic pots filled with 15 Kg sandy loam soil provided with farm manure at 91 ratio (30
water holding capacity) Sea salt solutions of different concentrations mentioned above
were used for irrigation The electrical conductivity of soil saturated paste (ECe) was also
determined at the end of the experiment (Table 18) Data on seedlings emergence was
recorded and their height were measured after 14 days of salinity treatment EC of the soil
(ECe) was initially 054 dSm-1 Statistical analyses were done according to the procedures
given in Experiment No 1
Since germination percentage of seeds pre-soaked in non-saline water was found
better under different concentrations of sea salt the seeds sown in soil for taking for
seedling establishment were pre-soaked in distilled water
29
142 Observations and Results
1421 Seedling establishment
Seedling emergence from soil was reduced significantly (p lt 0001) with increasing salt
concentration of irrigation water Not a single seedling emerged from soil in ge ECiw= 39
dSm-1 saline water irrigation However lower salinities (ECiw= 09 16 dSm-1) showed
slight decrease in seedling emergence with respect to controls Seedling emergence related
with salinity in accordance with a quadratic model as follows
Equation for seedling emergence () = 977751+ 44344 salt ndash 22215238 (salt)2 plusmn
6578 r = 09810 F = 15358 (p lt 00001)
Fifty percent reduction in seedling emergence was noticed at 016 sea salt
concentration (ECiw = 241 dSm-1 Figure 12 Appendix III)
1422 Shoot height
Shoot height was measured after fourteen days of irrigation Shoot length was
significantly decreased (p lt 0001) with increasing salinity A lower decrease was
observed in low sea salt salinity (ECiw= 09 and 16 dSm-1) compared to controls while
higher decrease in shoot height was noticed from ECiw= 2 dSm-1sea salt concentration
Shoot height related with salinity as follows
Equation for shoot height (cm) = 9116714 ndash 3420286 salt plusmn 09221 r = 0968 F =
128893 (p lt 0001)
Fifty percent reduction in shoot height was estimated at 013 sea salt concentration
(ECiw = 210 dSm-1) (Figure 12 Appendix III)
30
Table 18 Electrical conductivities of different Sea salt concentrations and ECe of soil saturated paste at the
end of experiment (ECe = 0447 + 1204 (salt ) plusmn 02797 R = 0987 F = 72301 (p lt
000001)
Sea salt () ECiw (dSm-1) ECe (dSm-1)
0 04 05
005 09 161
01 16 278
015 24 354
02 32 433
025 39 483
03 42 552
Electrical conductivity of soil saturated paste determined after 14 days of saline water irrigation in pots
Figure 12 Effect of irrigating water of different sea salt solutions on seedling emergence (A) and shoot
length (B) of C cajan (Bars represent means plusmn standard error of each treatment where similar
letters are not significantly different at p lt 005)
e f
Sea salt (ECiw = dSm-1
)
Contr
ol
16
27
8
35
4
43
3
48
3
Shoot le
ngth
(cm
)
0
2
4
6
8
10ab
c
de
Contr
ol
16
27
8
35
4
43
3
48
3Seedlin
g e
merg
ence (
)
0
20
40
60
80
100a
bb
c
d
A B
31
15 Experiment No 4
Growth and development of Cajanus cajan in Lysimeter (Drum pot
culture) being irrigated with water of different sea salt concentrations
151 Materials and methods
1511 Drum pot culture
A modified drum pot culture (lysimeter) installed by Ahmad amp Abdullah (1982) at
Biosaline research field (Department of Botany University of Karachi) was used in
present experiment Each drum pot (60 cm diameter 90 cm depth) was filled with 200 kg
of sandy loam mixed with cow-dung manure (91) having 28 water holding capacity
They are fixed at cemented platform at slanting position with basal hole to ensure rapid
drain Over irrigation was practiced to avoid the accumulation of salt in the root zone
1511 Experimental design
Growth and development of C cajan in drum pots was carried out in six different drum
pot sets (each in triplicate) and irrigated with sea salt of following concentrations
Drum pot Sets Sea salt
()
ECiw ( dSm-1) of
irrigation water
Resultant ECe (dSm-1) after
end of experiment
Set I Non saline (C) 04 05
Set II 005 sea salt 09 16
Set III 001 sea salt 16 28
Set IV 015 sea salt 24 35
Set V 02 sea salt 28 38
Set VI 025 sea salt 34 43
Note ECiw is the electrical conductivity of irrigation water and ECe is the electrical conductivity of the saturated soil extract taken after
eighteen weeks at the end of experiment
Ten surface sterilized seeds with 01 sodium hypochlorite were sowed in each
drum pot and were thinned to three healthy and equal size seedlings after two weeks of
establishment in their respective sea salt concentration Each drum pot was irrigated with
15 liters non-saline or respective sea salt solution at weekly intervals Electrical
conductivity of soil was measured by EC meter (Jenway 4510) using saturated soil paste
32
at the end of experiment Experiment was conducted for a period of 18 weeks (July to
November 2009) during which environmental data which includes average humidity
(midnight 76 and noon 54) temperature (low 23oC and high 33oC) wind velocity (14
kmph) and rainfall (~4 cm) was recorded (Pakistan Metrological Department Karachi) is
given in Figure 13Statistics were analysed according to the procedures given in
Experiment No 1
1512 Vegetative and Reproductive growth
Shoot height was measured at every two week interval after seedling establishment Fresh
and dry weight of shoot was recorded at final harvest (18th week when pods were fully
matured) Leaf succulence (dry weight basis Abideen et al 2014) Specific shoot length
(SSL Panuccio et al 2014) and relative growth rate (RGR Moinuddin et al 2014) were
measured using following equations
Succulence (g H2O gminus1 DW) = (FW minus DW) DW
SSL = shoot length shoot dry weight
RGR (g gminus1 dayminus1) = (lnW2 - lnW1) (t2 - t1)
Whereas FW fresh weight DW dry weight W1 and W2 initial and final dry weights and
t1 and t2 initial and final time of harvest in days
Reproductive data in terms of number of flowers number of pods number of seeds
and seed weight per plants was recorded during reproductive period
1513 Analysis on some biochemical parameters
Biochemical analysis of leaves was carried out at grand period of growth Following
investigations was undertaken at different biochemical parameters
i Photosynthetic pigments
Fresh and fully expended leaves (at 2nd3rd nodal part) samples (01g) were crushed in 80
chilled acetone and were centrifuged at 3000rpm for 10 minutes Supernatant were
separated and adjusted to 5ml final volume The absorbance was recorded at 663nm and
645 nm on spectrophotometer (Janway 6305 UVVis) for chlorophyll content while 480
33
and 510 nm for carotenoids Chlorophyll ab ratio was calculated after the amount
estimated The chlorophyll and carotenoid contents were determined according to Strain
et al (1971) and Duxbury and Yentsch (1956) respectively
Chlorophyll a (microgml) = 1163 (A665) ndash 239 (A649)
Chlorophyll b (microgml) = 2011 (A649) ndash 518 (A665)
Total Chlorophylls (microgml) = 645 (A665) + 1772 (A649)
Carotenoids (microgml) = 76 (A480) ndash 263 (A510)
ii Total soluble sugars
Dry leaf samples (01g) were homogenized in 5mL of 80 ethanol and were centrifuged
at 4000 g for 10 minutes 10 mL diluted supernatant in 5mL Anthronrsquos reagent was kept
to boil in 100oC water bath for 30 minutes and were cooled in running tap water Optical
density was taken at 620nm for the determination of soluble carbohydrates according to
Fales (1951)Total soluble carbohydrates was estimated against glucose as standard and
was calculated from the equation mentioned and expressed in mgg-1 dry weight
Total carbohydrates (microgmL-1) = 228462 OD 097275 plusmn004455
iii Protein content
Fresh and fully expended leaves at 2nd3rd nodal part were taken for protein estimation
The protein contents were measured according to Bradford Assay reagent method against
Bovine Serum Albumin as standards (Bradford 1976) Dye stock was made to dissolved
50mg comassie blue in 25 ml methanol The solution is added to 50ml of 85 phosphoric
acid and diluted to 100 ml with distilled water 02g fresh leaf samples were mills in 5 ml
phosphate buffer pH7 5ml of assay reagent (diluting 1 volume of dye stock with 4 volume
distilled water) were added in 01 ml leaf extract used for enzyme assay Absorbance was
recorded at 590nm and was expressed in mgg-1 fresh weight Proteins were calculated
from the following best fit standard curve equation
Protein (microgml-1) = -329196 + 1142755 plusmn 53436
34
152 Observations and Results
1521 Vegetative and Reproductive growth
Effect of sea salt on vegetative growth including height fresh and dry weight of Cajanus
cajan is presented in (Figure 14 and 15 Appendix-VI) Comparative analysis showed
that plant growth (all three parameters) was significantly increased with time (plt 0001)
however it was linearly decreased (plt 0001) with increasing salinity (Figure 16
Appendix-VI) shows the water content succulence relative growth rate (RGR) and
specific shoot length (SSL) of Cajanus cajan Under saline conditions all parameters were
significantly reduced in comparison to control however SSL showed decline after ECe38
dSm-1 Salt induced growth reduction was more pronounced at ECe 38 and 43 dSm-1 in
which plants died before reaching the reproductive maturity after 12 and 14 weeks at sea
salt treatments respectively Therefore further analysis was carried out in plant grown up
to ECe= 35 dSm-1 sea salt concentrations
Salinity significantly reduced (plt 0001) reproductive parameters including
number of flowers pods seeds and seed weight (Figure 17 Appendix-VII) Among all
treatments highest reduction was observed in 315 dSm-1 in which number of flowers and
pods reduced up to 7187 and 70 respectively Similar trend was observed in total
number and weight of seeds which showed 80 and 8793 reduction respectively
1522 Study on some biochemical parameters
i Photosynthetic pigments
Figure 18 Appendix-VII shows the effect of salinity on pigments (chlorophyll a b ab
ratio and carotenoids) of C cajan leaves A slight increase in total chlorophyll contents
(1828) and chlorophyll ab ratio (1215) was observed at low salinity (ECe= 16 dSm-
1) however they were significantly reduced (4125 and 3630 respectively) in high salt
treatment (plt 0001) Chlorophyll a was higher than chlorophyll b in all treatments
however chlorophyll b was un-affected by salinity whereas total chlorophyll content and
ab ratio was disturbed due to change in chlorophyll a This reduction was more
pronounced at high salinity (ECe= 35 dSm-1) in which chlorophyll a total chlorophylls
and ab ratio was decreased by 505 412 and 3630 respectively Carotenoid content
was maintained at ECe= 16 dSm-1 and decreased with further increase in salinity
35
ii Total soluble sugars
Total soluble sugars in leaves of C cajan is presented in Figure 19 Appendix-VII Total
leaf sugars in C cajan were remained un-affected at 16 dSm-1 and subsequently decreased
with further increase in medium salinity Although total sugars were decreased at ECe 28
and 35 dSm-1 a significant increase (~25) of soluble sugars was observed at higher
salinities However this increment was accounted for decrease (504 ) in insoluble sugar
content at that salinity levels
iii Protein
Total protein in leaves of C cajan is presented in Figure 19 Appendix-VII An increase
in leaf protein content in C cajan was found at lower salinity regime (ECe= 16 dSm-1)
which was followed by significant reduction with further increase in salinity This decline
was 2040 at 28 which was more pronounced (5646 ) at high salinity level (ECe=
35dSm-1)
36
Months (2009)
Jun Jul Aug Sep Oct Nov Dec
Valu
es
0
10
20
30
40
50
60
70
80
90
Rainfall (cm)Low Temp (
oC)
High Temp (oC)
Humidity at noon () Wind (kmph)
Humidity at midnight ()
Figure 13 Environmental data of study area during experimental period (July-November 2009)
Time (Weeks)
2 4 6 8 10 12 14 16 18
Pla
nt heig
ht (c
m)
0
30
60
90
120
150
180
210
43 38 35 28 16 Control
Figure 14 Effect of salinity using irrigation water of different sea salt concentrations on height of C cajan
during 18 weeks treatment (Lines represent means plusmn standard error of each treatment represents
significant differences at p lt 005)
37
Sea salt (ECe= dSm
-1)
Cont 16 28 35 38 43
Sea salt (ECe= dSm
-1)
Cont 16 28 35 38 43
Fre
sh w
eig
ht (g
)
0
5
10
15
20
25
30
35Initial Final
a
b b
c c cab b
c c cC 16 28 35 38 43
Fre
sh w
eig
ht
(g)
012345 a
bb
bc ca a ab b c c
Dry weightMoisture
Figure 15 Effect of salinity using irrigation water of different sea salt concentrations on initial and final
biomass (fresh and dry) of C cajan (Bars represent means plusmn standard error of each treatment Different
letters represent significant differences at p lt 005)
Mo
istu
re (
)
0
20
40
60
80
100
Succu
lance
(
)
0
20
40
60
80
100
Sea salt (ECe= dSm
-1)
Co
nt
16
28
35
38
43
RG
R (
)
0
20
40
60
80
100
Co
nt
16
28
35
38
43
SS
L (
)
0
20
40
60
80
100
Sea salt (ECe= dSm
-1)
ab
b b
c c
a
b bc c c
a
b b
c c c
a a a ab
c
Figure 16 Percent change (to control) in moisture succulence relative growth rate (RGR) and specific
shoot length (SSL) of C cajan under increasing salinity using irrigating water of different sea
salt concentrations (Bars represent means plusmn standard error of each treatment Different letters
represent significant differences at p lt 005)
38
Sea salt (ECe= dSm-1)
Control 16 28 35
Tota
l seeds (
Pla
nt-1
)
0
20
40
60
80
100
120
140 Seed w
eig
ht (g
pla
nt -1
)
0
5
10
15
20
25
Num
ber
10
20
30
40
50
60
70 a
b
cc
a
a
b
b
b c
c
a
b
a
c c
Flowers
Pods
Seed weightTotal seeds
Figure 17 Effect of irrigating water of different sea salt solutions on reproductive growth parameters
including number of flowers pod seeds and seed weight of C cajan (Values represent means
plusmn standard error of each treatment Different letters represent significant differences at p lt
005)
39
Sea salt (ECe=dSm-1
)
Control 16 28 35
Caro
tinoid
s (
mg g
-1 F
W)
000
005
010
015
020
025
030
Chlo
rophyll
(mg g
-1 F
W)
00
02
04
06
08
ab
ratio
00
05
10
15
20
25
ab
ab
b
a
cd
b
a
c
d
a
b
c
d
a
a
ab
b
Figure 18 Effect of irrigating water of different sea salt solutions on leaf pigments including chlorophyll a
chlorophyll b total chlorophyll and carotenoids of C cajan (Bars represent means plusmn standard
error of each treatment Different letters represent significant differences at p lt 005)
40
Figure 19 Effect of irrigating water of different sea salt solutions on total proteins soluble insoluble and
total sugars in leaves of C cajan (Bars represent means plusmn standard error of each treatment
Different letters represent significant differences at p lt 005)
Sea salt (ECe= dSm
-1)
C 16 28 35
Pro
tein
(m
g g
-1 F
W)
00
01
02
03
04
05
06
Su
gar
s (m
g g
-1 F
W)
00
02
04
06
08
a ab b
a a
b b
a ab b
a
b
ab
c
SoluableInsoluable
41
16 Experiment No 5
Range of salt tolerance of nitrogen fixing symbiotic bacteria associated
with root of Cajanus cajan
161 Materials and methods
1611 Isolation Identification and purification of bacteria
Nodules of C cajan grow in large clay pots and irrigated with running tap water at
biosaline agriculture research field were collected from the lateral roots (about 15 cm soil
depth) Nodules were surface sterilized with sodium hypochloride (2) for 5 min and
vigorously washed with sterilized distilled water Each nodule was crushed with sterilized
rod in 5 ml distilled water The bacterial suspension was streaked on yeast extract mannitol
agar (YEM) (K2HPO4 05 g MgSO 4 025g Na Cl 01 g Manitol 10g Yeast Extract 1g
Agar 20 g in 1000 ml of Distilled water) with the help of sterilized wire lope Colonies
were identified by studying different phenotypic characters as Rhizobium fredii
(Cappuccino and Sherman 1992 Sawada et al 2003) Pure culture of Rhizobium species
was stored at -20oC temperature
1612 Preparation of bacterial cell suspension
Bacteria were multiplied by growing in YEM broth for 48 hrs on shaking incubator (140
rpm) at 37oC in dark The culture in broth was centrifuged at 4000 rpm for 10 min to
obtained bacterial cell pellet Pellet was washed and centrifuged twice with sterilized
distilled water Pellet then re-suspended in sterilized distilled water before use
1613 Study of salt tolerance of Rhizobium isolated from root nodules of
C cajan
Assessment for salinity tolerance of Rhizobium species was assessed on YEM agar
Salinity levels of 0 05 10 15 20 25 and 30 having electrical conductivity 06 90
188 242 306 366 and 423 dSm-1 respectively were maintained with NaCl Bacterial
cell suspension of 01 ml (5times 103 colony forming unitsml) was poured in each sterilized
Petri dish 10 ml of molten YEM agar was poured immediately and shake well before
solidification of agar Petri plates were incubated at 37deg C in dark Colonies were observed
and counted in colony counter after 48 h and photographed (Dubey et al 2012 Singh and
42
Lal 2015) There were three replicates of each treatment and data were transformed to
log10 before analysis
162 Observations and Results
Colonies of Rhizobium on YEM agar at different salinity levels is presented in Figure 110
and 111 Appendix-VIII A significant decrease (plt0001) in rhizobial colonies was
observed with increasing salinity However the difference between non saline control and
90 dSm-1 and as that of 242 dSm-1 and 302 dSm-1 salt (NaCl) concentration showed
nonsignificant difference in rizobial colonies Whereas drastic decreased was observed on
further salinity levels Rhizobial colonies were not found at 423 dSm-1salt concentration
NaCl (ECw= dSm
-1)
06 9 188 242 306 366 423
Rh
izo
bia
l co
lonie
s (l
og
10)
0
1
2
3
4 a a
b
c c
d
e
Figure 110 Growth of nitrogen fixing bacteria associated with root of C cajan under different NaCl
concentrations (Bars represent means plusmn standard error of each treatment among the treatments
is recorded at p lt 005)
43
Figure 111 Photographs showing growth of Rhizobium isolated from the nodules of C cajan invitro on
YEM agar supplemented with different concentrations of NaCl (ECw)
188
423 90
Control
366
306 242
44
17 Experiment No 6
Growth and development of Ziziphus mauritiana in large size clay pot
being irrigated with water of two different sea salt concentrations
171 Materials and methods
1711 Experimental design
The grafted plants obtained from the local nursery of Mirpurkhas Sindh were transported
to the Biosaline Agriculture Research field Department of Botany University of Karachi
and were transplanted carefully in large earthen pots containing 20 Kg sandy loam soil
mixed with cow dung manure at 91 ratio having about 5 liters of water holding capacity
with a basal hole for drainage of excess salts to avoid accumulation in the rhizosphere
Over irrigation with about 15 liters of non-saline saline water was kept weekly in summer
and biweekly in winter to avoid accumulation of salts in rhizosphere Plants were irrigated
to start with non-saline tap water for about two weeks for establishment All the older
leaves were fallen and new leaves were developed during establishment period Following
irrigation schedule of non-saline (control) and saline water was selected in view of Z
mauritiana being moderately salt tolerant plant which includes both low and as well as
higher concentrations of the salt in irrigation
Sea salt () ECiw (dSm-1)
of irrigation water
Average resultant ECe (dSm-1) of soil
with some fluctuation often over
irrigation
Non saline (Control) 06 12
04 63 72
06 101 111
ECiw = Electrical conductivity of irrigation water ECe = Electrical conductivity of saturated soil
Healthy and well established plants were selected of nearly equal height and
divided into three sets each contain three replicates (total nine pots) Salinity was provided
through irrigation water of different sea salt concentrations All pots except non-saline
control were initially irrigated with 01 sea salt solution and then sea salt concentration
45
in irrigation medium was increased gradually upto the required salinity level The salinity
level of soil was monitored by taken the electrical conductivity of saturated soil paste the
end of experiment The electrical conductivity of soil (ECe) maintained at the level of 12
72 and 111 dSm-1 respectively as described by Mass and Hoffman (1977)
1712 Vegetative and reproductive growth
Vegetative growth in terms of shoot height fresh and dry weight of shoot and number of
branches were noted at destructive harvesting at initial (establishment) 60 and 120 days
of growth For dry weight shoots were dried in oven at 70˚C for three days Shoot
succulence specific shoot length (SSL) moisture percentage and relative growth rate
(RGR) was calculated at final harvest by using formulas given in Experiment No 4
Whereas number of flowers in reproductive data were recorded at onset of reproductive
period
As regard of fruit formation the duration of experiment was not sufficient for fruit
setting and furthermore the amount of sol in pots was not sufficient for healthy growth of
this plant Secondly flowering and fruiting is reported to be poor at the time of 1st initiation
of reproductive period (Azam-Ali 2006) Furthermore statistical significance of flower
and fruit count also become far less due to their excess dropping at early stage Hence it
was decided to proceed with study of fruit formation in forthcoming field trials of their
intercropping culture
1713 Analysis on some biochemical parameters
Biochemical analyses were performed at the grand period (at the time of flower initiation)
in fully expended fresh leaves Chlorophyll contents soluble sugar contents and soluble
proteins were analyzed Leaves samples taken from 3rd 4th node below the apex according
to the procedures given in Experiment No 4
46
172 Observations and Results
1721 Vegetative and Reproductive growth
Effect of sea salt on vegetative growth of Z mauritiana including height fresh and dry
weight is presented in (Figure 112 Appendix-IX) Comparative analysis showed that
plant growth (all three parameters) was significantly increased with time (plt 0001)
however number of branches was decreased (plt 0001) with increasing salinity
Figure 113 shows the moisture content succulence relative growth rate (RGR)
and specific shoot length (SSL) of Z mauritiana A non-significant difference in shoot
succulence SSL and moisture content was observed with time salinity and interaction of
both factors However RGR showed decline Salt induced growth reduction was more
pronounced at higher salinities
In Z mauritiana plants number of flowers showed significant decrease (plt0001)
with increasing salinity treatment Flower initiation seems non-significant at early growth
(60 days) period in controls and salinity treatments However drastic decrease was
observed with increasing salinity in 120 days of observation (Figure 114 Appendix-IX)
1722 Study on some biochemical parameters
i Photosynthetic pigments
The effect of Z mauritiana leaves pigments (chlorophyll a b ab ratio) on salinity shower
a slight difference in chlorophyll lsquoarsquo over control However chlorophyll lsquobrsquo contents
showed increase over control in both salinity treatments due to which the total chlorophylls
were also enhanced compared to controls Chlorophyll ab ratio was significantly
(plt0001) decreased in both salinities as compared to control (Figure 115 Appendix-IX)
ii Sugars and protein
In Z mauritiana plant soluble sugars were significantly decreased (plt0001) over controls
whereas proteins showed little decrease under salinity treatments compared to controls
(Figure 116 Appendix-IX)
47
Control 72 111
Fre
sh w
eig
ht (g
)
0
150
300
450
600
750
900
Sea salt (ECe= dSm
-1)
Control 72 111
Dry
weig
ht (g
)
0
150
300
450
600
750
900
Num
ber
of bra
nches
3
6
9
12
15
18
Heig
ht (c
m)
20
40
60
80
100
120
140
160
Initial 60 days 120 days
AcBb
Ba
AcBb Ba
AcBb Ba
Ac
BbBa
Figure 112 Effect of salinity using irrigation water of different sea salt concentrations on height number of
branches fresh weight and dry weight of shoot of Zmauritiana after 60 and 120 days of
treatment (Bars represent means plusmn standard error of each treatment Different letters represent
significant differences at p lt 005)
48
120 days 60 days InitialS
uccula
nce (
g g
-1 D
W)
00
03
06
09
12
Sea salt (ECe= dSm
-1)
SS
L (
cm
g-1
)
00
01
02
03
04
05
Control 72 111
Mois
ture
(
)
0
10
20
30
40
50
60
Control 72 111
RG
R (
mg g
-1 d
ay
-1)
0
5
10
15
20
a a aa a a a a a a
a aa a a a a a
a a aa a a a a a a a
b
b b
c
Figure 113 Effect of salinity using irrigation water of different sea salt concentrations on succulence
specific shoot length (SSL) moisture and relative growth rate (RGR) of Z maritiana (Bars
represent means plusmn standard error of each treatment Different letters represent significant
differences at p lt 005)
49
Sea salt (ECe= dSm
-1)
Control 72 111
Num
ber
of flow
ers
0
20
40
60
80
100
120
140 60 days120 days
Ac
BbBa
Figure 114 Effect of salinity using irrigation water of different sea salt concentrations on number of flowers
of Z mauritiana (Bars represent means plusmn standard error of each treatment Different letters
represent significant differences at p lt 005)
Sea salt (ECe= dSm
-1)
Control 72 111
Ch
loro
ph
yll
(mg g
-1)
00
03
06
09
12
15
18
bba
bba
bb
a
chl b chl a ab
ab
ra
tio
00
05
10
15
20
Figure 115 Effect of salinity using irrigation water of different sea salt concentrations on leaf pigments
including chlorophyll a chlorophyll b total chlorophyll and chlorophyll ab ratio of Z mauritiana (Values
represent means plusmn standard error of each treatment Different letters represent significant differences at p lt
005)
50
Figure 116 Effect of salinity using irrigation water of different sea salt concentrations on total sugars and
protein in leaves of Z mauritiana (Bars represent means plusmn standard error of each treatment
Different letters represent significant differences at p lt 005)
Sea salt (ECe= dSm
-1)
C 04 06
Pro
tein
s (m
g g
-1)
0
10
20
30
40
50
60
70
80
Solu
ble
sugar
s (m
g g
-1)
0
3
6
9
12
15
18a
a
bb
b b
Control 72 111
51
18 Discussion
Seed germination is the protrusion of radicle from the seed which is adversely affected by
salinity stress (Kaymakanova 2009) Salinity imposes the osmotic stress by accumulation
of Na+ and Cl- which decrease soil water potential that ultimately inhibits the imbibition
process (Othman 2005) Effect of seed germination against salinity is reported in linear
threshold response model of Maas and Hoffman (1977) The germination of a salt tolerant
desert legume Indigofera oblongifolia and a desert graminoid Pennisetum divisum are
also reported to behave to salinity in similar manner (Khan and Ahmad 1998 2007) Many
workers used chemical (organic inorganic) salt temperature biological and soil matrix
priming techniques to enhance seed germination percentage and especially germination
rate in saline medium (Ashraf et al 2008 Ashraf and Foolad 2005)Encouraging results
in most of the species of glycophytes and hydrophytes were found by presoaking in pure
water prior to germinating under saline condition Our study supports this finding and
seeds soaked in distilled water prior to germination performed better than those which
were presoaked in sea salt solutions Salinity adversely affects at all germination
parameters (germination percentage germination rate coefficient of germination velocity
and germination index) directly proportional with increasing salinity (Tayyab et al 2015)
With increase in time a delayed germination at higher salinity was found Higher sea salt
(168 dSm-1 for pure water presoaking and 35 dSm-1 for presoaking in respective
salinities) showed 50 or more reduction in all germination indices as compared to control
(Table 13-16 Figure 11)Our results are parallel with the finding of other workers such
as Kafi and Goldani (2001) who found the same trend in chickpea at higher salinities Pujol
et al (2000) reported that increased salinity inhibit the seed germination as well as delays
germination initiation in various halophyte species as well Similar response was also
found in some other crops such as pepper (Khan et al 2009) sunflower (Vashisth and
Nagarjan 2010) and eggplant (Saeed et al 2014) Salt tolerance within species may vary
at germination and other growth phases (Khan and Ahmad 1998)
According to our results C cajan appeared to be a salt sensitive in initial growth
phase specially when presoaked in saline medium (Figure 12) however at later growth
stages it proved relatively salt tolerant Salt stress delays or either seize the metabolic
activities during seed germination in salt sensitive and even in salt tolerant plants (Khan
and Ahmad 1998 Ali et al 2013b) Salinity also imposes the oxidative stress due to
52
overproduction of reactive oxygen species which may alter metabolic activities during
germination growth and developmental stages (Zhu 2001 Munns 2005
Lauchli and Grattan 2007)
In our study seeds of pigeon pea were unable to emerge beyond ECe39 dSm-1 sea
salt concentration Height of seedling was significantly affected by increasing salinity
(Figure 12) Similar results are also reported in Indian mustered (B juncea Almansouri
et al 2001) some Brassica species (Sharma et al 2013) and tomato cultivars (Jamil et
al 2005) Growth retardation with increasing salinity may be due to reduced
photosynthetic efficiency and inhibition of enzymatic and non-enzymatic proteins
(Tavakkoli et al 2011) Furthermore salt stress also limit the DNA and RNA synthesis
leads to reduced cell division and elongation during germination growth and
developmental stage
Khan and Sahito (2014) found variation in salt tolerance within species subspecies
and provenance level Furthermore the salt tolerance of a species may also vary at
germination and growth phases (Khan and Ahmad 1998 Ali et al 2013a) Srivastava et
al (2006) suggested that the genetic variability influences salinity tolerance eg wild
species like Cajanus platycarpus C scaraboides and C sericea showed better salt
tolerance than C cajan In this connection Wardill et al (2006) has also reported genetic
diversity in Acacia nilotica C cajan in this study appeared to be a salt sensitive at
germination in compression with later stages of growth Seedling establishment at saline
solution faces adverse effects when emerging radicle and plumule come in contact with
salt effected soil particle or saline water hence percent seedling establishment remains
less than germination percentage observed at petri plate Ashraf (1994) found that salinity
tolerance of different varieties of C cajan do not much differ at germination and early
growth stages whereas at adult growth stage show improvement in salt tolerance
Soil salinity is a major limiting factor for plant growth and yield production
particularly in leguminous plants (Guasch-Vidal et al 2013 Tayyab et al 2016) In
present study Plant height RGR fresh and dry biomass were severely reduced with
increasing salinity and plant was unable to grow after ECe= 43 dSm-1(Figure 14-16)
This growth inhibition of C cajan may be accounted for individual and synergistic effect
of water stress nutrient imbalances and specific ions toxicities (Hasegawa et al 2000
Silvera et al 2001) Salt induced ion imbalance results in lower osmotic potential which
53
alter physiological biochemical and other metabolic processes leading to overall growth
reduction (Del-Amor et al 2001) Excessive amount of salt in cytoplasm challenge the
compartmentalization capacity of vacuole and disrupts cell division cell elongation and
other cellular processes (Munns 2005 Munns et al 2006) Our results are parallel with
some other studies in which significant growth inhibition of peas chickpea and faba beans
have been reported against salt stress (El-Sheikh and Wood 1990 Delgado et al 1994)
Singla and Garg (2005) also observed a similar salt sensitive growth response in Cicer
arietinum In our study the fresh and dry biomass of C cajan also showed inhibitory
behavior to salt stress (Figure 15) Hernandez et al (1999) also found significant reduction
in dry biomass of pea plant and common bean (40 and 84 respectively) when grown
in saline medium Mehmood et al (2008) also found similar results in Susbania sasban
Salinity also has imposed deleterious effects on reproductive growth of C cajan
Production of flowers and pods are significantly decreased in response to salinity (Figure
19) Increase in flower shedding leads to decreased number of pods indicating salt
sensitivity of plant at reproductive phase which was more pronounced at high salinity
(Vadez et al 2007) Furthermore seed production and weight of seed per plant was also
linearly decreased Salt induced reduction of reproductive growth has also been found in
mung bean in which 60 and 12 less pods and seeds were produced respectively at 06
saline solution (Qados 2010) Similar results are reported in faba bean (De-Pascale and
Barbieri 1997) tomato (Scholberg and Locascio 1999) maiz sunflower (Katerji et al
1996) and watermelon (Colla et al 2006) Salinity reduces reproductive growth by
inhibiting growth of flowers pollen grains and embryo which leads to inappropriate ovule
fertilization and less number of seeds and fruits (Torabi et al 2013)
On biochemical parameters total chlorophyll and chlorophyll ab ratio has
increased in low salinity in contrast the adverse effect at higher salinity could be due to
high Na+ dependent breakdown of these pigments (Li et al 2010 Yang et al 2011)
Chlorophyll a is usually more prone to Na+ concentration and decrease in total chlorophyll
is mainly attributed to the destruction of chlorophyll a (Fang et al 1998 Eckardt 2009)
This diminution could be due to the destruction of enzymes responsible for green pigments
synthesis (Strogonov et al 1973) and increased chlorophyllase activity (Sudhakar et al
1997) Thus insipid of leaf was a visible indicator of salt induced chlorophyll damage
which was well correlated with quantified values as reported in other legume species
54
(Soussi et al 1998 Al-Khanjari et al 2002) In this study chlorophyll a was found to be
more sensitive than chlorophyll b (Figure 18) Garg (2004) also found similar reduction
in chlorophyll pigments (a b and total chlorophyll) in chickpea cultivars under salinity
stress
At low salinity (16 dSm-1) total carotenoids remained unaffected along with
increased total chlorophyll (Figure 18) which may suggest a role of carotenoids in
protection of photosynthetic machinery (Sharma et al 2012) Similar response was found
in Cajanus indicus and Sesamum indicum (Rao and Rao 1981) however
Sivasankaramoorthy (2013) and Ramanjulu et al (1993) reported slight increase of leaf
carotenoids in Zea maiz and mulberry when exposed to NaCl High salinity was destructive
for both leaf pigments (chlorophyll and carotenoids) of C cajan which was in accordance
with Reddy and Vora (1985) who found similar decrease in some other salt sensitive crops
Salinity led to the conversion of beta-carotene to Zeaxanthin which protect plants against
photo-inhibition (Sharma and Hall 1991)
In present study with increasing salinity water content and succulence of C cajan
were significantly reduced which indicated loss of turgor (Figure 16) Our data suggest
that decreased succulence by lowering water content may help in lowering leaf osmotic
potential when exposed to increasing salinity which is in agreement with findings of Parida
and Das (2005) and Abideen et al (2014) In addition increased production and
accumulation of organic substances is also necessary to sustain osmotic pressure which
provide osmotic gradient to absorb water from saline medium (Hasegawa et al 2000
Cha-um et al 2004) Compatible solutes including carbohydrates amino acids proteins
and ammonium compounds play important roles in water relations and cell stabilization
(Ashraf and Harris 2004) In this study C cajan produce more soluble sugars (Figure 18)
which is considered as a typical plant response under saline conditions (Murakeozy et al
2003) Sugars serve as organic osmotica and their available concentration is related to the
degree of salt stress and plantrsquos tolerance (Ashraf 1994 Murakeozy et al 2003) Sugars
are involved in osmoprotection osmoregulations carbon storage and radical scavenging
activities (Pervaiz and Satyawati 2008) On the other hand insoluble and total sugars were
reduced in higher salinity which is also supported by Parida et al (2002) and Gadallah
(1999) who found similar results in Bruguiera parviflora and Vicia faba
55
Total soluble proteins of C cajan were reduced due to deleterious effects of salinity
(Figure 18) The accumulation of Na+ in cytosol disrupts the protein and nucleic acid
synthesis (Bewley and Black 1985) Gill and Sharma (1993) and Muthukumarasamy and
Panneerselvam (1997) also reported decreased protein content with increasing salinity in
Cajanus cajan seedlings Similar results were found when tomato (Azeem and Ahmad
2011) Zingiber officinale (Ahmad et al 2009) and Sorghum bicolor (Ali et al 2013a)
were grown under variable salt concentrations (Figure 19)
Nodule formation of Rhizobium in Legume depends upon interaction between soil
chemistry of salt composition and osmotic regimes of salt and water (Velagaleti et al
1990 Zahran 1991 Zahran and Sprent 1986) Salinity reduces plant growth directly
through ion and osmotic effects and indirectly by inhibiting Legume-Rhizobium
association (El-Shinnawi et al 1989) Studies demonstrated a more sensitive response of
rhizobial N-fixing mechanism than growth of plant to abiotic stresses including salinity
(Mhadhbi et al 2004) In nodules metabolic disturbance initiated with the production of
ROS leading to tissues injury and loss of nodule function (Becana et al 2000) In general
it slow down the nitrogenase activity and decrease nodule protein and leghemoglobin
content which decreased becteroid development (Mhadhbi et al 2008) In consequence
plant suffer directly by salt induced ion toxicity low water uptake and photosynthetic
damage and indirectly through weak association of symbionts due to high energy demand
for nodule function (Pimratch et al 2008) In our study the isolated rhizobial strain from
nodules of C cajan was found to be tolerant to salinity even up to 2 (ECw= 306 dSm-1)
NaCl (Figure 110 and 111) Some of the other species of Rhizobium such as Brady
Rhizobium have been shown salt tolerant even at higher concentration than their
leguminous hosts (Zahran 1999) For instance a number of rhizobial species can tolerate
up to 06 NaCl (Yelton et al 1983) while Rhizobium meliloti can tolerate 175 to
40 NaCl and R leguminosarum can tolerate can tolerate upto 2 NaCl (Abdel-Wahab
and Zahran 1979 Sauvage et al 1983 Breedveld et al 1991 Helemish 1991
Mohammad et al 1991 Embalomatis et al 1994 Mhadhbi et al 2011) Rhizobia
isolated from soybean and chickpea can tolerate up to 2 NaCl with a difference of fast-
growing and slow growing strains (El-Sheikh and Wood 1990 Ghittoni and Bueno 1996)
Similarly Rhizobium from Vigna unguiculata can survive up to up to 55 NaCl
(Mpepereki et al 1997)
56
Present study shows an increase in vegetative growth in terms of plant height and
fresh and dry weight of shoot with increasing time under non-saline and saline conditions
but the increase was rapid at early period of growth (Figure 112) All the vegetative
growth parameters determined were reduced under salinity stress compared to non-saline
control Measurements of shoot moisture succulence specific shoot length and RGR
(Figure 113) indicate that Z mauritiana adjusted in its water relation over coming
negative water and osmotic potential with increase in salinity levels increased There is
evidence that water and osmotic potentials of salt tolerant plants become more negative in
higher salinities (Khan et al 2000) These altered water relations and other physiological
mechanisms help plants to get by adverse abiotic stress like that of drought and salinity
(Harb et al 2010) However the results clearly showed that salinity had an inhibitory
effect on growth but the decline was less at early sixty days and more during later 60-120
days in compression to controls Growth inhibition in shoot has been observed in number
of plants including different species of halophytes (Keiffer and Ungar 1997) chickpea
(Cicer arietinum Kaya et al 2008) and different wheat cultivars (Triticum aestivum
Moud and Maghsoudo 2008)
Salinity also caused reduction in the number of branches and the number of flowers
in Z mauritiana however reduction in the number of flowers is non-significant in ECe=
72 dSm-1 salinity treatment in comparison with non-saline control (Figure 114) The main
reason for this reduction could be attributed to suppression of growth under salinity stress
during the early developmental stages (shooting stage) of the plants These results are
similar to those reported by Ahmad et al (1991) and Khan et al (1998) As affirmed by
Munns and Tester (2008) suppression of plant growth under saline conditions may either
be due to osmotic effect of saline solution which decreases the availability of water for
plants or the ionic effect due to the toxicity of sodium chloride High salt concentration in
rooting medium also reduced the uptake of soil nutrients a phenomenon which affected
the plant growth thus resulting in less number of branches per plant Various abiotic
stresses such as temperature drought salinity light and heavy metals altered plant
metabolism which ultimately affects plant growth and productivity Amongst these
salinity stress is a major problem in arid and semiarid regions of the world (Kumar et al
2010) Salinity has an adverse effect on several plant processes including seed
germination seedling establishment flowering and fruit formation and ripening (Sairam
and Tyagi 2004) Salinity stress also imposes additional energy requirements on plant
57
cells and less carbon is available for growth and flower primordial initiation (Cheesman
1988) The lesser decrease in number of flowers at lower salinity (ECe= 72 dSm-1) has
been attributed to the fact that the cells of apex are un-vacuolated and the incoming salts
accumulated in the cytoplasm Munns (2002) further suggested a well-controlled phloem
transport of toxic ions from these cells prevented any change in reproductive development
Our findings showed an increase in total chlorophyll contents particularly
chlorophyll b contents were enhanced more than chlorophyll a contents under salinity
stress (Figure 115) In general the total chlorophyll contents decreased under high salinity
stress and this may be due to accumulation of toxic ions in photosynthetic tissues and
functional disorder of stomatal opening and closing (Khan et al 2009) The increase in
total chlorophylls appearing at salinity levels is considered as an important indicator of
salinity tolerance in plants (Katsuhara et al 1990 Demiroglu et al 2001) In another
study on Z mauritiana (cv Banara sikarka) the chlorophyll contents has shown decrease
with increasing salinity and sodicity but the seedlings treated with low salinity (ECe of 5
mmhoscm-1) shows slightly higher values than controls (Pandey et al 1991) Our study
also suggests that increase in total chlorophylls adapted this plant increased its tolerance
to salt stress
Slight decrease in protein has been shown under salinity treatments compared to
controls (Figure 16) Proteins play diverse roles in plants including involvement in
metabolic pathways as enzyme catalyst source of reserve energy and regulation of osmotic
potential under salt stress (Pessarakli and Huber 1991 Mansour 2000) Salts may
accumulate in cell cytoplasm and alter their viscosity depending on the response of plant
to salinity stress (Hasegawa et al 2000 Paravaiz and Satyawati 2008) The decrease in
protein contents under increasing salinity has also been documented in several plants
including Lentil lines (Ashraf and Waheed 1993) sorghum (Ali et al 2013a) and sugar
beet (Jamil et al 2014)
Soluble sugars were also decreased with increasing salinity treatments in our study
(Figure 16) Decrease in soluble sugars due to salinity has also been reported in Viciafaba
(Gadallah 1999) some rice genotypes (Alamgir and Ali 1999) Bruguiera parviflora
(Parida et al 2002) and Lentil (Sidari et al 2008) However the accumulation of soluble
sugars under salinity stress is considered as strategy to tolerate stress condition due to their
58
involvement in osmoprotection osmotic adjustment and carbon storage (Parida et al
2002 Parvaiz and Satyawati 2008)
From these experiments it is evident that C cajan is a salt sensitive plant at every
level of its life cycle starting from germination to growth phases Germination capacity
and salt tolerance ability of this species can be enhanced by water presoaking treatment
Growth reduction with increasing salinity could be attributed to physiological and
biochemical disturbances which ultimately affect vegetative and plant reproductive
growth Its roots are well associated with nitrogen fixing rhizobia and these
microorganisms were salt tolerant in in-vitro cultures Another fruit baring species of
marginal lands Z mauritiana showed growth improvement in lower salinity and its growth
was not much affected in high saline mediums owing to its controlled biochemical
responses
59
2 Chapter 2
Intercropping of Z mauritiana with C cajan
21 Introduction
Increasing soil salinity fresh water scarcity and agricultural malpractice creating shortage
of food crops for human and animal consumption (Bhandari et al 2014) and making
prices high Traditional agriculture which has been practiced since centuries using multi
species at a time in a given space could be a potential solution to narrow down the growing
edges of this supply demand scenario Plant species with innate resilience to abiotic
stresses like salinity and drought could be considered suitable to serve this purpose
especially for arid regions where marginal lands can be utilized to generate economy
Presence of such type of local systems in the region highlight their potential advantage in
crop production income generation as well as sustainability (Somashekar et al 2015)
For instance reports are available on successful intercropping of multipurpose trees
shrubs and grasses like millets pulses and some oil seed and fodder crops Green part of
these species usually mixed and used for cattle feed especially during the lean period The
utilization of the inter-row spaces of fruit trees like Ziziphus mauritiana for growing edible
legumes can generate further income by similar input (Dayal et al 2015) As an option
to this Cajanus cajan could serve as better intercropped as it provides protein rich food
nutritious fodder and wood for fuel which helped to uplift the socio-economic condition
of poor farmers Integrated agricultural practices improve the productivity of each crop by
keeping cost of production under sustainable limits (Arabhanvi and Pujar 2015)
Keeping in mind the above mentioned scenario in present study the possibility to
increase production of a non-conventional salt tolerant fruit tree (Z mauritiana) by
intercropping with a leguminous plant (C cajan) was investigated to produce edible fruits
and fodder simultaneously from salt effected waste lands
60
22 Experiment No 7
Physiological investigations on Growth of Ziziphus mauritiana and
Cajanus cajan intercropped in drum pot (Lysimeter) culture being
irrigated with water of sea salt concentration at two irrigation intervals
221 Materials and Methods
2211 Growth and Development
Experiment was designed to investigate the effect of intercropping on growth and
development of Z mauritiana (a fruit tree) and C cajan (a leguminous fodder) in drum
pot culture irrigated with water of 03 sea salt concentrations at two irrigation intervals
2212 Drum pot culture
Drum pot culture as recommended by Boyko (1966) and modified by Ahmed and
Abdullah (1982) was used for the present investigation as described in chapter 1
2213 Experimental Design
Three sets of 18 plastic drums (lysimeter) were used in this experiment One plant of Z
mauritiana were grown in each lysimeter Three replicates were kept for each treatment
comprising of 06 drums in each set which was further divided in two sub-sets First sub-
set was irrigated at every 4th and second subset at every 8th day
Set ldquoArdquo =Ziziphus mauritiana (Sole crop)
Set ldquoBrdquo = Cajanus cajan (Sole crop)
Set ldquoCrdquo = Ziziphus mauritiana + Cajanus cajan (intercropped)
The effect of salinity on sole crops of C cajan and Z mauritiana on salinity created
by various dilutions of sea salt has been investigated in chapter 1 Concentration of 03
sea salt considered equal level to its 50 reduction has been selected in present
experiment In addition irrigation was given in sub-sets in two intervals to investigate to
have some idea of its water conservation
61
2214 Irrigation Intervals
Sub-set 1 Irrigation was given every 4th day
Sub-set 2 Irrigation was given every 8th day
In set lsquoArsquo and lsquoCrsquo six month old saplings of Ziziphus mauritiana (vern grafted
ber) plants of nearly equal height and good health were transplanted in drum pots Plants
were irrigated to start with non-saline tape water for about two weeks for purpose of
establishment All the older leaves fell down and new leaves immerged during
establishment period
In set lsquoBrsquo and lsquoCrsquo Ten healthy sterilized seeds of Cajanus cajan imbibed in distill
water were sown in each drum pot and irrigated to start with tap water and after
establishment of seedlings only six seedlings of equal size with eqal distance (about one
feet) between C cajan and that of Z mauritiana were kept for further study The sowing
time of cajanus cajan seeds in both sets (B and C) was the same In drum pot lsquoCrsquo it was
sown when sapling of Z mauritiana have undergone two weeks of their establishment
period in tap water
When seedlings of C cajan reached at two leaves stage irrigation in all the sets
(ABC ) was started with gradual increase sea salt concentration till it reached to the
salinity level of treatment (03) in which they were kept up to end of experiment Each
drum was irrigated with enough water sea salt solution which retains 15 liters in soil at
field capacity Rest of water drain down with leaching of accumulated salt in root
rhizosphere
Vegetative growth of Z mauritiana plant was noted monthly in terms of height
volume of canopy while in C cajan height and number of branches was noted Shoot
length root length number of leaves fresh and dry weight of leaf stem and root leaf
weight ratio root weight ratio stem weight ratio specific shoot and root length plant
moisture leaves succulence and relative growth rate was observed and calculated at final
harvest in both the plant species growing individually (sole) or as intercropping at two
irrigation intervals
Investigations were undertaken on nitrate content relative water content and
electrolyte leakage at grand period of growth Amount of photosynthetic pigments soluble
62
carbohydrates proline content soluble phenols and Protein contents were also investigated
in fully expended leaves
Activity of catalase (CAT) ascorbate peroxidase (APX) guaiacol peroxidase
(GPX) superoxide dismutase (SOD) (Anti-oxidant enzymes) and nitrate reductase (NR)
activity was also observed in on both the Z mauritiana and C cajan leaves growing as
sole and as intercropped at two different irrigation intervals
The procedures of above mentioned analysis as follows
Leaf succulence (dry weight basis) Specific shoot length (SSL) and relative
growth rate (RGR) were measured according to the equations given in chapter 1
2215 Estimation of Nitrate content
NO3 was estimated through Cataldo et al (1975) 01g fresh leaf samples were boiled in
50 mL distilled water for 10 min 01mL of sample were added to mixed in 04 mL 50
salicylic acid (wv dissolved in 96 H2SO4 ) and allowed to stand for 20 min at room
temperature 95 mL of 2N NaOH was slowly mixed at last The samples were permissible
to cool NO3 concentration was observed at 410 nm and was calculated according to the
standard curve expressed in mg g-1 fresh weight
2216 Relative Water content (RWC)
Young and fully expended leaf was excise from each plant removing dust particles
preceding to Relative water content (RWC) Fresh weights (FW) were taken to all leaf
samples and were immersed in distilled water at 4 degC for 10 hours The soaked leaf samples
were taken out and surfeit water was removed by tissue paper Weighted again these leaf
samples for turgid weight (TW) and were oven dried at 70 degC Dry weight (DW) was
recorded after 24 hrs The RWC of leaf was calculated by the following formula
RWC () = [FW ndash DW] [TW ndash DW] x 100
2217 Electrolyte leakage percentage (EL)
EL was measured according to Sullivon and Ross (1979) Young and fully expended
leaves removing dust particles were taken 20 disc of 6mm diameter were made through
63
porer and were placed in the test tube containing 10ml de-ionized water First electrical
conductivity (EC lsquoarsquo) was record after shaken the tubes These test tubes now were placed
at 45-50oC warmed water bath for 30 min and observed second Electrical conductivity (EC
lsquobrsquo) Finally tubes were placed at 100oC water bath for ten min and obtained third and final
Electrical conductivity (EC lsquocrsquo) The electrolyte leakage was calculated in percentage by
using following formula
EL () = (EC b ndash EC a) EC b x 100
2218 Photosynthetic pigments
Photosynthetic pigments including chlorophyll a chlorophyll b total chlorophyll
chlorophyll ab ratio and carotinoids were estimated according to the procedure given in
chapter 1
2219 Total soluble sugars
Dry leaf samples (01g) were milled in 5mL of 80 ethanol and were centrifuged at 4000
g for 10 minutes and were estimated according to the procedure described in chapter 1
22110 Proline content
The proline contents were determined through Bates et al (1973) Each dried leaf powder
sample (01 g) was grinded and homogenized in 5 ml of 3 (wv) sulphosalicylic acid and
were centrifuged at 5000 g for 20 minutes 2ml supernatant was boiled by adding 2 ml
glacial acetic acid and 2 ml ninhydrin reagent (prepared by dissolving 125 g ninhydrin in
30 ml of glacial acetic acid and 20 ml 6 M phosphoric acid) in caped test tube The tubs
were kept in boiling water bath (100oC) for 1 hour After cooling 4 ml of toluene was
added to each tube and vortex Two layers were appeared the chromophore layer of
toluene was removed and their absorbance was recorded at 590nm against reference blank
of pure toluene The proline concentrations in leaves were determined from a standard
curve prepared from extra pure proline of (Sigma Aldrich) and were calculated from the
equation and were expressed in mgg-1 of leaf dry weight
Proline (microgmL-1) = -074092 + 1660767 (OD) plusmn054031
64
22111 Soluble phenols
The dried leaf powder (01g) was milled in 3ml of 80 methanol and was centrifuged at
10000g for 15 min (Abideen et al 2015) Final volume (5ml) were adjusted by adding
80 methanol Soluble phenols were determined by using Singleton and Rossi (1965) ie
5 ml of Folin-Ciocalteu reagent (19 ratio in distilled water) and 4 ml of 75 Na2CO3
were added to 01 ml supernatant The absorbance was recorded at 765 nm after incubation
of 30 minutes at room temperature The soluble phenols concentration in leaf tissues was
determined from a standard curved prepared from Gallic acid
22112 Total soluble proteins
The protein contents were measured according to Bradford Assay reagent method against
Bovine Serum Albumin as standards (Bradford 1976) Procedure was followed as given
in chapter 1
22113 Enzymes Assay
Enzyme extract prepared as given below was used for study of enzymes mentioned in text
The juvenile and expended leaf excised was frozen in liquid nitrogen and were stored at -
20 degC These leaf samples (100mg) was firmed in liquid nitrogen and were mills in 3 ml
of ice chilled potassium phosphate buffer (pH = 7 01 M) with 1mM EDTA and 1 PVP
(wv) The homogenate was filtered through a four layers of cheesecloth and were
centrifuged at 21000 g using refrigeration centrifuge (Micro 17 TR Hanil Science
Industrial Co Ltd South Korea) at 4 degC for 20 min The supernatant was separated and
stored at -20 degC and used for investigation on following enzymes
i Superoxide dismutase (SOD)
SOD (EC 11511) antioxidant enzymeactivity was measured through Beauchamp and
Fridovich (1971) derived on the inhibition of nitroblue tetrazolium (NBT) reduction by
produced O2minus using riboflavin photo-reduction 50 mM of pH 78 phosphate buffer (with
01mM EDTA 13 mM methionine) 75 microM nitroblue tetrazolium (NBT) 2 microM riboflavin
and 100 microl of enzyme extract was added to 3ml reaction mixture Riboflavin was added at
the last before the reaction was initiated under fluorescent lamps for 10 min Exposed and
un-exposed to florescence lamp without enzyme extract were used to serve as calibration
65
standards Activity was measured at 560nm Unit of SOD activity was defined as the
amount of enzyme required for 50 inhibition of NBT conversion
ii Catalase (CAT)
CAT (EC 11116) antioxidant enzyme activity was precise according to Aebi (1984)
derived on H2O2 reduction at 240nm for 30 s (ε = 36 M-1 cm-1)100mM potassium
phosphate buffer (pH=7) with 30mM H2O2 and 50 microl of diluted enzyme extract (adding in
last) was added to 3ml reaction mixture The decrease in absorbance due to H2O2 reduction
was measured at 240 nm and expressed in micromol of H2O2 reduced m-1g-1 fresh weight at 25
degC
iii Ascorbate peroxidase (APX)
Nakano and Asada (1981) method was used for APX (EC 111111) antioxidant
enzymeactivity by measuring the decrease in ascorbate oxidation by H2O2 The reaction
mixture (3ml) contained potassium phosphate buffer (50mM pH=7) 01mM H2O2 050
mM Ascorbate and 100 microl of enzyme extract and were observed 290 nm for 1 min 25 degC
(extinction coefficient 28 mM-1cm-1)
iv Guaiacol peroxidase (GPX)
GPX (EC 11117) antioxidant enzymeactivity was estimated through Anderson et al
(1995) 3ml of 50 mM potassium phosphate buffer (pH 7) guaiacol 75 mM H2O2 10 mM
reaction mixture with 20 microl of enzyme extract adding at last Increase in absorbance was
observed due to the formation of tetra-guaiacol at 470 nm for 2 min (extinction coefficient
266 mM-1cm-1)
v Nitrate reductase (NR)
The NR activity in leaves was observed through Long and Oaks 1990 Fresh leaf samples
(01g) were placed in 5ml of 100mM potassium phosphate pH 75 (added to 10
Isopropanol and 25mM KNO3) Tubes were vacuumed for 10 min to remove air from the
mixture and were placed in water bath shaker at 33oC for 60 min in dark The tubes were
placed in hot water (100oC) for 5 min 15 mL from the reaction mixture were added in 05
mL 20 sulphanilamide (wv dissolve in 5N HCl) and 025 mL 008 N-1-Napthylene-
66
diamine dihydrochloride Final volume up to 60 ml was made by adding distilled water
Color developed over the next 20 min Absorbance was measured at 540 nm using
spectrophotometer
67
222 Observations and Results
Sole and intercropped Ziziphus mauritiana
2221 Vegetative growth
Growth of Z mauritiana in terms of shoot root and plant length and number of leaves in
two different cropping system (sole and intercrop with C cajan) in two different irrigation
intervals has been presented in Figure 21 Appendix-XII A significant increase (plt0001)
in plant length was observed in 8th day irrigation in both the cropping systems in Z
mauritiana At 4th day of irrigation interval a non-significant increase in length was
observed in intercropped plants compared to sole crop Similarly at 8th day of irrigation
plants attain almost same heights in both the cropping systems
A significant increase (plt001) in root length was observed in sole Z mauritiana
at 8th day of irrigation compared to other treatments Smallest root length revealed in plants
that were irrigated at 4th day under sole crop system
The shoot length was significantly increase (plt0001) in plants which were
irrigated at 8th day under intercropped system However shoot length remains unaffected
when comparing the different cropping system at both the irrigation intervals
A significant increase (plt0001) in number of leaves was observed in intercropped
Z mauritiana plants compared to plants cultivated according to sole system However
more increase was observed in 4th day irrigated intercropped plant as compared to 8th day
The difference in number of leaves in sole crop at both irrigating intervals remains same
i Fresh weight
Figure 22 Appendix-XII showed fresh and dry weight of stem root and leaf of Z
mauritiana plant in two different cropping system (sole and intercrop with C cajan) in
two different irrigation intervals A significant increase (plt0001) in fresh weights of leaf
stem and root was observed in intercropping (with C cajan) 4th and 8th day of irrigation
interval compared to individual cropping of Z mauritiana In 4th day of irrigation the
increment was more pronounced in fresh weights of root (7848) leaves (4130) and
stem (4047) respectively with comparison to the crop growing alone Similarly
intercropping in 8th day of irrigation showed better growth of leaves (28) stem (12)
68
and root (31) against sole crop Whereas decrease in leaves 33 (plt005) stem 70
(plt0001) and root 60 (plt0001) fresh weights were observed in 8th day of irrigation
compared to 4th day intercropping However the difference was non-significant between
two sole crops irrigated at 4th and 8th day interval
ii Dry weight
Intercropping with comparison to the sole crop showed significant (plt0001) increase in
dry weights of leaves root and stem of Z mauritiana at 4th and 8th day of irrigation (Figure
22 Appendix-XII) At 4th day of irrigation intercropping showed an increment in dry
weights of Leaves (4366) stem (4109) and root (754) compared to the sole crop
Similar increase was observed in leaves (plt0001) stem (plt0001) and root (plt0001)
weights after 8th day of irrigation However intercropping at 8th day irrigation showed an
increment in root (19) stem (11) whereas a slight decrease (1) in leaves dry weight
When comparing irrigation time an increase in stem dry weight at 4th day whereas decline
in leaves dry weight was observed Root dry weights were more or less similar at both
irrigation intervals
iii Leaf weight ratio (LWR) root weight ratio (RWR) stem weight
ratio (SWR)
Leaf weight ratio (LWR) root weight ratio (RWR) stem weight ratio (SWR) of Z
mauritiana plant grown in two different cropping system (sole and intercrop with C cajan)
in two different irrigation intervals has been presented in Figure 23 Appendix-XII An
increased in LWR and SWR was recorded at 8th day of irrigation compared to 4th day of
irrigation in both cropping systems whereas decrease in RWR was observed LWR and
SWR remained un-change in sole and inter crop system However RWR increased in
intercrop system compared to sole crop system
iv Specific shoot length (SSL) specific root length (SRL)
Specific shoot length (SSL) specific root length (SRL) of Z mauritiana plant grown in
two different cropping system (sole and intercrop with C cajan) in two different irrigation
intervals has been presented in Figure 23 Appendix-XII SSL was observed higher in 8th
day of irrigation compared to 4th day in both the cropping systems However the increase
69
in SSL was lesser in sole crop compared to intercropping Similarly SRL was recorded
lesser in 4th day of irrigation compared to 8th day of irrigation in both cropping systems
Intercropped plants showed decline in SRL compared to sole crop plants Greatest SRL
revealed in plants that were irrigated after 8th day and planted according to sole crop
system
v Plant moisture
The moisture content of Z mauritiana plant grown in two different cropping system (sole
and intercrop with C cajan) in two different irrigation intervals has been presented in
Figure 23 Appendix-XII The moisture content of plants was significantly decreased
(plt005) in sole crop while increased (plt005) in intercropping at 8th day of irrigation
compared to 4th day At 4th day moisture remained same in both cropping system
However significant increase in moisture contents was observed in inter-crop system
compared to sole crop system after 8th day of irrigation
vi Plant Succulence
Succulence of Z mauritiana plant grown in two different cropping system (sole and
intercrop with C cajan) in two different irrigation intervals has been presented in Figure
23 Appendix-XII Plant succulence in 8th day was significantly reduced in sole crop
whereas increased in intercropping system In 4th day irrigated plants decrease in
succulence was noticed compared to plants that were irrigated at 8th day under sole crop
system However significant increase (plt0001) was observed in intercropped plants
irrigated at 4th day compared to 8th day
vii Relative growth rate (RGR)
Relative growth rate (RGR) of Z mauritiana plant grown in two different cropping system
(sole and intercrop with C cajan) in two different irrigation intervals has been presented
in Figure 23 Appendix-XII Relative growth rate remains unchanged at both irrigation
times under sole crop system However decline in 8th day was observed compared to 4th
day of irrigation under intercrop system Greatest RGR was recorded in plants that were
irrigated at 4th day under intercrop system
70
2222 Photosynthetic pigments
Photosynthetic pigments including Chlorophyll a chlorophyll b total chlorophyll
Chlorophyll ab ratio and carotinoids of Z mauritiana plant grown in two different
cropping system (sole and intercrop with C cajan) in two different irrigation intervals has
been presented in Figure 24 Appendix-XII
i Chlorophyll contents
A significant increase (plt0001) in chlorophyll a b and total chlorophyll was observed in
plants growing as sole crop compared to intercropped system at both the irrigation
intervals Higher chlorophyll contents were also recorded in plants that were irrigated at
8th day compared to 4th day of irrigation The chlorophyll ab ratio increased in 4th day
while decline in 8th day in intercropped system compared to sole crop However overall
results showed non-significant changes
ii Carotinoids
A significant increase (p lt 0001) in leaf carotinoids was observed in sole crop compare
to intercropped system at both irrigation times in Z mauritiana Least carotene content
was estimated in plants that were irrigated at 4th day under intercrop system
2223 Electrolyte leakage percentage (EL)
Electrolyte leakage percentage (EL) of Z mauritiana plant grown in two different
cropping system (sole and intercrop with C cajan) in two different irrigation intervals has
been presented in Figure 25 Appendix-XII A non-significant result was observed in
electrolyte leakage in plant growing at varying cropping system and irrigating intervals
2224 Phenols
Total phenolic contents in leaves of Z mauritiana plant grown in two different cropping
system (sole and intercrop with C cajan) in two different irrigation intervals has been
presented in Figure II25 Appendix-XII A significant increase (plt001) in total phenolic
contents was observed in intercropped growing at both irrigation interval compared to sole
crop However the increase was more pronounced at 8th day of irrigation Maximum
phenolic contents were measured in plants irrigated at 8th day under intercropped plants
71
2225 Proline
Total proline contents in leaves of Z mauritiana plant grown in two different cropping
system (sole and intercrop with C cajan) in two different irrigation intervals has been
presented in Figure 25 Appendix-XII A significant decreased (plt0001) was observed
in Z mauritiana cultivated according to intercropped system in both irrigation intervals
Maximum decrease was observed in intercropped plants irrigated at 8th day whereas
highest phenolic contents were observed in plants irrigated at 4th day under sole crop
system
2226 Protein and sugars
Protein and sugar contents in leaves of Z mauritiana plant grown in two different cropping
system (sole and intercrop with C cajan) in two different irrigation intervals has been
presented in Figure 26 Appendix-XII A nonsignificant difference in total protein and
sugar contents in Z mauritiana plants was observed in two different (4th and 8th day)
irrigation intervals However the interaction with time and irrigation interval also showed
nonsignificant result
2227 Enzyme essays
Antioxidant enzymes like Catalase (CAT) Ascorbate peroxidase (APX) Guaiacol
peroxidase (GPX) Superoxide dismutase (SOD) and Nitrate reductase activity in leaf of
Z mauritiana plant grown in two different cropping system (sole and intercrop with C
cajan) in two different irrigation intervals has been presented in Figure 27 and 28
Appendix-XII
i Catalase (CAT)
A significant decreased (plt0001) in catalase activities was observed in Z mauritiana
leaves in intercropped system in both time interval with compare to sole crop at 4th day
irrigated plant However maximum decline was in sole plants irrigated at 8th day interval
However their interaction with time was nonsignificant
72
ii Ascorbate peroxidase (APX)
A significant increase (plt0001) in APX activity was observed in 8th day irrigation in both
sole and intercropped plants with compare to sole and intercropped at 4th day irrigation
interval More increase (plt0001) was observed in intercropped Z mauritiana at 8th day
Whereas nonsignificant decrease was observed in two different cropping system in 4th day
irrigation interval However interaction between time and the treatments shows significant
values
iii Guaiacol peroxidase (GPX)
A significant (plt0001) increase in GPX was observed in 8th day intercropped Z
mauritiana plant with compare to irrigation intervals as well as cropping system However
at 4th day both cropping system showed nonsignificant difference Whereas more decline
was observed in 8th day sole crop The ANOVA reflects significant (plt005) interaction
between time and the cropped system
iv Superoxide dismutase (SOD)
A nonsignificant increase in SOD was observed in intercropped at 8th day irrigation
interval Whereas there was nonsignificant differences in 4th day intercropped and at both
time intervals of sole crop However interaction between time interval and the two
cropping system shows nonsignificant result
v Nitrate and Nitrate reductase
A significant increase (plt0001) in nitrate content and activity of nitrate reductase was
observed in intercropped plants of both irrigation intervals Increase in activity was
observed (plt0001) in intercropped Z mauritiana at 4th day
73
Sole and intercropped Cajanus cajan
2228 Vegetative growth
Growth of C cajan in terms of shoot root and plant length and number of leaves was
observed in two different cropping system (sole and intercrop with Z mauritiana) in two
different irrigation intervals has been presented in Figure 21 Appendix-XIII XIV A
significant increase (plt001) in plant length was observed in intercropped C cajan
compared to sole crop at both irrigation interval Whereas sole crop at 8th day interval
showed better results as compare to sole of 4th day Similarly root length remains
unaffected and showed non-significant change in both cropping systems and even at two
different irrigation intervals While shoot length was significantly (Plt001) decreased in
sole crop compared to intercropped at 4th day irrigation Whereas non-significant
difference be observed in rest of cropping systems growing at different irrigation interval
A significant increase (plt001) in leaves number was observed in intercropped
plants compared to sole crop at 4th and 8th day irrigation interval However most
significant decrease (plt0001) was observed in sole crop at 4th day
i Fresh weight
Figure 22 Appendix-XIV showed fresh and dry weight of stem root and leaf of C cajan
plant in two different cropping system (sole and intercrop with C cajan) in two different
irrigation intervals A significant increase (plt001) in fresh weight of leaf was observed in
intercropping (with Z mauritiana) at 4th and 8th day of irrigation interval compared to
individual cropping of C cajan The increase in intercropped system compared to sole
crop was more pronounced at 4th day (42) of irrigation than the 8th day (1701) Plants
showed higher leaves fresh weights in 8th day of irrigation compared to 4th day Similarly
the interaction between cropping system and the irrigation interval was significant
(Plt005)
An insignificant difference was observed in stem at 4th (15) and 8th (12) days
fresh weights in both intercropping system at two different irrigation intervals The
interaction between cropping system and the irrigation interval also showed non-
significant result
74
A non-significant difference in root fresh weight was observed in two different
cropping systems (sole and intercropped) in 4th and 8th day of irrigation intervals However
fresh weight of crop at 8th day irrigation interval was significantly increase (plt0001) over
4th day irrigation interval Similar pattern was observed in 4th day irrigated sole and
intercropped C cajan
ii Dry weight
A significant increase in leaves (42) stem (24) and root (18) dry weights were
observed in 4th day irrigation under intercropped system compared to sole However in 8th
day of irrigation this increase of dry weights was not much prominent Under sole crop
system dry weights of leaves stem and root was increased markedly in 8th day compared
to 4th day However in intercrop system the difference in dry weights was insignificant
between 8th and 4th day of irrigation
iii Leaf weight ratio (LWR) root weight ratio (RWR) stem weight
ratio (SWR)
Leaf weight ratio (LWR) root weight ratio (RWR) stem weight ratio (SWR) of C cajan
grown in two different cropping system (sole and intercrop with Z mauritiana) in two
different irrigation intervals has been presented in Figure 23 Appendix-XIV A
significant increase (plt0001) in LWR was observed at 8th day of irrigation compared to
4th day intercropped Similar pattern was noticed in RWR however SWR showed
insignificant difference between 4th and 8th day of irrigation A slight increase in LWR was
noticed in intercropped plants compared to sole Whereas RWR declined in intercrop
compared to sole and SWR remains un-changed
iv Specific shoot (SSL) root length (SRL)
Specific shoot length (SSL) specific root length (SRL) of C cajan grown in two different
cropping system (sole and intercrop with Z mauritiana) in two different irrigation
intervals has been presented in Figure 23 Appendix-XIV SSL and SRL were observed
to increase in sole crop compared to intercrop at 4th day of irrigation However increase
SSL and SRL was recorded in intercropped compared to sole at 8th day of irrigation A
general decline in SSL and SRL was noticed in 8th day of irrigation compared to 4th day
75
v Plant moisture
The moisture content of C cajan plant grown in two different cropping system (sole and
intercrop with Z mauritiana) in two different irrigation intervals has been presented in
Figure 23 Appendix-XIV The moisture content of plants was decreased significantly
(plt005) at 8th day irrigation interval compared to 4th day in sole crop Whereas non-
significant increase was observe in intercrop plants at 8th day of water irrigation
vi Plant succulence
Succulence of C cajan plant grown in two different cropping system (sole and intercrop
with Z mauritiana) in two different irrigation intervals has been presented in Figure 23
Appendix-XIV A significant increase (plt001) was observed in intercropped plants of C
cajan compared to sole crop at both irrigation interval However succulence increased in
sole crop and decreased in intercrop plants at 8th day of irrigation compared to 4th day
vii Relative growth rate (RGR)
Relative growth rate (RGR) of C cajan plant grown in two different cropping system (sole
and intercrop with Z mauritiana) in two different irrigation intervals has been presented
in Figure 23 Appendix-XIV A significant increase in RGR was observed in 8th day
compared to 4th day in both the cropping systems Highest increase was observed in
intercropped at 8th day irrigation At 4th day irrigation intervals intercropped plants
showed better RGR compared to Sole crop
2229 Photosynthetic pigments
Photosynthetic pigments including Chlorophyll a chlorophyll b total chlorophyll
Chlorophyll ab ratio and carotinoids of C cajan plant grown in two different cropping
system (sole and intercrop with Z mauritiana) in two different irrigation intervals has been
presented in Figure 24 Appendix-XIV
i Chlorophyll contents
A significant increase (plt005) in Chlorophyll a b and total chlorophyll was observed in
intercrop plants at 8th day irrigation interval Whereas at 4th day irrigation interval Sole
76
plants showed better results as compare to intercrop plants Plants at 8th day significantly
increase chlorophyll a b and total chlorophyll compared to 4th day of irrigation
Interactions between cropping systems and irrigation intervals were found significant
(chlorophyll a (plt001) chlorophyll b (plt001) and total chlorophyll (plt0001)
respectively) However the ratio of chlorophyll ab showed non-significant values in
cropping irrigation interval and their interaction
ii Carotenoids
A significant increase (plt001) in carotinoids was observed in intercropped C cajan at 8th
day of irrigation Whereas non-significant increase was observed in sole crop at 4th day
irrigation interval with compare to intercrop However the irrigation intervals showed
significant (plt0001) difference Whereas interaction of cropping system with irrigation
time also showed significant correlation (plt0001)
22210 Electrolyte leakage percentage (EL)
Electrolyte leakage percentage (EL) of C cajan plant grown in two different cropping
system (sole and intercrop with Z mauritiana) in two different irrigation intervals has been
presented in Figure 25 Appendix-XIV A non-significant increase in EL percentage was
observed in sole crop compared to intercrop plants growing at 4th and 8th day of irrigation
No significant change was noticed between the irrigation times to C cajan The interaction
between cropping system (sole and intercropped) and irrigation interval (4th and 8th day)
also showed non-significant
22211 Phenols
Total phenolic contents in leaves of C cajan plant grown in two different cropping system
(sole and intercrop with Z mauritiana) in two different irrigation intervals has been
presented in Figure 25 Appendix-XIV A nonsignificant result was observed in total
phenolic contents of C cajan growing as sole and intercropped system at two different
irrigation intervals However the interaction between irrigation intervals with crop system
showed significant (p lt 005) results
77
22212 Proline
Total proline contents in leaves of C cajan plant grown in two different cropping system
(sole and intercrop with Z mauritiana) in two different irrigation intervals has been
presented in Figure 25 Appendix-XIV Proline contents in leaves of C cajan showed
nonsignificant increase at 4th day of irrigation interval in both sole and intercropped
system Whereas the interaction between irrigation intervals showed significant (Plt001)
results
22213 Protein and Sugars
Protein and sugar contents in leaves of C cajan plant grown in two different cropping
system (sole and intercrop with Z mauritiana) in two different irrigation intervals has been
presented in Figure 26 Appendix-XIV A less significant difference (plt005) was
observed in two different (4th and 8th day) irrigation intervals However there was
nonsignificant difference in two cropped system More decrease was observed at 4th day
intercropped plants Whereas nonsignificant increase in 8th day intercropped and 4th day
sole plants were observed However interaction between crop and time of irrigation
showed significant results (plt0001)
22214 Enzyme assay
Antioxidant enzymes like Catalase (CAT) Ascorbate peroxidase (APX) Guaiacol
peroxidase (GPX) Superoxide dismutase (SOD) and Nitrate reductase activity in leaf of
C Cajan plant grown in two different cropping system (sole and intercrop with Z
mauritiana) in two different irrigation intervals has been presented in Figure II27
Appendix-XIV
i Catalase (CAT)
A significant increase (plt001) in catalase activity was observed in intercropped C cajan
at 8th day of irrigation with compare to other irrigation time and cropped system Whereas
increase was observed in sole crop at 4th day irrigation interval with compare to 8th day
However the irrigation intervals and the interaction between cropping system with
irrigation interval also showed nonsignificant correlation
78
ii Ascorbate peroxidase (APX)
A non-significant increase in APX was observed in intercropped plant in 4th and 8th day
irrigation interval with compare to sole crops Sole crop at 8th day showed maximum
decline However the difference between cropping system and their interaction with
irrigation interval also showed nonsignificant results
iii Guaiacol peroxidase (GPX)
A significant increase (plt005) in GPX activity was observed in 8th day sole crop
However there was nonsignificant difference among intercropped at two time interval and
sole crop at 4th day irrigation Whereas interaction with time to irrigation interval also
showed less significant results
iv Superoxide dismutase (SOD)
A significant decrease (plt0001) in SOD activity was observed in intercropped at 8th day
irrigation interval with compare to 4th day Maximum decrease was observed in 8th day
intercropped Whereas sole crop at 8th day also showed better result to 4th day sole crop
However ANOVA showed significant correlation among crop system at two time interval
and 4th day irrigation
v Nitrate and Nitrate reductase
Nitrate content and activity of nitrate reductase was nonsignificant in both cropping
system using both irrigation intervals However nonsignificant increase was observed in
nitrate content and activity of nitrate reductase in intercropped Z mauritiana at 8th day
79
Sole IntercropSole Intercrop
No o
f le
aves
0
20
40
60
Len
gth
(cm
)
0
40
80
120
160
200
2404
th day
Cajanus cajan
a
RootShoot
ab
a
a
b
a
a
8th
day
Figure 21 Vegetative parameters of Z mauritiana and C cajan at grand period of growth under sole and
intercropping system at 4th and 8th day irrigation intervals (Bars represent means plusmn standard error
of each treatment and significance among the treatments was recorded at p lt 005)
Sole IntercropSole Intercrop
No of
leav
es
0
200
400
600
Len
gth
(cm
)
0
40
80
120
160
200
240
Ziziphus mauritiana
RootShoot
4th
day 8th
days
b b
a a
a
b
cc
80
Sole Intercrop
Dry
wei
ght
(g)
50
100
150
200
250
300
Fre
sh w
eight
(g)
100
200
300
400
500
Sole Intercrop
4th
day 8th
day
a
b
c
a
b b aa
b
b
c c
a
bc
a
c
ba
b
c
a
b
c
Leaf Stem Root
Ziziphus mauritiana
Sole Intercrop
Dry
wei
ght
(g)
2
4
6
8
10
12
Fre
ah w
eight
(g)
5
10
15
20
25
30
35
40
Sole Intercrop
4th
day 8th
day
aa
b
a
a
b
a
b
c
a
b
c
a
c
b
a a
b
a
b
c
a
b
c
Leaf Stem Root
Cajanus cajan
Figure 22 Fresh and dry weight of Z mauritiana and C cajan plants under sole and intercropping system
at 4th and 8th day irrigation intervals (Bars represent means plusmn standard error of each treatment
and significance among the treatments was recorded at p lt 005)
81
Figure 23 Leaf weight ratio (LWR) root weight ratio(RWR) shoot weight ratio(SWR)specific shoot
length (SSL) specific root length (SRL) plant moisture Succulence and relative growth rate (RGR) of
Zmauritiana and C cajan grow plants under sole and intercropping system at 4th and 8th
day irrigation
intervals (Bars represent means plusmn standard error of each treatment and significance among the treatments
was recorded at p lt 005)
Sole Intercrop
Mo
istu
re (
)
0
20
40
60
80
SS
L (
cm g
-1)
01
02
03
04
05
06
RW
R (
g g
-1 D
W)
005
010
015
020
LW
R (
g g
-1 D
W)
01
02
03
04
05
06
07
Sole Intercrop
Su
ccu
lan
ce
(g H
2O
g-1
DW
)00
05
10
15
20
25
RG
R
(g g
-1 d
ay-1
)
001
002
003
004
005
SR
L (
cm g
-1)
05
10
15
20
25
SW
R (
g g
-1 D
W)
02
04
06
08
10
Ziziphus mauritiana
a a
bb
b
a
bb
a
b
aa
a aa
b
a
bb
c
b
a
bb
b
aa a
ba
bc
4th day
8th day
82
(Figure 23 continuedhellip)
Sole Intercrop
Mo
istu
re (
)
0
20
40
60
80
SS
L (
cm g
-1)
2
4
6
8
10
12
RW
R (
g g
-1 D
W)
002
004
006
008
010
012
014
LW
R (
g g
-1 D
W)
01
02
03
04
05
06
07
08
Sole Intercrop
Su
ccu
lan
ce
(g H
2O
g-1
DW
)
00
05
10
15
20
25
RG
R
(g g
-1 d
ay-1
)
001
002
003
004
005
SR
L (
cm g
-1)
5
10
15
20
25
SW
R (
g g
-1 D
W)
02
04
06
08
10
Cajanus cajan
a aab
a aaa
a
bba
a
b b
c
a aab
a
bbb
abbb
aa
bc
8th day
4th day
83
Sole Intercrop
Car
oti
noid
s (m
g g
-1 F
W)
00
01
02
03
04
05
Ch
loro
phyll
(m
g g
-1 F
W)
00
03
06
09
12
15
Sole Intercrop
4th
day 8th
day
Ch
loro
phyll
ab
rat
io
00
05
10
15
20
25Chl ab
Ziziphus mauritiana
a a
bb
a
b
a
b
a ab
b
Chl aChl b
Figure 24 Leaf pigments of Zmauritiana and C cajan grow plants under sole and intercropping system at
4th and 8th
day irrigation intervals (Bars represent means plusmn standard error of each treatment and
significance among the treatments was recorded at p lt 005)
Sole Intercrop
Car
oti
noid
s (m
g g
-1 F
W)
00
01
02
03
04
05
Ch
loro
phyll
(m
g g
-1 F
W)
00
03
06
09
12
15
18
Sole Intercrop
4th
day 8th
day
ab r
atio
00
05
10
15ab
ab
Cajanus cajan
bb b
a
a
b
cc
bb b
a
84
Ele
ctro
lyte
lea
kag
e(
)
0
5
10
15
4th
day 8th
dayP
hen
ols
(m
g g
-1)
0
5
10
15
20
25
30
Sole Intercrop
Pro
line
( g g
-1)
0
10
20
30
40
Sole Intercrop
Ziziphus mauritiana
a a a
a
b b ba
a
b
c
d
Figure 25 Electrolyte leakage phenols and prolein of Z mauritiana and C cajan at grand period of growth
plants under sole and intercropping system at 4th and 8
th day irrigation intervals (Bars represent
means plusmn standard error of each treatment and significance among the treatments was recorded at
p lt 005)
85
(Figure 25 continuedhellip)
E
lect
roly
te l
eakag
e(
)
0
20
40
60
80
4th
day 8th
day
Phen
ols
(m
g g
-1)
0
2
4
6
8
10
12
Sole Intercrop
Pro
line
( g g
-1)
000
003
006
009
012
015
018
Sole Intercrop
Cajanus cajan
a aa
a
a a aa
aa a
a
86
Sole Intercrop
Sugar
s (m
g g
-1)
0
20
40
60
Sole Intercrop
Pro
tein
(m
g g
-1)
00
02
04
06
4th
day 8th
day
Ziziphus mauritiana
a aa a
a
a a a
Sole Intercrop
Sugar
s (m
g g
-1)
0
10
20
30
Sole Intercrop
Pro
tein
(m
g g
-1)
00
02
04
06
08
10
4th
day 8th
dayCajanus cajan
ab
a
c
a
b
cc
Figure 26 Total protein and sugars in leaves of Z mauritiana and C cajan plants under sole and
intercropping system at 4th and 8th
day irrigation intervals (Bars represent means plusmn standard
error of each treatment and significance among the treatments was recorded at p lt 005)
87
Sole Intercrop
SO
D (
Unit
s m
g-1
)
0
2
4
6
8
10
12
14
Sole Intercrop
Cat
alas
e (U
nit
s m
g-1
)
0
5
10
15
20
25
AP
X (
Unit
s m
g-1
)
0
20
40
60
80
GP
X (
Unit
s m
g-1
)
00
01
02
03
04
05
4th
day 8th
day
Ziziphus mauritiana
a
bc
c
a
b
cc
a
c
b
b
b bb
a
Figure 27 Enzymes activities in leaves of Z mauritiana and C cajan plants under sole and intercropping
system at 4th and 8th
day irrigation intervals (Bars represent means plusmn standard error of each
treatment and significance among the treatments was recorded at p lt 005)
88
(Figure 27 continuedhellip)
Sole Intercrop
SO
D (
Unit
s m
g-1
)
0
1
2
3
4
5
Sole Intercrop
Cat
alas
e (U
nit
s m
g-1
)
0
2
4
6
8
4th
day 8th
dayG
PX
(U
nit
s m
g-1
)
00
05
10
15
20
25
Cajanus cajan
aA
PX
(U
nit
s m
g-1
)
0
20
40
60
80
100
bb
b
aaa
b
a
bbb
a
c
a
b
89
Sole Intercrop
NO
3 (
mM
ol
g-1
)
00
02
04
06
08
10
12
14
8th
day
Sole Intercrop
Nit
rate
Red
uct
ase
(mM
ol
g-1
)
0
1
2
3
4
4th
day
Nitrate reductaseNO
3
Ziziphus mauritiana
a
b
c
cb
b
b
a
Sole Intercrop
NO
3 (
mM
ol
g-1
)
00
02
04
06
08
10
12
8th
day
Sole Intercrop
Nit
rate
Red
uct
ase
(mM
ol
g-1
)
0
2
4
6
8
10
12
4th
dayCajanas cajan
a
bb
b
aa
aa
Nitrate reductase NO3
Figure 28 Nitrate reductase activity and nitrate concentration in leaves of Z mauritiana and C cajan plants
under sole and intercropping system at 4th and 8th
dayirrigation intervals (Values represent means
plusmn standard error of each treatment and significance among the treatments was recorded at p lt
005)
90
23 Experiment No 8
Investigations of intercropping Ziziphus mauritiana with Cajanus cajan
on marginal land under field conditions
231 Materials and Methods
2311 Selection of plants
Ziziphus mautitiana and Cajanus cajan were selected for this study as described in chapter
1
2312 Experimental field
Field of Fiesta Water Park was selected to investigate intercropping of Z mauritiana with
Ccajan It is situated about 50 km from University of Karachi at super highway toward
HyderabadThe area of study has subtropical desert climate with average annual rain fall
is ~20 cmmost of which is received during the monsoon or summer seasonSince summer
temperature (April to October) are approx 30-35 degC and the winter months (November to
March) are ~20 degC Wind velocity is generally high all the year Topography of the area
was uneven with clay- loam soil having gravels Xerophytic plants are pre-dominantly
present in the area including Prosopis spp Acacia spp Euphorbia spp Caparus
deciduas etc
2313 Soil analysis
Before conducting experiment soil of Fiesta Water Park field was randomly sampled at
three locationsatone feet of depthusing soil augerThese soil samples were analyzed in
Biosaline Research Laboratory Department of Botany University of Karachi to
determine its physical and chemical properties
i Bulk density
Bulk density was determinedin accordance with Blake and Hartge (1986) by using the
following formula
Bulk density = Oven dried soil (g) volume of soil (cm3)
91
ii Soil porosity
Soil porosity was calculated in accordance with Brady and Weil (1996) by using the
following formula
Soil porosity = 1- (bulk density Particle density) times 100
Where particle density = 265 gcm3
iii Soil texture and particle size
Soil particle size was determined by Bouyoucos hydrometric method in accordance with
Gee and Or (1986)On the basis of clay silt and sand percentages soil texture was
determined by using soil texture triangle presented in Figure 31
iv Water holding capacity
Water holding capacity in percentages was calculatedaccording to George et al (2013)
v pH and Electrical conductivity of soil (ECe)
Soil saturated paste was made with de-ionized water and leave for 24 hours Soil solution
was extracted through Buckner funnel and suction pump (Rocker 300) pH of soil
solution was taken on Adwa AD1000 pHMV meter and ECe was taken on electrical
conductivity meter (4510 Jenway)
2314 Experimental design
Six months old grafted Ziziphus mauritiana saplings were carefully transported in field of
Fiesta Water Park
Three equal size plots of 100times10 sq ft were prepared for this experiment
Plot ldquoArdquo = Ziziphus mauritiana (Sole crop)
Plot ldquoBrdquo = Cajanus cajan (Sole crop)
Plot ldquoCrdquo = Ziziphus mauritiana + Cajanus cajan (intercropped)
In plot lsquoArsquo and lsquoCrsquo pits of two cubic feet depth were prepared in two parallel rows
at a distance of 10 feet (Yaragattikar amp Itnal 2003)so that the distance of pits within the
row and the distance of pits between the rows were same Each row bears nine pits
Eighteen healthy saplings of nearly equal height and vigor of Z mauritiana were
92
transplanted in the pits and were fertilized with cow-dong manure Plants were irrigated
with underground (pumped) water initially on alternate day for two weeks older leaves
fall down completely and new leaves appeared in this establishment period Later the
irrigation interval was kept fortnightly Electrical conductivity of irrigated water (ECiw)
was 24 plusmn 05 dSm-1
After establishment of Z mauritiana water soaked seeds of intercropping plant (C
cajan) were sown in plot lsquoCrsquo Three vertical lines (strips design) of equal distance were
made between the rows of Z mauritiana The distance between the line was one feet
Eleven C cajan were maintained in each line at a distance of one feet which constitute a
total of 33 C cajan in 3 lines There were 264 plants of C cajan arranged in strip pattern
as intercrop for eighteen Z mauritiana A sole crop of C cajan in plot lsquoBrsquo was arranged
with the same manner to serve as control Similarly plot lsquoArsquo was served as control of Z
mauritianaThe experiment was observed up to reproductive yield of each plant
Field diagram Theoritical model of intercropping system used in this study showing sole crop in Plot lsquoArsquo
(Z Mauritiana) and Plot lsquoBrsquo (C cajan) while Plot lsquoCrsquo represents intercropping of both
species at marginal land
Six Z mauritiana plants were randomly selected from their two rows of block lsquoCrsquo
which were facing two rows of C cajan on either sides Similarly ten plants of C cajan
facing Z mauritiana were randomly selected for further study At the same manner six Z
mauritiana from block lsquoArsquo and ten C cajan from block lsquoBrsquo grown as sole crop were
selected as control for further study
93
2315 Vegetative and reproductive growth
Vegetative growth of Z mauritiana plant was noted in terms of height volume of canopy
while height and number of branches in Ccajan bimonthly after establishment Fresh and
dry weightsof leaves stem and root were observed at final harvest in both plant species
growing as sole or intercropping
Reproductive growth of Z mauritiana such as number length and diameter fruit
weight per ten plant and average fruit yield was measured at termination of the experiment
Whereas reproductive growth in C cajan was monitored in terms of number of pods
number of seeds weight of pods and weight of seed
2316 Analyses on some biochemical parameters
Following biochemical analysis was conducted in Fully expended leavesof Z mauritiana
and C cajan growing as sole and as intercropped at grand period of growth Additionally
fruits of Z mauritiana were also analyzed for their protein soluble and insoluble sugars
and total phenolic contents
i Photosynthetic pigments
Photosynthetic pigments including chlorophyll a chlorophyll b and total chlorophyll were
estimated in leaves of Z mauritiana and C cajan according to procedure described in
chapter 1
ii Protein in leaves
Protein contents were estimated in leaves of Z mauritiana and C cajan according to
procedure described in chapter 1
iii Total soluble sugars in leaves
Total soluble sugars were estimated in leaves of Z mauritiana and C cajanaccording to
procedure described in chapter 1
94
iv Phenolic contents in leaves
Phenolic content were estimated in leaves of Z mauritiana and C cajan according to
procedure described in chapter 1
2317 Fruit analysis
i Protein in fruit
Protein content in fruit of Z mauritiana was estimated according to procedure described
in chapter 1
ii Total soluble sugars in fruits
Total soluble sugars in ripe fruits of Z mauritiana were estimated according to procedure
described in chapter 1
iii Phenolic contents in fruits
Phenolic contents in fruits of Z mauritiana were estimated according to procedure
described in chapter 1
2318 Nitrogen estimation
Nitrogen was also estimated in root zone soil as well as in fully expended leaves of Z
mauritiana and C cajan plants
Total nitrogen in leaves and soil was estimated through AOAC method 95504
(2005) One g of dried powdered sample in round bottle flask was digested in presence of
20 mL H2SO4 15 mL K2SO4 and 07g CuSO4 at 400oC heating mental After digestion 80
ml distilled water was added in digest Then distillation was done at 100oC by adding 100
mL of 45 NaOH (drop wise) in digested solution Steam was collected in 35 mL of 01M
HCl in a flask Three samples of 10 mL each steam collected solution were taken and 2-3
drops of methyl orange was added as indicator Titration was made with 01M NaOH
Changeappearance of color indicates the completion of reactionPercent nitrogen was
calculated through following equation
N = (mL of acid times molarity) ndash (mL of base times molarity) times 14007
95
2319 Land equivalent ratio and Land equivalent coefficient
The LER defined the total land area needed for sole crop system to give yield obtained
mixed crop It is mainly used to evaluate the performance of intercropping (Willey 1979)
Land equivalent ratio (LER) of two crops was estimated according to (Willey 1979) by
using formula
Whereas partial LER of Z mauritiana calculated according to
Similarly Partial LER of Ccajan were calculated as
Land equivalent coefficient (LEC) an assess of dealings the effectiveness of relationship
of two crops (Alhassan et al 2012) was calculated by using (Adetiloye et al 1983)
equation as
Yield was calculated in gram fresh weight LER and LEC of height and total chlorophyll
were also calculated by using above formula by substituting their values with yield (fruits
of Z mauritiana and seeds of C cajan) to height fruits and chlorophyll respectively
23110 Statistical analysis
Data were analyzed by using (ANOVA) and the significant differences between treatment
means wereexamined by least significant difference (Zar 2010) All statistical analysis
was performed using SPSS for windows version 14 and graphs were plotted using Sigma
plot 2000
LER= Yield of Z mauritiana + Yield of C cajan (in intercropped) + Yield of C cajan + Yield of Z mauritiana (in intercropped)
Yield of Z mauritiana (sole) Yield of C cajan (sole)
Partial LER = Yield of Z mauritiana + Yield of C cajan (in intercropped)
Yield of Z mauritiana (sole)
Partial LER = Yield of C cajan + Yield of Z mauritiana (in intercropped)
Yield of C cajan (sole)
LEC = Partial LER of Z mauritiana times Partial LER of C cajan
96
232 Observations and Results
2321 Vegetative parameters
Vegetative growth parameters of Z mauritiana include plant height volume of canopy
grown individually as well as intercropped with C cajan is presented in Figure 29
Appendix-XV A significant increase in height and canopy volume of Z mauritiana with
time (p lt 0001) and cropping system (p lt 005) was observed However the interaction
between time and cropping system showed non-significant results In general the
intercropped plants were showed higher values in all vegetative parameters than sole crop
and this increase was more pronounced after 60 days
Figure 29 Appendix-XVII showed the vegetative growth parameters of C cajan
including height and number of branches Height of C cajan was significantly increased
(plt0001) with increasing time in plants growing sole and as intercropped with Z
mauritiana The interaction with time to crop height also showed significant (plt0001)
results in both cropping systems However slight decline in height of intercropped C
cajan was noticed at 120 days compared to sole crop Number of branches was significant
increased (plt0001) in both crops with increasing time The interaction of time with
branches also showed significant (plt0001) results in both cropping systems However
number of branches was slightly increased in intercropped plants at 120 days compared to
sole crop
2322 Reproductive parameters
i Fruit number and weight (fresh and dry)
Reproductive parameters of Z mauritiana and C cajan at grand period of growth under
sole and intercropping system has been presented in Figure 210 Appendix-XVI XVIII
Individual and interactive effect of time (p lt0001) and treatment (plt001) on number and
fresh weight of fruits of Z mauritiana was showed significant results Similarly plants
grown with C cajan showed significant increase (p lt0001) in fresh weight of fruits (p
lt005) whereas fruit dry weight and circumference was non-significant in comparison to
sole crop
97
In C cajan flowers were appeared only at blooming phase (during 60 days of treatment)
and no difference in number of flowers was observed in both cropping systems (sole and
with Z mauritiana (Figure 210 XVII)
Leguminous pods were initiated soon after flowering period (during 60 days) and
last till end of the experiment (120 days) A significant increase (plt0001) in pod numbers
was observed with increasing time in both sole and intercropped system But non-
significant differences in number of pods of both cropping system and their interaction
with time were observed
Similarly number and weight of C cajan seeds were showed non-significant difference
in both cropping systems
2323 Study on some biochemical parameters
i Photosynthetic pigments
Leaf pigments of Zmauritiana and C cajan grow plants under sole and intercropping has
been presented in Figure 211 Appendix-XVI XVIII In Z muritiana leaves A significant
increase (plt005) in chlorophyll a chlorophyll b total chlorophyll and carotinoids was
observed when grown as intercrop whereas the effect on chlorophyll ab ratio was non-
significant as that of sole one
In C cajan a slight decrease (plt005) in chlorophyll lsquobrsquo and total chlorophyll
(plt001) was observed in intercropped plants compare to sole one Whereas chlorophyll
lsquoarsquo chlorophyll ab ratio and carotinoids showed nonsignificant difference between sole
and intercropped C cajan
ii Total proteins sugar phenols
Sugars protein and phenols in leaves of Z mauritianaand C cajan at grand period of
growth under sole and intercropping system is presented in Figure 212 Appendix-XVI
XVIII Total proteins and soluble and insoluble sugar content of Z mauritiana leaves was
unaffected throughout the experiment However an increase in total phenolic content
(plt001) was observed in intercropped Z mauritiana plants than grown individually
98
In C cajan total soluble sugars protein and phenols in leaves showed non-
significant differences between sole to intercropped plants
Sugars protein and phenols in fruits of Z mauritiana grown under sole and
intercropping system is presented in Figure 213 Appendix-XVI A non-significant
increase was observed in phenolic as well as in soluble insoluble and total sugar contents
in fruits of Z mauritiana plants grown with C cajan (intercrop) as compare to the fruits
of sole crop
2324 Nitrogen Contents
Nitrogen in leaves and in soil of Z mauritiana and C cajan growing under sole and
intercrop system is presented in Figure 214 Appendix-XVI XVIII ANOVA showed a
non significant effect on nitrogen content of leaf as well as root zone soil of Z mauritiana
and C cajan grown individually or as intercropping system
2225 Land equivalent ratio (LER) and land equivalent coefficient
(LEC)
Land equivalent ratio (LER) Land equivalent coefficient (LEC) of height chlorophyll and
yield of of Z 98auritiana and C cajan growing as sole and intercropping system in has
been presented in Table 22 The LER using height of both species was nearly 2 in which
PLER of Z mutitania was 48 and PLER of C cajan was 519 Whereas the calculated
values of the land equivalent coefficient (LEC) of Z mauritiana and C cajan remained
9994
The LER using yield of both species was above 2 in which PLER of Z mauritiana
was 46 Whereas PLER of C cajan was 543 However the calculated values of LEC
of both species were 100
The LER using total chlorophylls of both species were more than 25 in which
PLER of Z mauritiana was 344 and as that of PLER of C cajan was 655 Whereas
the calculated values of LEC was 999 of both the species
99
Table 21 Soil analysis data of Fiesta Water Park experimental field
Serial number Parameters Values
1 ECe (dSm-1) 4266plusmn0536
2 pH 8666plusmn0136
3 Bulk density (gcm3) 123plusmn0035
4 Porosity () 53666plusmn1333
5 Water holding capacity () 398plusmn2811
6 Soil texture Clay loam
7 Sand () 385plusmn426
8 Silt () 3096plusmn415
9 Clay () 305plusmn1
Ece is the electrical conductivity of saturated paste of soil sample
Figure 29 Soil texture triangle (Source USDA soil classification)
100
Ziziphus mauritiana
Days
0 60 120
Volu
me
(m3)
0
10
20
30
Days
0 60 120
Hei
ght
(cm
)
0
50
100
150
200
250
Sole Intercrop
a
a
bb
c c
aa
bb
c c
Cajanus cajan
Days
0 60 120
Bra
nch
es (
)
0
10
20
30
Days
0 60 120
Hei
ght
(cm
)
0
50
100
150
200
250
300
Sole Intercrop
aa
bb
c c
aa
bb
c c
Figure 210 Vegetative growth of Z mauritiana and C cajan growing under sole and intercropping
system (Bars represent means plusmn standard error of each treatment and significance among the
treatments was recorded at p lt 005)
101
Ziziphus mauritiana
Fresh Dry
Fru
it w
eig
ht
(g)
0
50
100
150
200
Days
0 60 120 180
Nu
mb
er o
f F
ruit
s
0
100
200
300
Sole Intercrop
a
b
a
b
c
c
dd
Cajanus cajan
0 60 120
Num
ber
of
Pods
0
50
100
150
200
Days
0 60 120
Num
ber
of
Flo
wer
s
0
50
100
150
Sole Intercrop
Days
aa
bb
c c
Sole Intercrop
Num
ber
of
See
ds
0
100
200
300
400
500
See
d W
eight
(g)
0
10
20
30
40
50
60Number of seedsSeed weight
Figure 211 Reproductive growth of Z mauritiana and C cajan growing under sole and intercropping
system (Bars represent means plusmn standard error of each treatment and significance among the
treatments was recorded at p lt 005)
102
Ziziphus mauritiana
Cajanus cajan
Figure 212 Leaf pigments of Zmauritiana and C cajan growing under sole and intercropping (Bars
represent means plusmn standard error of each treatment and significance among the treatments was
recorded at p lt 005)
Sole Intercrop
Car
ote
noid
s (m
g g
-1)
00
01
02
03C
hlo
rophyl
l (m
g g
-1)
00
02
04
06
08
ab r
atio
00
05
10
15
20
25
ab
ab
Sole Intercrop
Car
ote
no
ids
(mg
g-1
)
00
01
02
03
Ch
loro
ph
yll
(m
g g
-1)
00
02
04
06
08
10
ab
rat
io
0
1
2
3
4ab
ab
103
Ziziphus mauritiana
Sole Intercrop
Lea
f P
hen
ols
(m
g g
-1)
0
2
4
6
8
10
12
Lea
f P
rote
ins
(mg
g-1
)
0
2
4
6
8
Lea
f S
ug
ars
(mg
g-1
)
0
5
10
15
20
25
30
35SoluableInsoluable
Figure 213 Sugars protein and phenols in leaves of Z mauritiana and C cajan at grand period of growth under
sole and intercropping system (Bars represent means plusmn standard error of each treatment and
significance among the treatments was recorded at p lt 005)
104
(Figure 212 continuedhellip)
Cajanus cajan
Sole Intercrop
Lea
f P
hen
ols
(m
g g
-1)
0
2
4
6
8
Lea
f P
rote
ins
(mg g
-1)
00
05
10
15
20
Lea
f S
ugar
s (m
g g
-1)
0
2
4
6
8
105
Ziziphus mauritiana
Sole Intercrop
Fru
it P
hen
ols
(m
g g
-1)
0
2
4
6
8
10
12
14
Fru
it P
rote
ins
(mg g
-1)
00
02
04
06
08
10
Fru
it S
ugar
s (m
g g
-1)
0
5
10
15
20
25
30
35 SoluableInsoluable
Figure 214 Sugars protein and phenols in fruits of Z mauritiana grown under sole and intercropping
system (Bars represent means plusmn standard error of each treatment and significance among the
treatments was recorded at p lt 005)
106
Z mauritiana
Sole Intercrop
Nit
rogen
(
)
0
1
2
3
4
5
6
7 LeafSoil
Cajanus cajan
Sole Intercrop
Nit
rogen
(
)
0
1
2
3
4
5
6
7 LeafSoil
Figure 215 Nitrogen in leaves and in soil of Z mauritiana and C cajan growing under sole and intercrop
system (Bars represent means plusmn standard error of each treatment and significance among the
treatments was recorded at p lt 005)
107
Table 22 Land equivalent ratio (LER) and Land equivalent coefficient (LEC) with reference to height chlorophyll and yield of of Z mauritiana and C cajan growing
under sole and intercropping system
Plant species Parameters Formulated with
reference to Height
Formulated with
reference to Total
Chlorophyll
Formulated with reference to Yield
(fresh weight of Z mauritiana fruit
and seed of C cajan)
Z mauritiana Partial LER 1027 1666 1159
C cajan Partial LER 0950 0877 0993
Intercropped
Total LER 1977 2543 2152
Z mauritiana amp C cajan
(Sole and intercropped) LEC 0975 1461 1151
107
108
24 Discussion
Intercropping is a common practice used to obtain better yield on a limited area through
efficient utilization of given resources which may not be achieved by growing each crop
independently (Mucheru-Muna et al 2010) In this system selection of appropriate crops
planting rates and their spatial arrangement can reduce competition for light water and
nutrients (Olowe and Adeyemo 2009) In general increased growth (biomass height
volume circumference biomass succulence SSL SRL SSR LWR SWR RWR and
RGR) of each species is a good indicator of successful intercropping The SRL and SSL
measure the ratio between the lengths of root or shoot per unit dry weight of respective
tissues (Wright and Westoby 1999) The weight ratio of leaf stem and root to total plant
weight (LWR SWR and RWR) describes the allocation of biomass towards each organ to
maximize overall relative growth rate (RGR) which explains how plant responds to certain
type of condition (Reynolds and Antonio 1996) In this study height and canopy volume
of Z mauritiana and height and branches of C cajan were increased when grown together
in comparison to sole crop in field experiment (Figure 29) Whereas in drum pot culture
biomass generally the length of plant canopy volume number of leaves RGR LWR
SWR RWR SSL and SRL were either higher or unaffected in both species growing in
intercropping at 4th and 8th days intervals (Figure 21-23) Similar beneficial effects on
growth of other intercrops have also been reported under different conditions (Yamoah
1986 Atta-Krah 1990 Kass et al 1992 Singh et al 1997) Dhyani and Tripathi (1998)
observed increased height stem diameter crown width and timber volume of three
intercropped species than sole crop Bhat et al (2013) also revealed significant
improvement in annual extension height and spread in apple plants intercropped with
leguminous plants
The increased growth of both intercropped plants of this study was well reflected
by their biochemical parameters Leaf pigments like chlorophyll a chlorophyll b and total
chlorophyll were either higher or remained unaffected (Figure 211) in both intercropped
plants than sole crops of field experiments Whereas in drum pot culture chlorophyll
content (Figure 24) was higher only in intercropped C cajan (specially in 8th days) Bhatt
et al(2008) and Massimo and Mucciarelli (2003) also reported the increased accumulation
of chlorophyll a b and total chlorophylls in leaves of soybean and peppermint when
109
grown with their respective intercrops Our results are also in agreement with Liu et al
(2014) and Otusanya et al (2008) reported similar results in Lycopersican esculentum and
later in Capsicum annum as well Some other reports are also available which shows non-
significant effect on leaf pigments in both cropping systems (Shi-dan 2012 Luiz-Neto-
Neto et al 2014)The synthesis and activity of chlorophyll depends on severity and type
of applied stress it generally increase in low saline mediums (Locy et al 1996) or
remained unaffected however sometimes stimulated (Kurban et al 1999 Parida et al
2004 Rajesh et al 1998)
Proteins and carbohydrates (sugars) perform vast array of functions which are
necessary for plant growth and reproduction (Copeland and McDonald 2012) Variation
in their contents helps to predict plant health which is usually decreased with applied stress
(Arbona et al 2013) Both are also the compulsory factors of animals diet since they
cannot manufacture sugars and some of the components of proteins which must be
obtained from food (Bailey 2012) In our experiment protein content was either remained
unchanged or increased which indicated a good coordination of both intercrops in field
and drum pot experiments (Figure 26 and 212) Liu et al (2014) also found that protein
and sugars were not affected in tomatogarlic intercrops In another experiment similar
results were found when corn was grown with and without intercropping (Borghi et al
2013)
Reactive oxygen species (ROS) are produced as a spinoff of regular metabolism
however under stress the overproduction of ROS may lead to oxidative damage (Baxter et
al 2014) In low concentrations ROS worked as messengers to regulate several plant
processes and also helps to improve tolerance to various biotic and abiotic stresses (Miller
et al 2009 Nishimura and Dangl 2010 Suzuki et al 2011) but when the concentration
goes beyond the critical limit ROS would become self-threatening at every level of
organization (Foreman et al 2003) To maintain a proper workable redox state an
efficient scavenging system of enzymatic (SOD CAT GPX and APX) andor non-
enzymatic (polyphenols sugars glutathione and ascorbic acid) antioxidants is required
which would be of critical importance when plant undergoes stress (Sharma et al 2012)
Among these enzymes SOD is a first line of defense which converts dangerous superoxide
radicals into less toxic product (H2O2) In further CAT APX and GPX worked in
association to get rid off from the excessive load of other oxygen radicals or ions (H2O2
110
OH- ROO etc) In this study antioxidant enzymes (SOD CAT GPX and APX) were
found to work in harmony which was not affected during 4th day treatment in both species
in comparison to sole crop (Fig 27) showing strong antioxidant defense which was not
compromised by cropping system When comparing in 8th day treatment a significant
general increase in all enzyme activities were observed in both species except for SOD
and GPX of C cajan (Fig 27) These results displayed relatively better performance and
tight control over the excessive generation of ROS which would be predicted in this case
due to less availability of water than in 4th day treatment (Karatas et al 2014 Doupis et
al 2013) Similarly by coping oxidative burst and maintaining cellular redox equilibrium
plants were able to improve growth performance especially in Z mauritiana (Fig 21)
Water deficit affect stomatal conductance which could bring about changes in
photosynthetic performance hence overproduction of ROS is usually found among
different crops (Moriana et al 2002 Miller et al 2010) As a response tolerant plants
overcome this situation by increased activity of antioxidant enzymes which was evident in
Wheat Rice olive etc (Zhang and Kirkham 1994 Sharma and Dubey 2005 Guo et al
2006 Sofo et al 2005)
Phenolic compounds despite their role in physiological plant processes are
involved in adsorbing and neutralizing reactive oxygen species (ROS Ashraf and Harris
2004) The overproduction of ROS may cause several plant disorders Plants produce
secondary compounds like polyphenols to maintain balance between ROS generation and
detoxification (Posmyk et al 2009) Increased synthesis and accumulation of phenolic
compounds is reported to safeguard cellular structures and molecules especially under
biotic abiotic constraints (Ksouri et al 2007 Oueslati et al 2010) In this study
intercropped Z mauritiana of field and both species in drum pot culture showed higher
phenolic content than individual crop (Figure 25 and 212) which may be attributed to
adaptive mechanism for scavenging free radicals to prevent cellular damage (Rice-Evans
1996)
In terms of fruit yield we observed that Z mauritiana is suitable for intercropping
as suggested by Yang et al (1992) Number of flowers fruits and fruit fresh weight of
both species either increased considerably or no-affected in intercropped plants compared
to individual ones (Figure 210) Moreover fruit quality of Z mauritiana includes proteins
phenols and soluble extractable and total sugars were also higher in intercropped plants
111
(Figure 213) Results of this study are better than other experiments reported by
Sharma (2004) Kumar and Chaubey (2008) and Kumar et al (2013) who did not find
influence of other understory forage crops (like Aonla) on the yield of Z mauritiana
However in other case the yield of intercropped ber was some time higher (Liu 2002)
Singh et al 2013 found no adverse effects on the yield of pigeonpea when intercropped
with mungbean however it improved the grain yield of associated species
A leguminous plant C cajan is used in this experiment as secondary crop which
can supplement Z mauritiana by improving soil fertility Results of both experiments
showed that the nitrogen was higheror un-affected (Figure 214) in soils of intercropped
plants which supports our hypothesis that leguminous intercrop increase N supply This
can be achieved by acquisition of limited resources to manage rootrhizosphere
interactions which can improve resource-use efficiency (Zhang et al 2010
Shen et al 2013 White et al 2013b Ehrmann and Ritz 2014 Li et al 2014) As a
consequence it impact on overall plant performance which starts from high photosynthetic
activity by increasing chlorophyll results in more availability of photoassimilate for
growth and reproductive allocation (Eghball and Power 1999) Use of C cajan in tree
intercropping proved beneficial for producing high yield crops and for the environment
(Gilbert 2012 Glover et al 2012)
Land equivalent ratio (LER) is commonly used to evaluate the effectiveness of
intercropping by using the resources of same environment compared with sole crop
(Vandermeer 1992 Rao et al 1990 1991 Cao et al 2012) It is the ratio of area for sole
crop to intercrop required to produce the equal amount of yield at the same management
level (Mead and Willey 1980 Dhima et al 2007) On the other hand land equivalent
coefficient (LEC) describe an association that concern with the strength of relationship It
is the proportion of biomassyield of one crop explained by the presence of the other crop
The LER 1 or more indicate a beneficial effect of both species on each other which increase
the yield of both crops as compare to single one (Zada et al 1988) In this experiment all
LER values were about 2 or more than 2 while LEC values were around 1 or more than
one in ZizyphusCajnus intercropping Both LER and LEC values were in descending
order of chlorophylls gt yield gt height (Table 22) However the partial LER was higher in
Zizyphus than Cajanus in all cases These results describe the superiority of intercropping
over sole cropping where LER values are even gt2 Some other studies reported LER from
112
09-14 (Bests 1976) 12-15 (Cunard 1976) and up to 2 (Andrews and Kassam 1976)
Similar results were reported in poplarsoybean system (Rivest et al 2010) black
locustMedicago sativa (Gruenewald et al 2007) wheatjujube (Zhang et al 2013)
Acacia salignasorghum (Droppelmann et al 2000 Raddad and Luukkanen 2007) The
high LER values in our system indicating a harmony in resource utilization in both species
which was also corroborated with their respective LEC values The greater LEC values (gt
025) suggesting an inbuilt tendency of studied crops to give yield advantage (Kheroar and
Patra 2013) Experiments based on traditional practices of growing legumes with cereals
demonstrated greater and continuous cash returns than individual-crops (Baker 1978) In
addition the same authors found further increase in cash returns by increasing the
proportion of cereal and incorporating maize with sorghum and millet In agreement with
our findings similar reports are also available from different intercropping systems
including sesamegreengram (Mandal and Pramanick 2014) maizeurdbean (Naveena et
al 2014) and pegionpeasorghum (Egbe and Bar-Anyam 2010)
After detailed investigations of both species using two different experiment designs
(drum pot and field) it is evident that intercropping had beneficial effects on growth
physiology biochemisty and yield of both species Furthermore by using this system
higher outcome interms of edible biomass and green fodder using marginal lands can be
obtained in a same time using same land and water resources which can help to eliminate
poverty and uplift socio-economic conditions
113
3 Chapter 3
Investigations on rang of salt tolerance in Carissa carandas
(varn karonda) for determining possibility of growing at waste
saline land
31 Introduction
Carissa carandas commonly known as Karonda or lsquoChrist thornrsquo belonging to family
Apocynaceae shows capability of growing under haloxeric conditions It is an important
plant which has established well at tropical and subtropical arid zone under high
temperatures It is large evergreen shrub and having short stem It has fork thorn and hence
used as hedges or fence around fields The leaves are oval or elliptic 25 to 75 cm long
dark green leathery and secrete white milk if detached The fruits are oblong broad- ovoid
or round 125- 25 cm long It has thin but tough epicarp Fruits are in clusters of 3-10
Young fruits are pinkish white and become red or dark purple on maturation
The plant is propagated through seed in August and September Budding and cutting
could also be undertaken Planting is started after first shower of monsoon Plants raised
from seeds are able to flower within two years Flowering starts in March and fruit ripen
from July to September (Kumar et al 2007) The fruit possess good amount of pectin and
acidity hence used in prickle jelly jam squash syrup and in chutney by the commercial
name lsquoNakal cherryrsquo (Mandal et al 1992) They are rich in vitamin C and good source
of Anthocyanin (Lindsey et al 2000) Its fruits also are one of the richest source of iron
(391 mg 100gm) (Tyagi et al 1999) Juice of its root is also used to treat various
microbial diseases such as diarrhea dysentery and skin disease (Taylor et al 1996)
Hence its range of salt and suitability for cultivation at waste saline land or with saline
water irrigation is being undertaken for commercial exploitation by preparing jams jellies
and prickles (Kumar 2014) Investigations on its growth and development at higher range
of salinities are being undertaken with an interest to cultivate it if profitable at highly saline
waste land
114
32 Experiment No 9
Investigation on the effect of higher range of salinities on growth of
Carissa carandas (varn karonda) created by irrigation of different
dilutions of sea salt
321 Materials and methods
3211 Drum Pot Culture
Drum pot culture as recommended by Boyko (1966) and modified by Ahmed and
Abdullah (1982) was used for the present investigation which was been already described
in Chapter 1 earlier
3212 Plant material
About six months old sapling of Carissa carandas (varn Karonda) having almost equal
height and volume poted in polythene bag in 3kg of soil fertilized with cow-dong manure
were purchased from the Noor nursery Gulshan-e-Iqbal Karachi Sindh and were
transported to the Biosaline research field department of Botany University of Karachi
3213 Experimental setup
Plants were transplanted in drum pot (Homemade lysimeter) filled with sandy loam mixed
with cow dung manure (91) Each drum pot was irrigated weekly during summer and
fortnightly during winter months with 20 liters tap water (Eciw= 0 6 dSm-1) or water of
sea salt concentrations of various ie 03 (Eciw = 42 dSm-1) 04 (Eciw =61 dSm-1)
06 (Eciw = 99 dSm-1) and 08 (Eciw = 129 dSm-1) The plants were established initially
by irrigation with tap water for two weeks and later salinity was gradually increased till
desired percentage is achieved for different treatments by dessolving of sea salt in
irrigation water Three replicates were maintained for each treatment Urea DAP and
KNO3 were the source of NPK provided in the ratio 312 50g granules Osmocot (Scotts-
Sierra Horticulture Products) and 50g Mericle-Gro (Scotts Miracle-Gro Products Inc)
were dissolved in irrigation water per drum after six months at six monthly intervals
Height and volume of canopy of these plants were recorded prior to the starting the
experiment and then after every six months interval
115
Since the vegetative growth performance in plants irrigated with 03 sea salt (Eciw = 42
dSm-1) was found comparatively better than control and only 26 decrease was noticed
in volume of canopy at plant irrigated with 04 sea salt (Eciw = 61 dSm-1) (Table III41)
the onward investigations were focused at higher salinity levels and plants were irrigated
with 06 (Eciw = 99 dSm-1) and 08 (Eciw = 129 dSm-1) sea salt in rest of experiment
3214 Vegetative parameters
Vegetative growth on the basis of plant height and volume were recorded while
reproductive growth was observed on the basis of number of flowers and number and
weight of fruits per plant Length and diameter of fruit were also recorded in ten randomly
selected fruits
3215 Analysis on some biochemical parameters
Following biochemical analysis of leaves was performed at grand period of growth (onset
of flowers)
i Photosynthetic pigments
Fresh fully expended leaves (01g) was crushed in 80 chilled acetone Further procedure
was followed described in chapter 1
ii Soluble sugars
Dry leaf samples (01g) were milled in 5mL of 80 ethanol and were centrifuged at 4000
g for 10 minutes Same procedure was followed as described in chapter 1
iii Protein content
The protein contents were measured according to Bradford Assay reagent method against
Bovine Serum Albumin which was taken for standard (Bradford 1976) as described in
chapter 1
iv Soluble phenols
The dried leaf powder (01g) was milled in 3ml of 80 methanol and was centrifuged at
10000g for 15 min Further procedure has been described in chapter 2
116
3216 Mineral Analysis
Estimation of Na+ and K+ were made according to Chapman and Pratt (1961) Oven dried
grinded Leaves (1g) furnace at 550ordmC for 6 hours and were digested in 5 ml of 2N HCl
Diluted and filtered solution was used to estimated Na+ and K+ in flame photometer
(Petracourt PFP I) The concentration of these ions was calculated against the following
standard curve equations
Na+ (ppm) = 0016135x1879824
K+ (ppm) = 0244346x1314603
117
322 Observations and Result
3221 Vegetative parameters
Vegetative growth in terms of height and volume of canopy of C carandas growing under
salinities created by irrigation of different dilutions of sea salt is presented in Table 32
Appendix-XIX A significant increase (plt0001) in plant height and volume of canopy
was observed with increasing time but the increase was rapid at early period of growth
However there was significant (plt0001) reduction under salinity stress The interaction
of time and salinity also showed significant (plt001) effect on plant parameters but the
increase in height and volume of canopy at Eciw= 42dSm-1of sea salt salinity was more
than control Plants irrigated with Eciw= 61 dSm-1 and Eciw= 99 dSm-1sea salt solution
showed decrease in height with respect to control but the difference between their
treatments was insignificantly higher decrease was observed in Eciw= 129 dSm-1 sea salt
irrigated plants
3222 Reproductive parameters
Reproductive growth in terms of flowers and fruits numbers flower shedding percentage
fresh and dry weight of ten fruit their length and diameter under salinities created by
irrigation of different dilutions of sea salt is presented in Table 33 Appendix-XX Number
of flowers and fruits significantly (plt0001) decreased with increasing salinity treatment
Difference in flower initiation seems non-significant at early growth period in controls and
salinity treatments However drastic decrease was observed in plants irrigated beyond
Eciw= 99 dSm-1 with increase in salinity
Flowers shedding percentage (Table 33 Appendix-XX) show an increase directly
proportional with increase in salinity however the difference in number of flowers
between the plants irrigated with Eciw= 99 dSm-1 and Eciw= 129 dSm-1 sea salt solution
is of little significance level (plt001)
Fresh and dry weight of average fruits (plt001) and their diameter (plt001) showed
decrease with increasing salinity whereas diameter and length of fruits showed non-
significant difference
118
3224 Study on some biochemical parameters
i Photosynthetic Pigments
Photosynthetic Pigments including Chlorophyll a chlorophyll b total chlorophyll
chlorophyll a b ratio and carotenoids of C carandas growing under salinities created by
irrigation of different dilutions of sea salt is presented in Figure 31 Appendix-XX The
chlorophyll contents of leaves significantly decreased (plt0001) over control with
increasing salinity however Chlorophyll rsquobrsquo at Eciw= 99 dSm-1salinity shows significant
increase (plt0001) over control Similarly Carotenoids at Eciw= 99 dSm-1 salinity show a
bit less significant increase (plt001) compare to control while at higher salinity (Eciw=
129 dSm-1) the decline is observed at all above mentioned parameters
iii Protein Sugars and phenols
Some biochemical parameters including Protein sugars and phenolic contents of C
carandas growing under salinities created by irrigation of different dilutions of sea salt is
presented in Figure 31 Appendix-XX Soluble proteins in leaves show non-significant
decrease at Eciw= 99 dSm-1salinity as compared with controls but a significant decrease
(plt005) was noted at Eciw= 129 dSm-1 salinity Sugars also showed non-significant
decrease at both the salinity whereas on contrary soluble phenols showed significant
increase (plt0001) with increasing salinity
3225 Mineral analysis
Mineral analysis including Na and K ions performed in leaves of C carandas growing
under salinities created by irrigation of different dilutions of sea salt is presented in Figure
32 Appendix-XX Sodium significantly increased (plt0001) all the way with increasing
salinity of growth medium Whereas significant decrease (plt0001) was observed in
Potassium with increasing salinity K+Na+ ratio show continuous increase with increasing
salinity
119
Table 31 Electrical conductivities of different sea salt concentration used for determining
their effect on growth of C carandas
Treatment
Sea salt ()
ECiw of irrigation water (dSm-1) ECe of soil saturated paste
(dSm-1)
Non-saline control 06 09
03 42 48
04 61 68
06 99 112
08 129 142
Whereas ECiw and ECe are the electrical conductivities of irrigation water and soil saturated past measured in deci semen per meter
120
Table 32Vegetative growth in terms of height and volume of canopy of C carandas growing under salinities created by irrigation of different dilutions of
sea salt
Treatment
Sea salt
(ECiw dSm-1)
Initial values prior to
starting saline water
irrigation
Growth at different salinities after 06 months
Height Volume Height Volume of canopy
cm m3 cm
increase
over initial
values
increase
decrease over
control
m3 increase over
initial values
increase
decrease
over control
Control 3734plusmn455 0029plusmn0001 8227plusmn4919 5363plusmn830 - 014plusmn0015 7952plusmn269 -
42 3674plusmn1415 0026plusmn0003 9930plusmn6142 6280plusmn205 +1710 019plusmn0017 8593plusmn098 +806
61 3752plusmn1243 0026plusmn0001 6490plusmn5799 4132plusmn485 -2305 012plusmn0010 7740plusmn117 -282
99 3819plusmn4499 0028plusmn0005 5793plusmn5821 3123plusmn1446 -4185 009plusmn0008 6759plusmn377 -1499
129 3676plusmn3114 0026plusmn0008 5250plusmn4849 2775plusmn1276 -4836 006plusmn0005 5690plusmn1110 -2844
LSD0 05
Salinity
Time Fisherrsquos least significant difference
91
172
002
0005
Values represent means plusmn standard error of each treatment and significance among the treatments was recorded at p lt 005
120
121
Table 33 Vegetative growth in terms of height and volume of canopy of C carandas growing under salinities
created by irrigation of different dilutions of sea salt
Treatment
Sea salt
(ECiw dSm-1)
Growth at different salinities after 12 months
Height Volume of canopy
cm
increase
over initial
values
increase
decrease over
control
m3
increase
over initial
values
increase
decrease over
control
Control 16214 plusmn633 7674plusmn307 - 077plusmn012 9689plusmn449 -
99 9736plusmn1048 6056plusmn561 -2109 034plusmn006 9367plusmn412 -333
129 6942plusmn565 4741plusmn480 -3822 022plusmn002 9064plusmn623 -645
Table 33 continuedhellip
Treatment
Sea salt
(ECiw= dSm-1)
Growth at different salinities after 18 months
Height Volume of canopy
Cm
increase
over initial
values
increase
decrease over
control
m3
increase
over initial
values
increase
decrease over
control
Control 1676plusmn1135 7776plusmn756 - 094plusmn011 9701plusmn578 -
99 10547plusmn842 6351plusmn666 -1833 045plusmn010 9445plusmn1024 -264
129 7581plusmn593 5154plusmn716 -3372 030plusmn003 9318plusmn580 -395
Table 33 continuedhellip
122
Table 33 continuedhellip
Treatment
Sea salt
(ECiw= dSm-1)
Growth at different salinities after 24 months
Height Volume of canopy
Cm
increase
over initial
values
increase
decrease over
control
m3
increase
over initial
values
increase
decrease over
control
Control 1911plusmn6
05 8055plusmn941 - 121plusmn015 9837plusmn522 -
99 1110plusmn5
31 6557plusmn543 -1859 053plusmn002 9509plusmn1032 -334
129 8754plusmn10
67 5990plusmn801 -2564 040plusmn008 9287plusmn745 -560
Table 33 continuedhellip
Treatment
Sea salt
(ECiw= dSm-1)
Growth at different salinities after 30 months
Height Volume of canopy
Cm
increase
over initial
values
increase
decrease over
control
m3
increase
over initial
values
increase
decrease over
control
Control 2052plusmn1126 8182plusmn676 - 146plusmn029 9873plusmn729 -
99 11700plusmn816 6743plusmn610 -1759 070plusmn011 9565plusmn850 -312
129 9628plusmn552 6189plusmn573 -2436 050plusmn004 9417plusmn1011 -462
LSD0 05 Salinity 77 007
Time 168 016
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and significance among the treatments was recorded at p lt 005
123
Table 34 Reproductive growth in terms of flowers and fruits numbers flower shedding percentage fresh and dry weight of ten fruit and their totals
perplant fruit length and diameter of C carandas growing under salinities created by irrigation of different dilutions of sea salt
Treatment
Sea salt
(ECiw= dSm-1)
Flower Fruits Flower
shedding
Weight of
Ten
fruit(fresh)
Weight of
Ten
fruit(dry)
Weight of
total fruitplant
(fresh)
Weight of
total fruitplant
(dry)
length
fruit
diameter
fruit
Numbers Numbers g g g g mm mm
Control 19467plusmn203 16600plusmn231 1468plusmn208 2282plusmn022 605plusmn009 37891plusmn891 10047plusmn283 1800plusmn003 1423plusmn006
99 12050plusmn202 7267plusmn491 3980plusmn307 1880plusmn035 530plusmn029 13695plusmn1174 3880plusmn469 1732plusmn037 1297plusmn011
129 12567plusmn549 6967plusmn203 4449plusmn082 1541plusmn023 435plusmn026 10742plusmn470 3041plusmn268 1711plusmn015 1233plusmn038
LSD0 05 Salinity 1514 1417 929 115 097 3785 1494 0971 097
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and significance among the treatments was recorded at p lt 005
123
124
Sea Salt (ECiw
= dSm-1
)
Cont 99 129
Car
ote
nio
ds
(mg
g-1
)
00
01
02
03
04
Ch
loro
ph
yll
(m
g g
-1)
00
01
02
03
04
05
06
ab
rat
io
00
05
10
15
20
25
30
35
ab
Chl a Chl b
a
a
a a
b
bcbc
a
b
c
a a
b
Figure 31 Chlorophyll a chlorophyll b total chlorophyll chlorophyll a b ratio carotenoids contents of C
carandas growing under salinities created by irrigation of different dilutions of sea salt (Bars
represent means plusmn standard error of each treatment and significance among the treatments was
recorded at p lt 005)
125
Sea Salt (ECiw
= dSm-1
)
Cont 99 129
Ph
eno
ls (
mg
g-1
)
0
5
10
15
20
Pro
tein
s (m
g g
-1)
0
1
2
3
4
Su
gar
s (m
g g
-1)
0
30
60
90
120
150Soluble Insoluble
a
a
a
a
a
a
b
b
b
c
ab
a
a
b
Figure 32 Total protein sugars and phenolic contents of C carandas growing under salinities created by
irrigation of different dilutions of sea salt (Bars represent means plusmn standard error of each treatment
and significance among the treatments was recorded at p lt 005)
126
Sea Salt (ECiw
= dSm-1
)
Cont 99 129
Ions
(mg
g-1
DW
)
0
20
40
60
80
100
120
KN
a ra
tio
00
01
02
03
04
05
06
07
Na K KNa
c
a
b
b
a
c
a
b
c
Figure 33 Mineral analysis including Na and K ions was done on leaves of C carandas growing under salinities
created by irrigation of different dilutions of sea salt (Bars represent means plusmn standard error of each
treatment and significance among the treatments was recorded at p lt 005)
127
33 Discussion
The volume and height of plants were increased per unit time under saline conditions This
increase was observed after six months in 03 sea salt (ECiw = 42 dSm-1) treated plants in
comparison to control (Table 32) Slight decrease was observed at 04 sea salt
(ECiw=61dSm-1) irrigation after which (Eciw= 99 dSm-1 and Eciw = 129 dSm-1sea salt) the
growth was significantly inhibited (Table 33) Noble and Rogers (1994) also noticed a general
decrease in growth of some of the glycophytes Humaira and Ahmad (2004) and Rivelli et al
(2004) also reported a proportional decrease in height of canola with increasing salinity
Cotton plants irrigated with saline water or those grown at saline soil are reported to increase
Na+ content in leaves accompanied by significant reduction in vegetative biomass (Meloni et
al 2001) Bayuelo-Jimenez et al (2003) observed salt induced growth inhibition of tomato
plant which was higher in shoot than root
Reproductive growth in terms of number of flowers number of fruits fruit length and
diameter were decreased and percent flower shedding increased with increasing salinity
(Table 34) These effects were higher at Eciw= 99 dSm-1and then maintained with further
salinity increment However weight of fruits (fresh and dry) and total fruits per plant were
linearly decreased with increasing medium salt concentrations A decrease in different phases
of reproductive growth like flowering fertilization fruit setting yield and quality of seeds etc
are reported to be seriously affected at different level of salinity by various workers (Lumis et
al 1973 Waisel 1991 Shannon et al 1994 Tayyab et al 2016) Cole and Mclead (1985)
and Howie and Lloyd (1989) reported severe effects of different salinity treatments on
flowering intensity fruit setting and number of fruits of Citrus senensis Walker et al (1979)
also reported reduction in the fruit weight during early ripening stage of Psidium guajava
Decrease in fruit diameter of strawberries (Fragaria times ananassa) has been reported with
salinity (Ehlig and Bernstein 1958)
In this study photosynthetic pigments of C carandas were decreased with salinity and
this decrease was more sever at Eciw = 129 dSm-1sea salt salinity (Figure 31) Such a decline
in amount of leaf pigments across different salinity regimes was also reported in cotton
(Ahmed and Abdullah 1979) Pea (Hernandez et al 1995 and Hernandez et al 1999) Vicia
128
faba (Gadallah 1999) Mulberry genotype (Agastian et al 2000) and B parviflora (Parida et
al 2004)
Leaf sugars and protein were decreased in both salinity levels (Figure 32) which could
be attributed to inhibition in transport of photosynthetic product (Levit 1980) Decrease
synthesis and mobilization of glucose fructose and sucrose has been demonstrated in number
of plants growing under salt stress (Kerepesi and Galiba 2000) Inhibition in the protein and
nucleic acid synthesis in Pisum sativum and Tamarix tetragyna plants were also reported by
Bar-Nun and Poljahoff-Mayber (1977) Melander and Harvath (1977) suggested that salt
induced reduction in protein is due to increase in protein hydrolysis
A significant increase in leaves phenol with increase in salinity (Figure 32) was
observed in present investigation was also demonstrated previously in Achilleacollina (Giorgi
et al 2009) Lactuca sativa (Kim et al 2008) and B parviflora (Parida et al 2004)
Inspite of over irrigation of saline water and maintaining leaching fraction of about
40 in drum pots accumulation of salts in rhizosphere soil was not completely avoided which
was evident in the differences between ECiw and ECe values (Table 31) Deposition of salts
in rhizosphere soil interferer absorption of minerals in plants For instance leaf Na+ content
of C carandas was significantly increased while K+ decreased with increasing soil salinity
(Figure 33) Over accumulation of toxic ions disturbed plant water status which directly
affects plant growth (Flowers et al 1977 Greenway and Munns 1980) A negative
relationship between Na+ and K+ concentration in roots and leaves of guava was also reported
by Ferreira et al (2001) Increase in Na+ content decreased K+ availability and K+Na+ ratio
in Vicia taba (Gadallah 1999) and also affect the uptake of other essential minerals in
Casurina equsetifolia (Dutt et al 1991)
Carissa carandas found to be a good tolerant to salinity and drought and it can produce
edible fruits from marginal lands of arid areas Fruits of this species can be consumed in a raw
form as well as in industrial products like pickles jams jellies and marmalades
129
4 Conclusions
In the light of above mentioned investigations it appears that pre-soaking treatment of Cajanus
cajan seeds has initiated metabolic processes at faster rate earlier which has helped seeds to
start germinative metabolism prior to be effected by toxic Na+ ions at higher salinities Cajanus
cajan and Ziziphus mauritiana were found to be the good companions for intercropping These
species synergistically enhanced the growth and biochemical performance of each other by
improving fertility of marginal land and maintaining harmony among different physiological
parameters which was missing in their sole crop Their intercropping could produce fodder
and delicious fruits even from under moderately saline substrate up to profitable extant
Carissa carandas also tolerated low and moderately salinities well by adjusting proper
regulation of physiological and biochemical parameters of growth It can provide protein rich
edible fruits jams jellies and pickles of commercial importance for benefit of poor farmer
from moderately saline barren land
130
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12
141
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with soybean increases soil microbial biomass mineral N supply and tree
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Santa-Cruz MM F Martinez-Rodriguez R Perez-Alfocea R Romero-Aranda and MC
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167
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168
6 THESIS APENDECES
Appendix-I One way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution of seed germination of pre-soaked seeds of C cajan in non-saline water prior to germination under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Mean
germination rate
(GR)
Salinity treatment 4422 20 221133 21015 0000
Error 441949 42 10522
Total 4864 62
Mean germination
velocity (GV)
Salinity treatment 418813 20 20941 51836 0000
Error 169671 42 40398
Total 588484 62
Mean
germination
time (GT)
Salinity treatment 0271 20 0013 8922 0000
Error 0064 42 0002
Total 0335 62
Mean germination
Index (GI)
Salinity treatment 4422 20 221133 21015 0000
Error 441949 42 10523
Total 4864607 62
Final
germination
(FG)
Salinity treatment 32107 20 1605397 25285 0000
Error 2666 42 63492
Total 34774 62
Appendix-II Two way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution of seed germination of pre-soaked seeds of C cajan in non-saline water prior to germination under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Germination percentage per
day
Salinity treatment 509583 20 25479 19187 0000
Time 53156 9 5906 4663 0002
Salinity treatment times time 251743 180 1398576 1053 ns
Error 531130 400 1327825
Total 1375283 629
Germination
rate per day
Salinity treatment
Time 761502 9 84611 83129 0000
Salinity treatment times time 442265 20 22113 24630 0000
Error 359117 400 0898
Total 2108622 629
Appendix-III One way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution of seed
germination of pre-soaked seeds of C cajan in respective saline water prior to germination under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Final mean germination
velocity (GV)
Salinity treatment 0538 6 0089 35585 0000
Error 0035 14 0003
Total 0573
Final mean
germination time (GT)
Salinity treatment 20862 6 3477 26256 0000
Error 1854 14 0132
Total 22716 20
Final mean germination
index (GI)
Salinity treatment 110514 6 18419 190215 0000
Error 1356 14 0097
Total 111869 20
Final
germination percentage (GP)
Salinity treatment 6857 6 1142857 40 0000
Error 400 14 28571
Total 7257 20
Appendix-IV Two way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution of seed
germination of pre-soaked seeds of C cajan in respective saline water prior to germination under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Germination percentage per
day
Salinity treatment 86644 6 14440816 505428 0000
Time 23378 6 3896 136373 0000
Salinity treatment times time 2717 36 75472 2641 0001
Error 2800 98 28571
Total 115540 146
Germination rate
per day
Salinity treatment 117386 6 19564 360762 0000
Time 128408 6 21401 394636 0000
Salinity treatment times time 58747 36 1632 30091 0000
Error 5314 98 0054
Total 309855 146
169
Appendix-V One way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution on seedling
emergence and height of germinating seeds of C cajan under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Seedling height of C cajan
Salinity treatment 200822 5 40056 169666 0000
Error 2833 12 0236
Total 203115 17
Seedling
emergence of C cajan
Salinity treatment 24805 6 4134 6381 000
Error 9070 14 647867
Total 33875 20
Appendix-VI Two way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution on growth and
development of C cajan in lysemeter (Drum pot) under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Plant height of
C cajan
Salinity treatment 261079 5 52215 720259 0000
Time 126015 8 15751 132488 0000
Salinity treatment times time 76778 40 1919 16144 0000
Error 11413 96 118893
Total 477028 161
Appendix-VII One way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution on growth
and development of C cajan in lysemeter (Drum pot) under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Number of
Flowers of C
cajan
Salinity treatment 3932 3 131075 39719 0000
Error 264 8 33
Total 419625 11
Number of pods
of C cajan
Salinity treatment 1473 3 491 23105 0000
Error 170 8 2125
Total 1643 11
Number of
seedspod of C cajan
Salinity treatment 3 3 1
Error 0 8 0
Total 3 11
Number of seeds plant of
C cajan
Salinity treatment 19332 3 6444 45621 0000
Error 1130 8 14125
Total 20462 11
Weight of
seeds plant of C cajan
Salinity treatment 592976 3 197658 85572 0000
Error 18478 8 2309
Total 611455 11
Chlorophyll a
of C cajan
Salinity treatment 0117 3 0039 81241 0000
Error 0004 8 0000
Total 0121 11
Chlorophyll b
of C cajan
Salinity treatment 0004 3 0001 15222 0001
Error 0001 8 0000
Total 0005 11
Total chlorophyll of
C cajan
Salinity treatment 0160 3 0053 164401 0000
Error 0002 8 0000
Total 0162 11
Chlorophyll a b
ratio of C cajan
Salinity treatment 242 3 0806 9327 0005
Error 0692 8 0086
Total 3112 11
Carotenoids of
C cajan
Salinity treatment 0015 3 0005 4510 0039
Error 0009 8 0001
Total 0025 11
Soluble sugars
of C cajan
Salinity treatment 0043 3 0014 6515 0015
Error 00178 8 0002
Total 0061 11
Insoluble
sugars of C
cajan
Salinity treatment 0118 3 0039 36262 0000
Error 0008 8 0001
Total 0127 11
Total sugars of
C cajan
Salinity treatment 0019 3 0006 4239 0045
Error 0012 8 0001
Total 0031 11
Protein of C cajan
Salinity treatment 0212 3 0070 15735 0001
Error 0036 8 0004
Total 0248 11
170
Appendix-VIII One way ANOVA for completely randomized design for range of salt tolerance of nitrogen fixing symbiotic bacteria
associated with root of C cajan
Variables Source Sum of Squares df Mean Square F-value P
Nodule
associated
Rhizobial colonies of C
cajan
Salinity treatment 35927 2 17963 229402 0000
Error 1409 18 0078
Total 37337 20
Appendix-IX Two way ANOVA for completely randomized design for growth and development of Z mauritiana in large size clay pot being irrigated with water of two different sea salt concentration
Variables Source Sum of Squares df Mean Square F-value P
Height of
Z mauritiana
Time 91030 2 45515 839 0000
Salinity treatment 3268 2 1634 10 0000
Time times Salinity treatment 1533 4 383 238 ns
Error 6751 42 161
Total 104554 71
Number of
branches of
Z mauritiana
Time 25525 2 127625 25333 0000
Salinity treatment 86333 2 43166 11038 0000
Time times Salinity treatment 27416 4 6854 1752 ns
Error 16425 42 3910
Total 6575 71
Number of
flowers of
Z mauritiana
Time 73506 2 36753 167777 0000
Salinity treatment 12133 2 6066 25061 0000
Time times Salinity treatment 27824 4 6956 28736 0000
Error 10166 42 242063
Total 127759 71
Fresh weight of
Shoot of
Z mauritiana
Time 3056862 2 1528431 340777 0000
Salinity treatment 107829 2 53914 12020 0000
Time times Salinity treatment 51303 4 12825 2859 0031
Error 251167 56 4485
Total 3515820 71
Dry weight of Shoot of
Z mauritiana
Time 784079 2 392039 338932 0000
Salinity treatment 26344 2 13172 11387 0000
Time times Salinity treatment 13042 4 3260 2818 0033
Error 64774 56 1156690
Total 913855 71
Succulence of
Z mauritiana
Time 0002 2 0001 0214 ns
Salinity treatment 0006 2 0003 0682 ns
Time times Salinity treatment 0007 4 0002 0406 ns
Error 0199 45 0004
Total 51705 54
Spacific shoot
length of Z mauritiana
Time 0000 2 914 0176 0000
Salinity treatment 0002 2 0001 2096 ns
Time times Salinity treatment 0003 4 0001 1445 ns
Error 0023 45 0001
Total 6413 54
Moisture
contents of Z mauritiana
Time 1264 2 0632 0243 ns
Salinity treatment 3603 2 1801 0691 ns
Time times Salinity treatment 4172 4 1043 0400 ns
Error 117146 45 2603
Total 131675 54
Relative growth
rate of Z mauritiana
Time 1584206 1 1584206 532968 ns
Salinity treatment 18921 2 9460 3183 ns
Time times Salinity treatment 61624 2 30812 10366 0000
Error 89172 30 2972
Total 4034 36
Appendix-X One way ANOVA for completely randomized design for growth and development of Z mauritiana in large size clay pot
being irrigated with water of two different sea salt concentration
Variables Source Sum of Squares df Mean Square F-value P
Chlorophyll a
of Z mauritiana
Salinity treatment 0004 2 0002 7546 0003
Error 0006 21 0000
Total 0010 23
Chlorophyll b of Z mauritiana
Salinity treatment 0037 2 0018 4892 0018
Error 0080 21 0003
Total 0117 23
171
Total
chlorophyll of
Z mauritiana
Salinity treatment 0144 2 0072 39317 0000
Error 0038 21 0002
Total 0182 23
Chlorophyll ab ratio of
Z mauritiana
Salinity treatment 1499 2 0749 33416 0000
Error 0471 21 0022
Total 1969 23
Total soluble
sugars of
Z mauritiana
Salinity treatment 378271 2 189135 36792 0000
Error 107952 21 5140
Total 486223 23
Total protein contents of
Z mauritiana
Salinity treatment 133006 2 66502 5861 0009
Error 238268 21 11346
Total 371274 23
Appendix-XI Three way ANOVA for split-split plot design for physiological investigations on growth of Z mauritiana and C cajan in
drum pot being irrigated with water of sea salt concentration at two irrigation intervals
Variables Source Sum of Squares df Mean Square F-value P
Height of
Z mauritiana
Time 4499 2 2249 28888 0004
Crop 448028 1 448028 2208 ns
Irrigation intervals 2523 1 2523 2774 ns
Time times Crop 928088 2 464044 2288 ns
Time times irrigation interval 1120400 2 560200 0615 ns
Crop times irrigation interval 2690151 1 2690 2957 ns
Time times Crop times irrigation interval 171927 2 85963 0094 ns
Error 10916 12 909732
Total 35
Canopy volume of Z mauritiana
Time 7943 2 3971 6554 ns
Crop 0382 1 0382 0579 ns
Irrigation intervals 0068 1 0069 0103 ns
Time times Crop 0265 2 0133 0201 ns
Time times irrigation interval 1142 2 0571 0852 ns
Crop times irrigation interval 0722 1 0722 1077 ns
Time times Crop times irrigation interval 1998 2 0999 1491 ns
Error 8043 12 0670
Total 29439 35
Appendix-XII Two way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C
cajan in drum pot being irrigated with water of sea salt concentration at two irrigation intervals
Variables Source Sum of Squares df Mean Square F-value P
Plant length of
Z mauritiana
Crop 2986 1 2986 75322 0000
Irrigation interval 2986 1 2986 75322 0000
Crop times Irrigation interval 15336 1 153367 3868 ns
Error 317166 8 39645
Total 292428 12
Shoot length of
Z mauritiana
Crop 1069741 1 1069741 30890 0000
Irrigation interval 1069741 1 1069741 30890 0000
Crop times Irrigation interval 253001 1 253001 73058 0026
Error 27704 8 3463
Total 103376 12
Root length of
Z mauritiana
Crop 19763 1 19763 2671 ns
Irrigation interval 481333 1 481333 65059 0000
Crop times Irrigation interval 800333 1 800333 108177 0000
Error 59186 8 7398
Total 49165 12
Main branches
of Z mauritiana
Crop 33333 1 33333 5797 0042
Irrigation interval 48 1 48 8347 0020
Crop times Irrigation interval 0333 1 0333 0057 ns
Error 46 8 575
Total 2888 12
Lateral
branches of Z mauritiana
Crop 1344083 1 1344083 41356 0000
Irrigation interval 54675 1 54675 16823 0000
Crop times Irrigation interval 784083 1 784083 24125 0000
Error 26 8 325
Total 22465 12
Leaf numbers of
Z mauritiana
Crop 22465 12 98283 96482 0000
Irrigation interval 25025 1 25025 24566 0001
Crop times Irrigation interval 11907 1 11907 11688 0009
Error 8149 8 1018667
172
Total 2037850 12
Shootroot ratio
of Z mauritiana
Crop 0027 1 0027 1842 ns
Irrigation interval 0001 1 0001 0097 ns
Crop times Irrigation interval 0825 1 0825 54909 0000
Error 0120 8 0015
Total 27776 12
Plant fresh
weight of Z mauritiana
Crop 398107 1 398107 577818 0000
Irrigation interval 139514 1 139514 20249 0000
Crop times Irrigation interval 146898 1 146898 21321 0000
Error 5511 8 688982
Total 7248659 12
Plant dry weight of Z mauritiana
Crop 87808 1 87808 471436 0000
Irrigation interval 57893 1 57893 31082 0000
Crop times Irrigation interval 61132 1 61132 32821 0000
Error 14900 8 186257
Total 1875710 12
Stem fresh
weight of
Z mauritiana
Crop 46687 1 46687 227539 0000
Irrigation interval 17933 1 17933 87402 0000
Crop times Irrigation interval 20180 1 20180 98351 0000
Error 16414 8 205185
Total 1718530 12
Root fresh weight of
Z mauritiana
Crop 58450 1 58450 2295 0000
Irrigation interval 42186 1 42186 165641 0000
Crop times Irrigation interval 37307 1 37307 146487 0000
Error 203746 8 25468
Total 357145 12
Leaf fresh weight of
Z mauritiana
Crop 29970 1 29970 19089 0000
Irrigation interval 117018 1 1170187 7453 0025
Crop times Irrigation interval 2310 1 2310 14714 0004
Error 125596 8 15699
Total 699711 12
Stem dry weight
of Z mauritiana
Crop 13587 1 13587 216591 0000
Irrigation interval 11856 1 11856 18899 0000
Crop times Irrigation interval 6787763 1 6787 108197 0000
Error 50188 8 62735
Total 4689795 12
Root dry weight
of Z mauritiana
Crop 1358787 1 13587 216591 0000
Irrigation interval 1497427 1 14974 118615 0000
Crop times Irrigation interval 128773 1 12877 1020052 0000
Error 100993 8 12624
Total 124421 12
Leaf dry weight
of Z mauritiana
Crop 2374 1 2374 135380 0000
Irrigation interval 8748 1 8748 4987 ns
Crop times Irrigation interval 26403 1 2640 150539 0000
Error 140313 8 17539
Total 127170 12
Plant moisture of Z mauritiana
Crop 22082 1 22082 5608 0045
Irrigation interval 38702 1 38702 9830 0013
Crop times Irrigation interval 44406 1 44406 11279 0009
Error 31496 8 3937
Total 29872 12
Stem moisture of Z mauritiana
Crop 0005 1 0005 0000 ns
Irrigation interval 110663 1 110663 12023 0008
Crop times Irrigation interval 0897 1 0897 0097 ns
Error 73633 8 9204
Total 28532 12
Root moisture of Z mauritiana
Crop 235266 1 235266 16502 0003
Irrigation interval 3923 1 3923 0275 ns
Crop times Irrigation interval 0856 1 0856 0060 ns
Error 114051 8 14256
Total 17572 12
Leaf moisture
of Z mauritiana
Crop 130413 1 130413 47746 0000
Irrigation interval 22256 1 22256 8148 0021
Crop times Irrigation interval 210662 1 210662 77127 0000
Error 21850 8 2731
Total 38888 12
173
Relative growth
rate of Z mauritiana
Crop 0000 1 0000 287467 0000
Irrigation interval 0000 1 0000 164217 0000
Crop times Irrigation interval 0000 1 0000 179626 0000
Error 0000 8 0000
Total 0009 12
Relative water
contents of Z
mauritiana
Crop 37381 1 37381 1380 ns
Irrigation interval 49871 1 49871 1841 ns
Crop times Irrigation interval 13496 1 13496 0498 ns
Error 216649 8 27081
Total 50855 12
Chlorophyll a of Z mauritiana
Crop 0103 1 0103 32466 0000
Irrigation interval 0003 1 0003 1075 ns
Crop times Irrigation interval 0000 1 0000 0187 ns
Error 0025 8 0003
Total 1498 12
Chlorophyll b
of Z mauritiana
Crop 0027 1 0027 196164 0000
Irrigation interval 0002 1 0002 15656 0004
Crop times Irrigation interval 0006 1 0006 45063 0000
Error 0001 8 0000
Total 0456 12
Total chlorophyll
of Z mauritiana
Crop 0257 1 0257 53469 0000
Irrigation interval 0001 1 0001 0315 ns
Crop times Irrigation interval 0002 1 0002 0442 ns
Error 0038 8 0004
Total 3736 12
Chlorophyll a b ratio of
Z mauritiana
Crop 0002 1 0002 0028 ns
Irrigation interval 0169 1 0169 1696 ns
Crop times Irrigation interval 1064 1 1064 10643 0011
Error 0799 8 0099
Total 43067 12
Carotenoids of
Z mauritiana
Crop 0018 1 0018 42747 0000
Irrigation interval 0002 1 0002 5298 0050
Crop times Irrigation interval 0003 1 0003 8118 0021
Error 0003 8 0000
Total 0451 12
Phenol of
Z mauritiana
Crop 24641 1 24641 13168 000
Irrigation interval 5078 1 5078 2714 ns
Crop times Irrigation interval 10339 1 10339 5525 0046
Error 14969 8 1871
Total 6289 12
Proline of Z mauritiana
Crop 0001 1 0001 52288 0000
Irrigation interval 0000 1 0000 6972 0029
Crop times Irrigation interval 0000 1 0000 0358 ns
Error 0000 8 0000
Total 0005 12
Protein of Z mauritiana
Crop 200001 1 200001 296 ns
Irrigation interval 69264 1 69264 102 ns
Crop times Irrigation interval 4453 1 4453 006 ns
Error 540367 8 67545
Total 814086 11
CAT enzyme of
Z mauritiana
Crop 74171 1 74171 11404 0009
Irrigation interval 299930 1 299930 46117 0000
Crop times Irrigation interval 15336 1 15336 2358 ns
Error 52029 8 65036
Total 441467 11
APX enzyme of
Z mauritiana
Crop 191918 1 191918 6693 0032
Irrigation interval 4665 1 4665 162723 0000
Crop times Irrigation interval 336912 1 336912 11750 0009
Error 229383 8 28672
Total 5423 11
GPX enzyme of
Z mauritiana
Crop 0000 1 0000 0020 ns
Irrigation interval 0103 1 0103 5893 0041
Crop times Irrigation interval 0109 1 0109 6220 0037
Error 0140 8 0017
Total 0353 11
SOD enzyme Crop 8471 1 8471 1364 ns
174
of
Z mauritiana
Irrigation interval 6220 1 6220 1001 ns
Crop times Irrigation interval 21142 1 21142 3405 ns
Error 49664 8 6208
Total 85498 11
NR enzyme of
Z mauritiana
Crop 7520 1 75208333333 37253364154 0003
Irrigation interval 1360 1 1360 6737 0318
Crop times Irrigation interval 0016 1 0016 0079 ns
Error 1615 8 0201
Total 10512 11
Nitrate of
Z mauritiana
Crop 003 1 003 3028 ns
Irrigation interval 0018 1 0018 1831 ns
Crop times Irrigation interval 0003 1 0003 0336 ns
Error 0079 8 0009
Total 0130 11
Appendix-XIII Three way ANOVA for split-split design for physiological investigations on growth of Z mauritiana and C cajan in drum
pot being irrigated with water of sea salt concentration at two irrigation intervals
Variables Source Sum of Squares df Mean Square F-value P
Height of
C cajan
Time 14990 2 7495 235059 0000
Crop 7848 1 7848 42235 0000
Irrigation intervals 749056 1 749056 9676 0009
Time times Crop 2638 2 1319140 7098 00262
Time times irrigation interval 309932 2 154966 2001 ns
Crop times irrigation interval 9127 1 9127 0117 ns
Time times Crop times irrigation interval 31974 2 15987 0206 ns
Error 928935 12 77411
Total 29065 35
Apendix-XIV Two way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C
cajan in drum pot being irrigated with water of sea salt concentration at two irrigation intervals
Variables Source Sum of Squares df Mean Square F-value P
Plant length of C cajan
Crop 1056563 1 1056563 12331 0007
Irrigation interval 21675 1 21675 2529 ns
Crop times Irrigation interval 137363 1 137363 1603 ns
Error 68544 8 8568
Total 334030 12
Shoot length of C cajan
Crop 808520 1 808520 36580 0000
Irrigation interval 165020 1 165020 7466 0025
Crop times Irrigation interval 285187 1 285187 12902 0007
Error 17682 8 22102
Total 224013 12
Root length of C cajan
Crop 16567 1 16567 0674 ns
Irrigation interval 3520 1 3520 0143 ns
Crop times Irrigation interval 26700 1 26700 1087 ns
Error 196453 8 24556
Total 11133 12
Main branches
of C cajan
Crop 80083 1 80083 64066 0000
Irrigation interval 10083 1 10083 8066 0021
Crop times Irrigation interval 075 1 075 06 ns
Error 10 8 125
Total 335 12
Letral branches
of C cajan
Crop 0 1 0
Irrigation interval 0 1 0
Crop times Irrigation interval 0 1 0
Error 0 8 0
Total 0 12
Leaf numbers
of C cajan
Crop 1776333 1 1776333 16679 0003
Irrigation interval 972 1 972 9126 0016
Crop times Irrigation interval 176333 1 17633 1655 0234
Error 852 8 1065
Total 22342 12
Shootroot ratio of C cajan
Crop 0385 1 0385 0638 0447
Irrigation interval 0007 1 0007 0011 0916
Crop times Irrigation interval 2669 1 2669 4424 0068
Error 4825 8 0603
Total 264061 12
Crop 76816 1 76816 7494853 0025
175
Plant fresh
weight of
C cajan
Irrigation interval 730236 1 730236 7124832 0028
Crop times Irrigation interval 266869 1 266869 2603812 0145
Error 81993 8 102491
Total 25941 12
Plant dry weight of C cajan
Crop 38270 1 38270 1150145 0009
Irrigation interval 53046 1 53046 15942 0003
Crop times Irrigation interval 20202 1 20202 6071 0039
Error 26619 8 3327
Total 4150 12
Stem fresh weight of
C cajan
Crop 16100 1 16100 1462 ns
Irrigation interval 9900 1 9900 0899 ns
Crop times Irrigation interval 00675 1 0067 0006 ns
Error 8806 8 11007
Total 3318 12
Root fresh weight of
C cajan
Crop 0190 1 0190 0248 ns
Irrigation interval 27331 1 27331 35753 0000
Crop times Irrigation interval 2698 1 2698 3529 0097
Error 6115 8 0764
Total 432050 12
Leaf fresh
weight of C cajan
Crop 541363 1 541363 13825 0005
Irrigation interval 347763 1 347763 8881 0017
Crop times Irrigation interval 208333 1 208333 5320 0049
Error 313246 8 39155
Total 7236 12
Stem dry weight
of C cajan
Crop 10323 1 10323 11530 0009
Irrigation interval 0452 1 0452 0505 ns
Crop times Irrigation interval 0232 1 0232 0259 ns
Error 7162 8 0895
Total 125151 12
Root dry weight
of C cajan
Crop 0007 1 0007 012 ns
Irrigation interval 0607 1 0607 972 0014
Crop times Irrigation interval 0367 1 0367 588 0041
Error 05 8 0062
Total 3515 12
Leaf dry weight
of C cajan
Crop 9363 1 9363 15649 0004
Irrigation interval 34003 1 3400 5683 0000
Crop times Irrigation interval 11603 1 11603 19392 0002
Error 4786 8 0598
Total 95072 12
Plant moisture of C cajan
Crop 199182 1 19918 6011 0039
Irrigation interval 272215 1 27221 8215 0020
Crop times Irrigation interval 76654 1 76654 2313 0166755
Error 265079 8 33134
Total 38272 12
Stem moisture
of C cajan
Crop 100814 1 10081 3290 0107246
Irrigation interval 53460 1 53460 1744 0223065
Crop times Irrigation interval 19778 1 1977 0645 0444938
Error 245119 8 30639
Total 31036 12
Root moisture
of C cajan
Crop 26266 1 26266 1389 ns
Irrigation interval 223809 1 223809 11836 0008
Crop times Irrigation interval 0097 1 0097 0005 ns
Error 151272 8 18909
Total 58346 12
Leaf moisture
of C cajan
Crop 2623 1 2623 39350 0000
Irrigation interval 1765 1 1765 26477 0000
Crop times Irrigation interval 1425 1 1425452 21378 0001
Error 533411 8 66676
Total 36263 12
Relative growth
rate of C cajan
Crop 0000 1 0000 17924 0002
Irrigation interval 0000 1 0000 21296 0001
Crop times Irrigation interval 0000 1 0000 88141 0017
Error 0000 8 0000
Total
Crop 256935 1 256935 1560 ns
Irrigation interval 268827 1 26882 1633 ns
176
Electrolyte
leakage of C
cajan
Crop times Irrigation interval 30379 1 30379 0184 ns
Error 1316923 8 16461
Total 50381 12
Chlorophyll a
of C cajan
Crop 0101 1 0101 7957 0022
Irrigation interval 0062 1 0062 4893 ns
Crop times Irrigation interval 0199 1 0199 15600 0004
Error 0102 8 0012
Total 5060 12
Chlorophyll b
of C cajan
Crop 0017 1 0017 7758 0023
Irrigation interval 0027 1 0027 12389 0007
Crop times Irrigation interval 0056 1 0056 25313 0001
Error 0017 8 0002
Total 1727 12
Total
chlorophyll of C cajan
Crop 0178 1 0178 14819 0004
Irrigation interval 0198 1 0198 16520 0003
Crop times Irrigation interval 0509 1 0509 42379 0000
Error 0096 8 0012
Total 13217 12
Chlorophyll a b
ratio of C cajan
Crop 0065 1 0065 0691 ns
Irrigation interval 0033 1 0033 0357 ns
Crop times Irrigation interval 0016 1 0016 0173 ns
Error 0756 8 0094
Total 35143 12
Carotenoids of C cajan
Crop 0021 1 0021 19599 0002
Irrigation interval 0028 1 0028 26616 0000
Crop times Irrigation interval 0041 1 0041 38531 0000
Error 0008 8 0001
Total 1443 12
Phenol of C cajan
Crop 0799 1 0799 3171 ns
Irrigation interval 0040 1 0040 0159 ns
Crop times Irrigation interval 0911 1 0911 3617 ns
Error 2016 8 0252
Total 970313 12
Proline of C cajan
Crop 0008 1 0008 14867 0004
Irrigation interval 0019 1 0019 34536 0000
Crop times Irrigation interval 0008 1 0008 14969 0004
Error 0004 8 0000
Total 0155 12
Protein of C
cajan
Crop 116376 1 116376 3990 ns
Irrigation interval 434523 1 434524 14899 0048
Crop times Irrigation interval 33166 1 33166 1137 ns
Error 233303 8 29163
Total 817371 11
CAT enzyme
of C cajan
Crop 0249 1 0249 0121 ns
Irrigation interval 2803 1 2803 13702 ns
Crop times Irrigation interval 92392 1 9239 4517 ns
Error 16362 8 2045
Total 28654 11
APX enzyme
of C cajan
Crop 855939 1 855939 4073 ns
Irrigation interval 1078226 1 1078226 5130 ns
Crop times Irrigation interval 13522 1 13522 64349 000
Error 1681112 8 210139
Total 17137 11
GPX enzyme
of C cajan
Crop 0965 1 0965 9265 0160
Irrigation interval 1167 1 1167 11195 0101
Crop times Irrigation interval 0887 1 0887 8514 0194
Error 0833 8 0104
Total 3854 11
SOD enzyme
of C cajan
Crop 4125 1 4125 9731 0142
Irrigation interval 4865 1 4865 11477 0095
Crop times Irrigation interval 20421 1 20421 48172 0001
Error 3391 8 0423
Total 32804 11
Nitrate
reductase
enzyme
Crop 0053 1 0053 0034 ns
Irrigation interval 0001 1 0001 0000 ns
Crop times Irrigation interval 10329 1 10329 6650 0327
177
of C cajan Error 12424 8 1553
Total 22808 11
Nitrate of
C cajan
Crop 0039 1 0039 0576 ns
Irrigation interval 0083 1 0083 1222 ns
Crop times Irrigation interval 0003 1 0003 0005 ns
Error 0545 8 0068
Total 0668 11
Appendix-XV Two way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C
cajan intercropped on marginal land under field condition
Variables Source Sum of Squares df Mean Square F-value P
Height of Z mauritiana
Time 79704 3 26568 77303 0000
Treatment 979209 1 979209 4702 0455
Time times Treatment 756019 3 252006 1210 3381 ns
Error 3332 16 208259
Total 90366 39
Canopy volume of Z mauritiana
Time 1049 3 3498 115444 0000
Treatment 3509 1 3509 5966 0266
Time times Treatment 3374 3 1124 1911 1684 ns
Error 9413 16 5883
Total 1284 39
flowers numbers of Z
mauritiana
Time 1794893 3 598297 770043 0000
Treatment 19980 1 19980 10152 0057
Time times Treatment 21017 3 7005 3559 0381
Error 31488 16 1968
Total 1882468 39
Fruits numbers
of Z mauritiana
Time 324096 3 108032 297941 0000
Treatment 10824 1 10824 64081 0000
Time times Treatment 7141 3 2380 14093 0001
Error 2702 16 168913
Total 351833 39
Appendix-XVI One way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C cajan intercropped on marginal land under field condition
Variables Source Sum of Squares df Mean Square F-value P
Weight of ten
fruits (FW) of
Z mauritiana
Treatment 557113 1 557113 6663 0032
Error 668923 8 83615
Total 1226036 9
Weight of ten fruits (DW) of
Z mauritiana
Treatment 4356 1 4356 0321 ns
Error 10862 8 13577
Total 112976 9
diameter of fruit of Zmauritiana
Treatment 0534 1 0534 0946 ns
Error 4514 8 0564
Total 5048 9
Fruit weight per plant of
Z mauritiana
Treatment 0739 1 0739 4022 ns
Error 1471 8 0184
Total 2211 9
Fruit sugar
(soluble) of
Z mauritiana
Treatment 5041 1 5041 0081 ns
Error 497328 8 62166
Total 502369 9
Fruit sugar (extractable) of
Z mauritiana
Treatment 32041 1 32041 0424 ns
Error 604384 8 75548
Total 636425 9
Total fruit
sugars of Z mauritiana
Treatment 16 1 16 0780 ns
Error 164 8 205
Total 18 9
Chlorophyll a of
Z mauritiana
Treatment 0082 1 0082 1384 0020
Error 0024 4 0006
Total 0105 5
Chlorophyll b
of Z mauritiana
Treatment 0011 1 0011 8469 0043
Error 0005 4 0001
Total 0016 5
Total chlorophyll of
Z mauritiana
Treatment 0152 1 0152 11927 0025
Error 0051 4 0013
Total 0203 5
Treatment 0015 1 0015 0867 ns
Error 0067 4 0017
178
Chlorophyll a b
ratio of Z mauritiana
Total 0082 5
Carotinoids of Z mauritiana
Treatment 0011 1 0011 9719 0035
Error 0004 4 0001
Total 0015 5
Leaf protein of
Z mauritiana
Treatment 0106 1 0106 4 ns
Error 0106 4 0027
Total 0213 5
Leaf sugars
(soluble) of
Z mauritiana
Treatment 054 1 054 0025 ns
Error 848 4 212
Total 8534 5
Leaf sugars
(Extractable) of Z mauritiana
Treatment 486 1 486 8055 0046
Error 2413 4 0603
Total 7273 5
Total sugars in
leaf of Z
mauritiana
Treatment 216 1 216 0104 ns
Error 83333 4 20833
Total 85493 5
Leaf phenols of
Z mauritiana
Treatment 8166 1 8166 5665 ns
Error 5766 4 1442
Total 13933 5
Leaf nitrogen of Z mauritiana
Treatment 15 1 15 1939 ns
Error 3093 4 0773333
Total 4593 5
Soil nitrogen of
Z mauritiana
Treatment 0375 1 0375 21634 ns
Error 0693 4 0173
Total 1069 5
Appendix-XVII Two way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C
cajan intercropped on marginal land under field condition
Variables Source Sum of Squares df Mean Square F-value P
Height of Ccajan
Time 700196 2 350098 2716 0000
Treatment 594405 1 594405 16017 0000
Time times Treatment 488829 2 244415 6586 0004
Error 1001996 27 37111
Total 705495 59
Number of branches of
Ccajan
Time 8353 2 4176 1050050 0000
Treatment 24066 1 24066 18672 0000
Time times Treatment 24133 2 12066 9362 0000
Error 348 27 1288
Total 8572 59
Number of flowers of
Ccajan
Time 289297 2 144648 301277 0000
Treatment 365066 1 365066 0701 ns
Time times Treatment 730133 2 365066 0701 ns
Error 14059 27 520733
Total 317415 59
Number of pods
of Ccajan
Time 347682 2 173841 70559 0000
Treatment 159135 1 159135 1558 ns
Time times Treatment 8167 2 40835 0399 ns
Error 27574 27 1021276
Total 447407 59
Appendix-XVIII One way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C
cajan intercropped on marginal land under field condition
Variables Source Sum of Squares df Mean Square F-value P
Shoot weight
(FW) of
Ccajan
Treatment 0 1 0 0 ns
Error 87444 4 21861
Total 87444 5
Shoot weight
(RW) of Ccajan
Treatment 0 1 0 0 ns
Error 13808 4 3452
Total 13808 5
Number of
seeds of
Ccajan
Treatment 245 1 245 0005 ns
Error 940182 18 52232
Total 940427 19
Weight of seeds
of Ccajan
Treatment 02 1 02 0000 ns
Error 7585 18 421406
Total 7585 19
179
Chlorophyll a of
Ccajan
Treatment 0001 1 0001 5442 ns
Error 0001 4 0000
Total 0002 5
Chlorophyll b
of Ccajan
Treatment 0006 1 0006 9079 0039
Error 0002 4 0001
Total 0008 5
Total
chlorophyll of
Ccajan
Treatment 0017 1 0017 51558 0001
Error 0001 4 0000
Total 0019 5
Chlorophyll a b ratio of
Ccajan
Treatment 0183 1 0183 5532 ns
Error 0132 4 0033
Total 0316 5
Leaf protein of Ccajan
Treatment 0001 1 0001 0017 ns
Error 0228 4 0057
Total 0228 5
Leaf sugars of
Ccajan
Treatment 0015 1 0015 0003 ns
Error 1624 4 406
Total 16255 5
Leaf phenols of
Ccajan
Treatment 0201 1 0201 0140 ns
Error 5746 4 1436
Total 5948 5
Leaf nitrogen
of Ccajan
Treatment 1306 1 1306 3062 ns
Error 1706 4 04266
Total 3013 5
Appendix-XIX Two way ANOVA for completely randomized design for investigations on determining range of salt tolerance in Carissa
carandas
Variables Source Sum of Squares df Mean Square F-value P
Height of C carandas
Time 72042 5 14408 55957 0000
Salinity treatment 49345 2 24672 196775 0000
Time times Salinity treatment 16679 10 1667920 13302 000
Error 3009 24 125385
Total 143777 53
Volume of
canopy of
C carandas
Time 3329 4 0832 38126 000
Salinity treatment 1393 2 0696 67129 000
Time times Salinity treatment 0813 8 0102 9792 000
Error 0207 20 0010
Total 5969 44
Appendix-XX One way ANOVA for completely randomized design for investigations on determining range of salt tolerance in Carissa carandas
Variables Source Sum of Squares df Mean Square F-value P
Number of
flowers of C carandas
Salinity treatment 10288 2 5144194 1342937 0000
Error 229833 6 38305
Total 10518 8
Number of fruits of
C carandas
Salinity treatment 18000 2 9000 268215 0000
Error 201333 6 33555
Total 18201 8
Flower shedding
percentage of C carandas
Salinity treatment 1541647 2 770823 53455 0000
Error 86519 6 144199
Total 1628166 8
Weight of ten fruits (FW) of
C carandas
Salinity treatment 82632 2 41316 187678 0000
Error 1321 6 0220
Total 83953 8
Weight of ten
fruits (DW) of
C carandas
Salinity treatment 4355 2 2177 13753 0005
Error 095 6 0158
Total 5305 8
Fruits per plant
(FW) of
C carandas
Salinity treatment 133127 2 66563 278148 0000
Error 1435861 6 239310
Total 134563 8
Fruits per plant
(DW) of C carandas
Salinity treatment 8782 2 439117 117790 0000
Error 223677 6 37279
Total 9006 8
Size of fruits of C carandas
Salinity treatment 1301 2 0651 4125 ns
Error 0946 6 0158
Total 2248 8
Salinity treatment 5607 2 2804 17592 0003
180
Diameter of fruit
of C carandas
Error 0956 6 0159
Total 6563 8
Chlorophyll a of C carandas
Salinity treatment 0112 2 0056 119786 0000
Error 0003 6 0000
Total 0115 8
Chlorophyll b of
C carandas
Salinity treatment 0005 2 0002 434 0000
Error 0000 6 0000
Total 0005 8
Total chlorophyll of C carandas
Salinity treatment 0159 2 0079 104188 0000
Error 0005 6 0001
Total 0164 8
Chlorophyll a b
ratio of C carandas
Salinity treatment 9661 2 4831 324691 0000
Error 0089 6 0015
Total 9751 8
Carotenoids of C carandas
Salinity treatment 0029 2 0014 28822 0000
Error 0003 6 0001
Total 0032 8
Leaf Protein of
C carandas
Salinity treatment 2722 2 1361 98 0012
Error 0833 6 0138
Total 3555 8
Soluble sugar of
C carandas
Salinity treatment 234889 2 117444 12735 0006
Error 55333 6 9222
Total 290222 8
In soluble sugars
of C carandas
Salinity treatment 595395 2 297698 39094 0000
Error 45689 6 7615
Total 641085 8
Total sugar of
C carandas
Salinity treatment 1576898 2 788448 39201 0000
Error 120676 6 20113
Total 1697574 8
Phenols of C carandas
Salinity treatment 14675 2 7338 74202 0000
Error 0593 6 0099
Total 15268 8
Leaf Na+ of
C carandas
Salinity treatment 1346 2 673 673 0000
Error 6 6 1
Total 1352 8
Leaf K+ of C carandas
Salinity treatment 798 2 399 133 0000
Error 18 6 3
Total 816 8
Leaf K+ Na+
ratio of C carandas
Salinity treatment 0305 2 0153 654333 0000
Error 0001 6 0000
Total 0307 8
181
7 Publications
iv
CERTIFICATE
It is hereby certified that this thesis is based on the results of the experimental work carried
out by Mr TAYYAB SO MUHAMMAD HANIF under my supervision on the topic
ldquoInvestigation on intercropping of Ziziphus mauritiana with Cajanus cajan for fruit
and fodder at marginal land and cultivation of Carissa carandas for fruits through
saline water irrigationrdquo
Mr TAYYAB had been enrolled under my guidance for the award of PhD in
Department of Botany University of Karachi I have personally checked all the research
work reported in the thesis and certify its accuracyvalidity It is further certified that the
materials included in this thesis have not been used partially or fully in a manuscript
already submitted or in the process of submission in partialcomplete fulfillment for award
of any other degree from any other university Mr TAYYAB has fulfilled requirements of
the University of Karachi for the submission of this dissertation and I endorse its
evaluation for the award of PhD Degree
RESEARCH SUPERVISOR
PROF DR RAFIQ AHMAD
FPAS FTWAS
Professor (Retd) Botany (Plant Physiology)
PI Biosaline Research Projects
Department of Botany
University of Karachi
Karachi-75270 Pakistan
v
DEDICATED TO MY FAMILY
MUHAMMAD HANIF (MY FATHER)
MRS ARIFA (LATE)
(MY BELOVED MOTHER)
SHAHEEN TAYYAB (MY WIFE)
vi
ACKNOWLEDGMENTS
All the praises for almighty Allah and all respects for Prophet Muhammad (Peace be Upon
Him) who has shown me the straight path
I am grateful to my supervisor Prof Dr Rafiq Ahmad for his keen interest
patronage and guidance during this research work which made successful submission of
this thesis
I also obliged to Prof Dr Ehtesham Ul Haque and Prof Dr Javed Zaki (Present
and Former Chairmen Department of Botany respectively) for providing me all the
necessary facilities and administrative support
Being employed as lecturer in Department of Botany Govt Islamia Science
College Karachi I am also thankful to Education and literacy Department Govt of Sindh
(Pakistan) for providing me facilities to perform this study
Thanks are due to Dr D Khan in assessing statistical data analysis and colleague
of Biosaline lab Dr M Azeem Dr Naeem Ahmed and M Wajahat Ali Khan for their
cooperation throughout the course of study
I am also gratefully acknowledged to Mr Noushad Raheem and Mr Noor Uddin
of Fiesta Water Park for providing field plot and facilities to perform this study I am also
thankful to Pakistan Metrological Department for providing environmental data
I am also obliged to Dr M Qasim and Dr M Waseem Abbasi for their suggestions
and support in writing this thesis
Assistance of Abbul Hassan (Lab attendant) Tajwar Khan (Biosaline field
Attendant) and Mr Wahid (Plant Physiology Lab Assistant) is also acknowledged
Thanks are also due to my friends Dr Rafat Saeed Dr Kabir Ahmad Dr Zia Ur
Rehman Farooqi Dr Noor Dr M Yousuf Adnan Asif Bashir Dr A Rauf A Hai Faiz
Ahmed MA Rasheed Jallal Uddin Saadi Ahsan Shaikh Saima Fehmi A Mubeen
Khan Dr Noor Ul Haq Saima Ahmad S Safder Raza SM Akber and my college
colleagues for giving me encouragement during this research work
vii
I can never forget the support and encouragement and good wishes of Mr M
Wilayat Ali Khan Mrs Shahnaz Rukhsana Mr Mansoor Mrs Rabia Mansoor Mrs
Chand Bibi and Mrs Saeeda Anwar
In the last I am highly grateful to my beloved father Muhammad Hanif my loving
mother Arifa (when she alive) my caring wife Shaheen and sweet childrenrsquos Sara and
Sarim my supportive brothers and sisters and all family members for their prayers love
sacrifices and encouragements provided during course of this research work
viii
TABLE OF CONTENTS
No Title Page no
Acknowledgement vi
Summary xix
Urdu translation of summary xxi
General introduction 1
Layout of thesis 11
1 Chapter 1 13
11 Introduction 13
12 Experiment No 1 15
121 Materials and methods 15
1211 Seed collection 15
1212 Experimental Design 15
122 Observations and Results 17
13 Experiment No 2 22
131 Materials and methods 22
1311 Seed germination 22
132 Observations and Results 23
14 Experiment No 3 28
141 Materials and methods 28
1411 Seedling establishment 28
142 Observations and Results 29
1421 Seedling establishment 29
1422 Shoot height 29
15 Experiment No 4 31
151 Materials and methods 31
1511 Drum pot culture 31
1512 Experimental design 31
1513 Vegetative and Reproductive growth 32
1514 Analysis on some biochemical parameters 32
152 Observations and Results 34
1521 Vegetative and Reproductive growth 34
ix
No Title Page no
1522 Study on some biochemical parameters 34
16 Experiment No 5 41
161 Materials and methods 41
1611 Isolation Identification and purification of bacteria 41
1612 Preparation of bacterial cell suspension 41
1613 Study of salt tolerance of Rhizobium isolated from root
nodules of C cajan
41
162 Observations and Results 42
17 Experiment No 6 44
171 Materials and methods 44
1711 Experimental design 44
1712 Vegetative and reproductive growth 45
1713 Analysis on some biochemical parameters 45
172 Observations and Results 46
1721 Vegetative and Reproductive growth 46
1722 Study on some biochemical parameters 46
18 Discussion (Chapter 1) 51
2 Chapter 2 59
21 Introduction 59
22 Experiment No 7 60
221 Materials and Methods 60
2211 Growth and Development 60
2212 Drum pot culture 60
2213 Experimental Design 60
2214 Irrigation Intervals 61
2215 Estimation of Nitrate content 62
2216 Relative Water content (RWC) 62
2217 Electrolyte leakage percentage (EL) 62
2218 Photosynthetic pigments 63
2219 Total soluble sugars 63
22110 Proline content 63
22111 Soluble phenols 64
x
No Title Page no
22112 Total soluble proteins 64
22113 Enzymes Assay 64
222 Observations and Results 67
2221 Vegetative growth 67
2222 Photosynthetic pigments 70
2223 Electrolyte leakage percentage (EL) 70
2224 Phenols 70
2225 Proline 71
2226 Protein and sugars 71
2227 Enzyme essays 71
2228 Vegetative growth 73
2229 Photosynthetic pigments 75
22210 Electrolyte leakage percentage (EL) 76
22211 Phenols 76
22212 Proline 77
22213 Protein and Sugars 77
22214 Enzyme assay 77
23 Experiment No8 90
231 Materials and Methods 90
2311 Selection of plants 90
2312 Experimental field 90
2313 Soil analysis 90
2314 Experimental design 91
2315 Vegetative and reproductive growth 93
2316 Analysis on some biochemical parameters 93
2317 Fruit analysis 94
2318 Nitrogen estimation 94
2319 Land equivalent ratio and Land equivalent coefficient 95
23110 Statistical analysis 95
232 Observations and Results 96
2321 Vegetative parameters 96
2322 Reproductive parameters 96
xi
No Title Page no
2323 Study on some biochemical parameters 97
2324 Nitrogen Contents 98
2325 Land equivalent ratio land equivalent coefficient 98
24 Discussion (Chapter 2) 108
3 Chapter 3 113
31 Introduction 113
32 Experiment No 9 114
321 Materials and methods 114
3211 Drum Pot Culture 114
3212 Plant material 114
3213 Experimental setup 114
3214 Vegetative parameters 115
3215 Analysis on some biochemical parameters 115
3216 Mineral Analysis 116
322 Observations and Result 117
3221 Vegetative parameters 117
3222 Reproductive parameters 117
3223 Study on some biochemical parameters 118
3224 Mineral analysis 118
33 Discussion (Chapter 3) 127
4 Conclusion 129
5 References 130
6 Appendices 168
7 Publications 181
xii
LIST OF FIGURES
Figure Title Page no
11 Effect of irrigation water of different sea salt solutions on seed
germination indices of C cajan
27
12 Effect of irrigating water of different sea salt solutions on
seedling emergence (A) and shoot length (B) of C cajan
30
13 Environmental data of study area during experimental period
(July-November 2009)
36
14 Effect of salinity using irrigation water of different sea salt
concentrations on height of C cajan during 18 weeks treatment
36
15 Effect of salinity using irrigation water of different sea salt
concentrations on initial and final biomass (fresh and dry) of C
cajan
37
16 Percent change in moisture succulence relative growth rate
(RGR) and specific shoot length (SSL) of C cajan under
increasing salinity using irrigating water of different sea salt
concentrations
37
17 Effect of irrigating water of different sea salt solutions on
reproductive growth parameters including number of flowers
pod seeds and seed weight of C cajan
38
18 Effect of irrigating water of different sea salt solutions on leaf
pigments including chlorophyll a chlorophyll b total
chlorophyll and carotenoids of C cajan
39
19 Effect of irrigating water of different sea salt solutions on total
proteins soluble insoluble and total sugars in leaves of C cajan
40
110 Growth of nitrogen fixing bacteria associated with root of C
cajan under different NaCl concentrations
42
111 Photographs showing growth of Rhizobium isolated from the
nodules of C cajan in vitro on YEM agar supplemented with
different concentrations of NaCl
43
xiii
Figure Title Page no
112 Effect of salinity using irrigation water of different sea salt
concentrations on height number of branches fresh weight and
dry weight of shoot of Z mauritiana after 60 and 120 days of
treatment
47
113 Effect of salinity using irrigation water of different sea salt
concentrations on succulence specific shoot length (SSL)
moisture and relative growth rate (RGR) of Z mauritiana
48
114 Effect of salinity using irrigation water of different sea salt
concentrations on number of flowers of Z mauritiana
49
115 Effect of salinity using irrigation water of different sea salt
concentrations on leaf pigments including chlorophyll a
chlorophyll b total chlorophyll and chlorophyll ab ratio of Z
mauritiana
49
116 Effect of salinity using irrigation water of different sea salt
concentrations on total sugars and protein in leaves of Z
mauritiana
50
21 Vegetative parameters of Z mauritiana and C cajan at grand
period of growth under sole and intercropping system at two
irrigation intervals
79
22 Fresh and dry weight of Z mauritiana and C cajan plants under
sole and intercropping system at 4th and 8th day irrigation
intervals
80
23 Leaf weight ratio (LWR) root weight ratio (RWR) shoot weight
ratio (SWR)specific shoot length (SSL) specific root length
(SRL) plant moisture Succulence and relative growth rate
(RGR) of Z mauritiana and C cajan grow plants under sole and
intercropping system at 4th and 8th day irrigation intervals
81
24 Leaf pigments of Z mauritiana and C cajan grow plants under
sole and intercropping system at 4th and 8th day irrigation
intervals
83
xiv
Figure Title Page no
25 Electrolyte leakage phenols and proline of Z mauritiana and C
cajan at grand period of growth plants under sole and
intercropping system at 4th and 8th day irrigation intervals
84
26 Total protein in leaves of Z mauritiana and C cajan plants
under sole and intercropping system at 4th and 8th day irrigation
intervals
86
27 Enzymes activities in leaves of Z mauritiana and C cajan plants
under sole and intercropping system at 4th and 8th day irrigation
intervals
87
28 Nitrate reductase activity and nitrate concentration in leaves of
Z mauritiana and C cajan plants under sole and intercropping
system at 4th and 8th day irrigation intervals
89
29 Soil texture triangle (Source USDA soil classification) 99
210 Vegetative growth of Z mauritiana and C cajan growing under
sole and intercropping system
100
211 Reproductive growth of Z mauritiana and C cajan growing
under sole and intercropping system
101
212 Leaf pigments of Z mauritiana and C cajan growing under sole
and intercropping
102
213 Sugars protein and phenols in leaves of Z mauritiana and C
cajan at grand period of growth under sole and intercropping
system
103
214 Sugars protein and phenols in fruits of Z mauritiana grown
under sole and intercropping system
105
215 Nitrogen in leaves and in soil of Z mauritiana and C cajan
growing under sole and intercrop system
106
31 Chlorophyll a chlorophyll b total chlorophyll chlorophyll a b
ratio carotenoids contents of C carandas growing under
salinities created by irrigation of different dilutions of sea salt
124
xv
Figure Title Page no
32 Total protein sugars and phenolic contents of C carandas
growing under salinities created by irrigation of different
dilutions of sea salt
125
33 Mineral analysis including Na and K ions was done on leaves of
C carandas growing under salinities created by irrigation of
different dilutions of sea salt
126
xvi
LIST OF TABLES
Table Title Page no
11 Electrical conductivities of different sea salt solutions
used in germination of C cajan
18
12 Effect of irrigation water of different sea salt solutions
on germination percentage (GP) per day of C cajan
seeds pre-soaked in non-saline water prior to
germination with duration of time under various salinity
regimes
19
13 Effect of irrigation water of different sea salt solutions
on germination rate (GR) per day of seeds C cajan pre-
soaked in non-saline water prior to germination with
duration of time under various salinity regimes
20
14 Effect of irrigation water of different sea salt solutions
on mean germination rate (GR) coefficient of
germination velocity (GV) mean germination time
(GT) mean germination index (GI) and final
germination (FG) of C cajan seeds pre-soaked in non-
saline water prior to germination under various salinity
regimes
21
15 Electrical conductivities of different sea salt solutions
used in germination of C cajan
24
16 Effect of irrigation water of different sea salt solutions
on germination percentage (GP) per day of C cajan
seeds pre-soaked in respective sea salt concentrations
with duration of time
25
17 Effect of irrigation water of different sea salt solutions
on germination rate (GR) per day of C cajan seeds pre-
soaked in respective sea salt concentrations with
duration of time
26
xvii
Table Title Page no
18 Electrical conductivities of different Sea salt
concentrations and ECe of soil saturated paste at the end
of experiment
30
21 Soil analysis data of Fiesta Water Park experimental
field
99
22 Land equivalent ratio (LER) and Land equivalent
coefficient (LEC) with reference to height chlorophyll
and yield of Z mauritiana and C cajan growing under
sole and intercropping system
107
31 Electrical conductivities of different sea salt
concentration used for determining their effect on
growth of C carandas
119
32 Vegetative growth in terms of height and volume of
canopy of C carandas growing under salinities created
by irrigation of different dilutions of sea salt
120
33 Vegetative growth in terms of height and volume of
canopy of C carandas growing under salinities created
by irrigation of different dilutions of sea salt
121
34 Reproductive growth in terms of flowers and fruits
numbers flower shedding percentage fresh and dry
weight of ten fruit and their totals per plant fruit length
and diameter of C carandas growing under salinities
created by irrigation of different dilutions of sea salt
123
xviii
LIST OF ABBREVIATIONS
APX Ascorbate peroxidase
CAT Catalase
DAP Diammonium Phosphate (fertilizer)
dSm-1 Deci Siemens per meter
ECe Electrical conductivity of the Soil saturated extract
ECiw Electrical conductivity of the irrigation water
GPX Guaiacol Peroxidase
GR Glutathione reductase
GSH Reduced glutathione
LEC Land equivalent coefficient
LER Land equivalent ratio
NPK Nitrogen Phosphate Potash (fertilizer)
NR Nitrate reductase
RGR Relative growth rate
ROS Reactive oxygen species
RWR Root weight ratio
SOD Superoxide dismutase
SRL Specific Root Length
SSL Specific Shoot Length
SWR Shoot weight ratio
xix
Summary
Salinity is a growing threat to crop production which affects sustainability of agriculture
in aridsemiarid areas Growth responses of plant to salinity vary considerably among
species Cajanus cajan Ziziphus mauritiana and Carissa carandas are sub-tropical crops
grown worldwide particularly in Asian subcontinent for edible and fodder purposes but
not much is known about their salinity tolerance and intercropping
Effect of salinity has been initially studied in present work at germination of C cajan
under different sea salt salinities using presoaked seeds with water and respective salt
solutions Seed germination decreased with increasing salinity and it was more sever in
presoaking under water of different salinities The 50 threshold reduction started at
ECiw= 35 dSm-1 sea salt in presoaking treatments However this threshold was decreased
up to ECiw= 168 dSm-1 sea salt at further seedling establishment stage Growth experiment
of C cajan in drum pot culture (Lysimeter) also showed a salt induced growth reduction
in which plant tolerate salinity up to 42 dSm-1 At this salinity leaf pigments (chlorophylls
and carotenoids) proteins and insoluble sugars decreased up to 50 whereas soluble
sugars were increased (~25) Reproductive growth was also affected at this salinity in
which at least 70 reduction in flowers pods and seeds were observed
Salt tolerance of symbiotic nitrogen fixing bacteria associated with root of C cajan
showed salinity tolerance up to ECw= 366 dSm-1 NaCl salinity invitro environment For
intercropping experiments Ziziphus mauritiana (grafted variety) was selected with C
cajan Preliminary investigations showed a growth promotion in Z mauritiana at low
salinity (ECe= 72 dSm-1) and growth was remained unaffected up to ECe= 111 dSm-1
Intercropping of C cajan with Z mauritiana was primarily done in drum pot (Lysimeter)
culture Result showed better growth responses of both species when growing together as
intercrops than sole in which encouraging results were found in 8th day irrigation interval
rather than of 4th day Biochemical parameters eg photosynthetic pigments protein
phenols electrolyte leakage and sugars of these species displayed increase or decrease
according to their growth responses Increased activity of antioxidant enzymes and that of
nitrate reductase and its substrate (NO3) also contributed in enhancement of growth
Field experiment of intercropping of above mentioned plants at marginal land
irrigated with underground water (Eciw= 28 dSm-1) showed better vegetative growth of
xx
both species than sole crop The overall reproductive growth remained unaffected
although the numbers size and weight of fruit were better in intercropping system
Photosynthetic pigments were mostly increased whereas leaf protein and sugars remained
unchanged In addition higher values of LER and LEC (gt 1) indicated the success of
intercropping system
Experiment on salinity tolerance of Carissa carandas (varn karonda) using drum
pots culture showed improvement at low salinity (up to ECiw= 42 dSm-1 sea salt) whereas
higher salinity (ECiw= 129 dSm-1 sea salt) adversely affected vegetative and reproductive
growth Plant managed to tolerate up to ECiw= 99 dSm-1 sea salt Salinity severely affected
biochemical parameters including photosynthetic pigments proteins and sugars whereas
leaf phenolics were increased Leaf accumulated high amount of Na+ whereas affect
absorption of essential minerals like K+ was decreased
In the light of above mentioned investigations it appears that C cajan can be
propagated in saline soils with good presoaking techniques in non-saline water which
would helped to grow at moderately saline conditions It could be a good option used as
intercrop species because of its ability to improve soil fertility even under water deficit
conditions The proposed Cajanus-Ziziphus intercropping system could help poor farmers
to generate income from unproductive soils by obtaining sufficient fodder from C cajan
for their cattle and producing delicious edible fruits from Z mauritiana for commercial
purposes Carissa carandas could also be introduced as new crop for producing fruits from
moderate saline waste lands and used for preparing prickle jam and jelly for industrial
purposes
xxi
لاصہ خ
کا عمل ے ں ب ڑھئ لف پ ودوں می ی ےمخ طرہ ہ
وا خ ا ہ ے ب ڑھی لئ داوار کے ی ں زرعی ب وں می
ر علاق ج
ن ی م ب
ر و ب ج ن کھاری پ ن کھاری پ ن ب
دا کروت ی ر اور ر ب ے ارہ ا ہ وت لف ہ ی ی مخ کاف ں ودگی می اص Subtropical کی موج ا اور خ ی و پ وری دب ں ج ی ں ہ صلی
کی ف طے
خ
وراک و ں ج می
ی ملکوں
ائ ی ش کھاکر ای کی ی ان پ ودوں کم لوگ ہ ہت کن ب ں لی ی ی ہ
وئ عمال ہ
ارے کے طور ب ر است ری پ ن سے خ
ں ی ے ہ ں علم رکھئ ارے می ے عمل کے ت گئ ے گائ
کر ا ھ ملا
ی سات ک ہ رواداری اور ات
وں ج ن ر کےب ے ارہ
ھگوئ ہلے سے ت ں ب کاز والے محلول می لف ارت ی
مک کے مخ
دری ں ں سمی ی مطالعہ می
دائ ی کھاری اب کا
کہ پ ن کے و ی ج وئ ع ہ
کمی واف ں ی ت می ب
کی طن وں ج ن
ھ ب ہ کے سات
اف ں اض کھاری پ ن می ا گی ا کی دہ اہ کا مش رات
iwEC =اب
1-35 dSm می خ ی کہ ت ی ج مک کے ب راب ررہ
دری ں زی سمی کا
ہ ارت ں ی ام می ی ت صدی dSm= iwEC 168-1پ ودوں کے ق
ق
ی ک رہ ں Lysemeterت ے والے پ ودوں می ڑھئ ں ب روان چ می 1-dSm 24 ں جوضلہ مک محلول می
دری ں زی سمی کا
ارت
ں کر می ر خل ب زب ر س ی
ات اور غ روز مادوں لمخی
گ اف الت ف کے رت ی ت
ائ ی ں ض کھاری پ ن می ی اس
گئ کھی
ت ت د زا ب رداش
ت صدی 05اف
ق
ی ش کم وب ں کر می ی کہ خل ب زب ر س ں 50کمی ج وں می ج ن
ھلی اورب ھول ت ں ت ن می ری ج دی ب ڑھوب ولی
ا پ ا رہ مات
ہ ں اف ت صدی اض
05ق
ی گئ کھی
ت ح طور د
کمی واض ت صدی
ق
ی وی شلک سہب ڑ سے می کی چ ر مک (Symbiotic)ارہ
کی ں ا رت ی
کٹ ی ے والے ب
کرئ مد خ
ن من روج ی
اب سے (NaCl)ت
ی ر کے سا dSmwEC 366 =-1رواداری ں ب ری ہ می ج ے عمل کے ت گئ ے
گائ
کر ا ھ ملا
ی سات ک ہ یات
گئ کھی
ت ک د ر ت ھ ارہ
ت
بی ق کے ب
حق ی ت دائ ی ا اب گی ا ی
کھاری پ ن کو ج کم ں ے می ج ں dSme (Ec 72 =-1(ن ی کہ می ری ج ں ب ڑھوب ی ر می e (Ec =ب
)1-111 dSm ہل ہلے ب ے عمل ب گئ ے
گائ
کر ا ھ ملا
ی سات ک ہ کو ات ر ی ر اور ب ی ارہ
ر رہ اب ر می ی
ک غ کی خد ت
Lysemeter ج ب رآم ت ا ی زا ب ی کے جوضلہ اف
اش ی ے سے آب
ف ف ھ دن کے و
سی ت آت
کی ی ار دن ی خ
گئ کی ں ں دمی ن می ے ج
وئ ہ
ے عمل گئ ے گائ
کر ا ھ ملا
ی سات ک ہ سی ت ات
کی ی ے پ ودوں
گائ
ن ہا ا کی پ ودوں ب شام
وں اق
ے دوپ ج گئ
ت ا ی زا ب ادہ جوضلہ اف ں زت می
ی ول ب ات ف روزمادوں لخمی
گ اف الت ف کے رت ی ت
ائ ی ضلاات می درخ ی می
ائ کی می ی
ائ ےجی
وئ Electrolyteب رآمد ہ
Leakage کی کر ں س ی وں می ب ی ان پ ودوںاور ب
ی ش کمی ب ں دار می ی دپ ں مق
ں دکھائ ر می
اظ ی ری کے ب
کے ب ڑھوب
xxii
Antioxidant ی ظرح سے ہ اور اس ہ اف ں اض کی سرگرمی وں می امروں
اور اس کے Nitrate Reeducatesخ
Substrate )3(NO ا ی کا سی ب ب ہ اف ں اض ما می وں
ش ھی ی
ت
ےdSmiw(Ec 28 =-1(معمولی گئ ے ئ کب راب ں سی ی می ائ ہ ت والے ت درج ں می ری ہ می ج
ی ت ئ ن ہا زمب کی ب الا پ ودوں
ے عمل گئ ے گائ
کر ا ھ ملا
ی سات ک ہ سی ت ات
کی ی ے پ ودوں
ادوں ب ر لگائ ی
ب ما ب وں
ش دی ی ولی
ے پ
وئ ج خاضل ہ
ت ا ی ر بہی ادہ ب ں زت می
ےض ر رہ ہی ں ب ام می ط ے ت گئ ے
گائ
کر ا ھ ملا
ی سات ک ہ شامت اور وزن ات عداد ج
کی ت ھلوں ی کہ ت ی ج ر رہ اب ر می ی
الت ف ی غ ی ت
ائ
ی وئ ں ہ ہی
ع ب ی دت لی واف ی ب
کوئ ں دار می کی مق کر
ات اور س ں لمخی ی وں می ب ی کہ ب ہ ج
اف ا اض مات
ں ں روزمادوں می
گ اف د کے رت LER مزت
ے LEC (gt1)اور ی ہ کرئ ارہ کی ظرف اس ی ائ کامی کی ام
ط ے ت گئ ے
گائ
کر ا ھ ملا
ی سات ری ات ک ہ
کی ب ڑھوب
ک دا کروت ں ری ہ می ج کھاری پ ن ) Lysemeterو کھاری پ ن روداری کے ت ا کم گی ا ں اگات iwEC = 142می
1-dSm ( کھاری پ ن ادہ ی کہ زت ی ج وئ ری ہ ہی ں ب مک( می
دری ں زی سمی کا
زی dSm= iwEC 129-1 ارت کا دری ارت سمی
ی وئ ر ہ
اب ری ب ری ظرح می
دی ب ڑھوب ولی
ی اور پ
ائ علی
ں ف مک( می
ی کہ ں ک dSm9= iw(Ec 9-1(ج مک ت
دری ں زی سمی کا
ارت
ت کب رداش ات اور س روز مادوں لخمی گ اف الت ف کے رت ی ت
ائ ی ضلاات می درخ ی می
ائ کی می ی
ائ ےجی اب رہ کامی ں ےمی
ر ب ری ظرح کرئ
ں ی وں می ب وا ب ہ ہ
اف ں اض ی ول می ب
ں ف ی وں می
ب ی کہ ب ں ج ی
وب ر ہ اب می
+Na ہ سے کی وج مع ی ج اف رلز کے K+اض روری می
ی سے ض ج
ی وئ ر ہ
اب کی ضلاجی ت می ے
کرئ زب چ
ا ت ق حق الا ت ہ ت درج ے ظر می
وئ ےہ
ھگوئ ں ت ی می
ائ ہلے سے ت کہ ب ی
ے آئ مئ ں ی ہ ت ات سا ی می
ئ کی روش ر ت ہ سے ارہ کی وج ے
ت ف
ھی مدد دے س ں ت ے می گئ ں ا ن می ن زمی مکی دل ں وکہ معی ے ج ا ہ اسکی ا خ ھی لگات
ں ت ن خالات می مکی کو ں وں ج ن
وزہ کے ب ے مج ا ہ کی
داواری ی ر ب ی ے عمل غ گئ ے
گائ
کر ا ھ ملا
ی سات ک ہ ی ر ات ر اور ب ی ضلاجی ت والی ارہ
اف ے اض لئ وروں کے
اپ کی صور ت خ ر ن ارہ زمی
ھی دا ت کروت ے ا ہ وسکی ت ہ اب کا ذرت عہ ت ے ی ب ڑھائ
کی آمدئ وں
کشاپ ی صورت
ارئ ح کی ت ل
ھ ی ت وردئ دار ج ی ر سے مزت ارہ اور ب ی خ
عئصت
صل کے طور ب ی ف ئے ب لئ ے کے
کرئ دا ی ھل ب ن سے ت کارآمد زمی ر ی
ن اور غ مکی
دل ں ے معی
لئ اضد کے ے رمق ا ہ اسکی ا خ کی ی ش ب
1
General Introduction
Intercropping is a major resource conservation technique for sustainable agriculture under
various climatic conditions (Zhang et al 2010 Li et al 2014) It can reduced operational
cost for the production of multiple crops with maintained or even higher level of
productivity (Vandermeer 2010 Perfecto and Vandermeer 2010) It can enhance the
water use efficiency by saving 20 to 40 irrigation water with improved fertilizer
management (Fahong et al 2004 Jat et al 2005 Jani et al 2008) Intercropping system
is more suitable in marginal areas with lower mechanization and cultivation input by
farmers on small tracts of farmlands (Ngwira et al 2012) It can enhance the cumulative
production per unit area and protect the small farmers against market fluctuations or crop
failure ensure the income improve soil fertility and food demands (Rusinamhodzi et al
2012) In this system dominating more compatible and productive species are selected or
replaced in which complementarity effects and beneficial interactions resulting enhanced
yield as compared to monoculture (Huston 1997 Loreau and Hector 2001) It was
estimated that in species diverse systems biomass production is 17 times higher as
compared to monoculture (Cardinale et al 2007)
It is suggested that intercropping is the best suitable cropping system which can
improve the resource-use efficiency by procurement of limiting resources enhanced
phyto-availability and effective plants interactions (Marschner 2012 White and
Greenwood 2013 Ehrmann and Ritz 2014) It is widespread in many areas of world
particularly in latin America it is estimated about 70-90 by small farmers which mainly
grow maiz potatoes beans and other crops under this system whereas intercropping of
maiz with different crops is estimated about 60 (Francis 1986) Additionally
agroforestry is more than 1 billion ha in this area (Zomer et al 2009) The land used for
intercropping system of various crops is greatly varied from 17 in India to 98 in Africa
(Vandermeer 1989 1992 Dupraz and Liagre 2011)
In intercropping system two or more crops or genotypes coexist and growing
together at a same time on a similar habitat (Li et al 2013) It may be divided into various
types such as in mixed intercropping system two or more crops simultaneously growing
without or with limited distinct arrangements whereas in relay intercropping system
second crop is planted when the first is matured while in strip intercropping both the crops
2
are simultaneously growing in strips which can facilitate the cultivation and crop
interactions (Ram et al 2005 Sayre and Hobbs 2004)
Several less-conventional fruit tress including Manilkara zapota (Chicko)
Ziziphus mauritiana (Jujubar) Carissa carndas (Karanda) Annona squamosa (Sugar
apple) and Grewia asiatica (Falsa) has been reported with high nutritional value with
capability to grow at marginal lands (Mass and hoffman 1997) Qureshi and Barrett-
Lennard (1998) suggested few grafted plants that can widely use to improve the quality
and productivity of fruits Grafting is also used to induce stress tolerance in plants against
various abiotic and biotic stresses including salinity stress (Rivero et al 2003) Both root
stocks and shoot stocks contribute to increase the tolerance level of plants Root stocks
represent the first part of defense to control the uptake and translocation of nutrients and
salts throughout the plant (Munns 2002 Santa-cruz et al 2002 Zrig et al 2011) while
shoot stocks develops physiological and biochemical changes to promote plant growth
under stress conditions (Moya et al 2002 Chen et al 2003)
Ziziphus mauritiana Lamk (varn grafted ber) belongs to the family Rhamnaceae
grows widely in most of the dry tropical and subtropical regions around the world Various
grafting methods are used for their propagation including wedge and whip or tongue
methods (Nerd and Mizrahi 1998) Intercropping of these grafted fruit trees with various
leguminous crops is also being successfully practiced in many countries thought the world
Leguminous crops are considered excellent symbiotic nitrogen fixing crops It can
effectively improve soil fertility and offset the critical problems of sub-tropical areas to
fight against desertification and soil degradation These plants are considered as an
excellent source of proteins for humans and animals They can fix the 90 of atmospheric
nitrogen and contribute 40 nitrogen to the soil thus increase the soil fertility (Peoples et
al 1995) However most of the leguminous plants are not salt tolerant while some
species are better drought tolerant and effectively contribute in marginal lands (Zahran
1999)
Among the leguminous plants Pigeon pea (Cajanus cajan (L) Millspaugh) of the
family Fabaceae is widely grown for food fodder and fuel production particularly in
semiarid areas The salinity tolerance of this specie is not well documented both at
germination and seedling stages This crop is still underexploited due to its edible and
3
economic importance While limited investigations has been made to uncover its
nutritional quality medicinal uses and drought tolerance
The identical physiological traits are important in both the mono and intercropping
systems to maximize the resource acquisition The exploitation of best possible
combination of traits of different plants in intercropping system is very important to
maximize the overall performance in intercropping system It depends on the above ground
beneficial plant interactions for light space and optimal temperatures (Wojtkowski 2006
Zhang et al 2010 Shen et al 2013 Ehrmann and Ritz 2014) as well as the
complementary below ground plant interactions with soil biotic factors (Bennett et al
2013 Li et al 2014)
Water is also a major limiting factor intercropping can enhanced the acquisition
of water by root architecture and distribution in the soil profile for effective utilization of
rainfall (Zegada-Lizarazu et al 2006 De Barros et al 2007) and enhanced the water use
efficiency for effective hydraulic redistribution by deep rooted crops and water stored in
the soil profile (Morris and Garrity 1993 Xu et al 2008) Mycorrhizal networks around
the roots of intercrop plants also enhanced the availability of water and available resources
and reduced the surface runoff (Caldwell et al 1998 Van-Duivenbooden et al 2000
Prieto et al 2012)
Intercropping with leguminous plants can enhanced the agricultural productivity in
less productive soils due to enhanced nitrogen availability and also improved the soil
fertility by effective nitrogen fixation (Seran and Brintha 2010 Altieri et al 2012) Due
to weaker soil nitrogen competition intercropping with legumes enhanced the nitrogen
availability to the non-leguminous intercrop which also absorbs the additional nitrogen
released in the soil or root nodules of the leguminous plant (Li et al 2013 White et al
2013a) The use of legumes in many intercropping systems is pivotal According to the
listing of Hauggaard-Nielsen and Jensen (2005) seven out of ten are the legumes among
the most frequently used intercrops around the world
The ecological range of adaptability of legumes reaches from the inner tropics to
arctic regions with individual species expressing tolerance to drought temperature
nutrient deficiency in soil water logging salinity and other environmental conditions
(Craig et al 1990 Hansen 1996) The woody perennial leguminous plants have a number
4
of purposes they can be used to reclaim degraded wastelands retard erosion and provide
shade fuel wood timber and green manure (Giller and Wilson 1991)
Trees with nitrogen fixing capability play an important role to offset the critical
problems of tropical and sub-tropical regions in their fight against desert encroachment
and soil impoverishment These plants are capable to live in N-poor soils through their
association with Rhizobium that fix atmospheric nitrogen Nitrogen fixing activity in the
field depends both on their N2-fixing potential and on their tolerance to existing
environmental stresses (Galiana et al 2002) Symbiotic N2 fixation in leguminous plants
can mainly be considered an excellent source of protein supply for human and animal
consumption They range from extensive pasture legumes to intensive grain legumes and
are estimated to contribution up to 40 of their nitrogen to the soil (Simpson 1987)
The traits in the monocropping system in the selected crop extensively exploit the
acquisition of limiting resources in the environment and continuously focused on the
availably of similar resources for the successful crop production (White et al 2013 ab)
whereas in intercropping with different crops cycling of resources can be optimized to
the complementarity or facilitation traits (Costanzo and Barberi 2014) to overcome
resource limitations during the growing season (Hill 1996 George et al 2014)
For the long term sustainable agriculture and food production in resource limiting
areas with lower input Intercropping systems have the potential to increase the
productivity With efficient mechanization cultural practices and optimized nutrient
management rapid improvements are also possible through this system In future
perspective intercrops with higher resource use efficiency through plant breeding and
genetics is likely to be the most effective option for sustainable agriculture and
development
Increase of world population and demand of additional food production
The demand and production gap of food fodder fuel wood and livestock products is
increasing day by day due to global population which will increase from about 7 billion
(FAO 2014) to 9 billion by 2050 (Haub 2013) The increasing urbanization further
intensifies the problem which will increase from 54 to 66 expected in 2050 (UN
2014) Majority of this rise in urbanization will occur in developing countries around the
5
globe The major problem is to meet the challenge of increasing food demand for this ever
growing population up to 70 more food crops to feed the additional 23 billion population
worldwide by 2050 (FAO 2010 2011) Hence there is great need to increase the re-
vegetation for fuel wood and fodder production (Thomson 1987) An increase in
production could be envisaged through increasing the yield of already productive land or
through more extensive use of unproductive land The high concentration of salts in soil
or water does not let the conventional crops grow and give feasible economic return
Hence it is necessary to search for unconventional crops for foods fodder and fuel which
could give profitable yield under saline conditions (Ahmad and Ismail 1993) Reclamation
of this land through chemical and engineering treatments is very expensive The most
appropriate use of saline wasteland is the production of high yielding salt tolerance fuel
wood timber and forage species (Qureshi et al 1993) Therefore the most attractive
option is to screen a range of species and identify those which have potential of being
commercially valuable for the degraded environments (Ismail et al 1993)
Pakistan is in semi-arid region and the 6th most populated county of the world
Population drastically increased in Pakistan which was 80 million in 1980 and annual
increase in population is about 4 million (UNDES 2011) This is continuously
overburdened and it is estimated that in 2025 it will reach to 250 million and 335 million
in 2050 which decrease the available water per capita to less than 600 m3 resulting 32
shortfall of water requirements causing an alarming condition particularly for Pakistan
Furthermore this shortfall in 2050 leading to severe food shortage upto 70 million tones
which indicates the further development and serious measures for the new resources
(ADB 2002) Subsequent severe food and fodder crises along with all the resource
limitations with continuous increase in urbanization from the current 35 to 52 in 2025
will further intensity the agriculture production and demand
Shortage of good quality irrigation water
On earth surface the major resources of available fresh water is deposited in the form of
ponds lakes rivers ice sheets and caps streams and glaciers whereas underground water
as underground streams and aquifers With the drastic increase in population the water
consumption rise as the twice of the speed of population growth The scarcity of water is
widespread to many countries of different regions Majority of population in developing
countries suffering from seasonal or year round water shortage which will increase with
6
expected climatic changes Currently almost 50 countries around the globe are facing
moderate to severe shortage of water
Due to the greenhouse effect it is estimated that since the start of 20th century 14
degF temperature is already risen which will likely rise at least another 2degF and over the next
100 years it is estimated about more than 11degF due to the consequences of biogenic gases
(El-Sharkawy 2014) This is mainly due to the product of human activities including
industrial malpractices excess fossil fuel consumption deforestation poor land use and
cultural practices
Rising in atmospheric CO2 concentration which probably reached 700 μmol (CO2)
molminus1 resulting severe climatic changes It will accelerate the melting of ice and glacier
resulting the rising rainfall and storms in tropics and high latitude consequently 06 to 1
meter rise in sea level on the expense of costal lowlands across the continents After this
initial high flows the decrease in inflow was very terrifying Due to these climatic changes
humans suffering from socioeconomic changes including degradation of lands with lower
agricultural output and degradation of natural resources will further enhanced the poverty
and hunger resulting dislocation and human migrations (Randalls 2010)
In the mean while scarcity of good quality water is increasing day by day with the
demands of water for domestic agricultural and industrial utilization which will further
increase up to 10 of the total available resources as estimated by 2025 which needs
serious water managements (Bhutta 1999) It is very challenging for the modern
agriculture to ensure the increasing demand of more arable and overburdened population
with the limiting resources including the unavailability of good quality water and
deterioration of even previously productive land (Du et al 2015)
In Pakistan Indus River basin is the back bone of agriculture and socioeconomic
development which contributes 65 of the total river flows and 90 for the food
production with a share of 25 to the GDP It is estimated that about 30-40 of its surface
storage capacity will reduce by 2025 due to siltation of reservoirs and climatic changes It
will impose serious threat to irrigated agriculture in near future consequently with
decreases in groundwater resources resulting shortage of fresh water and 15-20
reduction in grain yield in Pakistan (World Bank 2006)
7
Spread of saline soil and reduction in agricultural yield
Along with scarcity of water soil salinity is one of the major environmental stresses which
severely threaten the agriculture The damages of salinity is widespread around the world
which is so far effected the more than 800 million hectare (more than 6) of land
worldwide including 397 million ha by salinity associated with 434 million ha by sodicity
(FAO 2010) The out of total 230 million hactares of irrigated land more than 45 million
hactares (20) is so far effected by salinity which is about the 15 of total cultivated land
(Munns and Tester 2008)
In Pakistan out of 2036 million hectares of cultivated land more than 6 million
hectares is affected by salinity and water logging of various degrees (Qureshi et al 2004)
About 16 million hectares of tropical arid plains which have been put under crop
cultivation depend extensively on canal irrigation network This area (about 60) is now
seriously affected by water logging and salinity (Qureshi et al 2004) The rise of subsoil
water levels accompanied by its subsequent decline due to irrigation combined with
insufficient drainage has led to salinization of valuable agricultural land in arid zones all
over the world (Ahmad and Abdullah 1982) The dominated cation in salt-affected soil is
Na+ followed by Ca2+ and Mg2+ while the anions Cl and SO4 are almost equal in
occurrence (Qureshi et al 1993) Salt content varies in different regions of the salt-
affected areas but at certain sites could reach up to an ECe of 90-102 dSm-1 (Ahmad and
Ismail 1993)
Salinity is a chief anxiety to meet the ever growing demands of food crops Salinity
adversely affects the plant growth and productivity Plants differentially respond to salt
stress and categories into four classes Salt sensitive moderately salt sensitive moderately
salt tolerant and highly salt tolerant plants on the basis of their tolerance limits Whereas
mainly plants are divided into halophytes (salt tolerant) and glycophytes (salt sensitive) on
the basis of adaptive evolution (Flowers 2004 Munns and Tester 2008) Unfortunately
majority of cultivated crops are not able to withstand in higher salinity regimes and
eventually die under higher saline conditions which proposed serious attentions to manage
the dissemination of salinity (James et al 2011 Rozema and Flowers 2008)
Excessive accumulation of salts in rhizosphere initially reduced the water
absorption capacity of roots leading to hyperosmotic stress followed by specific ion
8
toxicity (Munns 2008 Rahnama et al 2010) Plants initially manage the overloaded salt
by various excluding and avoidance mechanisms depending on their tolerance levels The
management of salt inside the cytosol is depends on the compartmentalization capacity of
plants followed by osmotic adjustments and efficient antioxidant defense mechanisms
Whereas higher salt beyond the tolerance impose injurious effects on various
physiological mechanisms These are including disruption of membrane integrity
increased membrane injuries nutrient ion imbalances osmotic disturbance
overproduction of reactive oxygen species (ROS) compromised photosynthesis and
respiration due to stomatal closure and damages of enzymatic machinery (Munns and
Tester 2008) In specific ion toxicity Na+ and Cl- are the chief contributors in
physiological disorders Excessive Na+ in rhizosphere antagonize the uptake of K+
resulting lower growth and productivity (James et al 2011) Salt load in the cytosol trigger
the overproduction of ROS including H2O2 OH- super oxides and singlet oxygen They
are involved in sever oxidative damages to various vital cellular components including
DNA RNA lipids and proteins (Apel and Hirt 2004 Ahmad and Umar 2011)
Strategies to cope up the salinity problem
The development and cultivation of highly salt tolerant crop varieties for salt affected areas
is the major necessity to meet the future demands of food production whereas the majority
of available food crops are glycophytes Therefore it is an emergent need of crop
improvement methods which are more efficient cost effective and grow on limiting
resource The use of poor quality water for irrigation is also very important under the
proposed shortage of fresh water in near future For the development of salt tolerant
varieties more understanding of stress mechanisms are required at whole plant molecular
and cellular levels
The variability in stress tolerance of salt sensitive genotypes (glycophytes) and
highly salt tolerant plants (halophytes) showed genetic basis of salt tolerance It indicate
that salt tolerance is a multigenic trait which involves variety of gene expressions and
related mechanisms Salt stress induces both the qualitative and quantitative changes in
gene expression (Manchanda and Garg 2008) These multigenetic expressions play a key
role in upregulation of various proteins and metabolites responsible for the management
of anti-stress mechanisms (Bhatnagar-Mathur et al 2008) Plant breeding and transgenic
strategies are intensively used for decades to improve the crop performance under salinity
9
and aridity conditions Few stress tolerant varieties are so far released for commercial
production whereas in natural condition where plant exposed to variety of climatic
conditions the overall performance of plant have changed as compared to controlled in
invitro conditions (Schubert et al 2009 and Dodd and Perez-Alfocea 2012) The success
stories about transgenic approaches for crop improvement under stressful environments
are still very scanty because of the insufficient understanding about the sophisticated
mechanisms of stress tolerance (Joseph and Jini 2010) It indicates that there is less
correlation between the assessment of stress tolerance in invitro and invivo conditions
Although there have been some achievement in this connection in some model plants
including rice tobacco and Arabidopsis (Grover et al 2003) which proposed the
possibilities of success in other crops in future Variety of technicalities and associated
financial challenges are still associated with this strategy
In conventional cultivation practices continuous irrigation with poor quality water
can enhanced the salinization due to evapotranspiration leading to increased saline andor
sodic soils This problem can be cope up by intercropping system in which high salt
tolerant or salt accumulator plants are intercropped with salt sensitive crops which can
accumulate salt thus can reduce the risk of salt increment in soil Additionally better
cultivation practices including the micro-jet or drip irrigation and partial root zone drying
technique is also very fruitful to optimize the water requirements and avoid the risks
associated with conventional flooding irrigation system
In dry land agriculture plantation of deep rooted perennials during off season or
annuals can reduced the risk of salinization They continuously grown and utilize excess
amount of water create a balance between water utilization and rail fall Thus prevent the
chance of salt accumulation on soil surface due to increased water table and
evapotranspiration (Manchanda and Garg 2008) The efficient irrigation and
intercropping strategy is seemed quite attractive cost effective and very beneficial in less
mechanized poor marginal areas It can ameliorate the injurious effects of salinity and
increased production per unit area thus ensure the sustainable agriculture in semi-arid or
marginal lands (Venkateswarlu and Shanker 2009)
A number of plant species are available that are highly compatible with saline
sodic and marginal lands The cultivation of these species with proposed intercropping
system is economically feasible to grow in marginal soil Some plants including Carissa
10
carandus Ziziphus mauritiana and Cajanus cajan was selected to revealed their potential
for intercropping under saline marginal lands These are important plants which can
established well at tropical and subtropical arid zone under high temperatures Hence their
range of salt tolerance and suitability for cultivation at waste saline land or with saline
water irrigation is being undertaken for commercial exploitation
Objective of present investigation
The plan of present investigation has been worked out to look into possibility of increasing
production of an unconventional salt tolerant fruit tree (Z mauritiana) by intercropping
with a legume ( C cajan) which apart from increasing fertility of soil could be able to
provide fodder for grazing animals from salt effected waste land Possibility of making
use of saline water for irrigation has also been considered for growing leguminous plant
(C cajan) and salt tolerant unconventional fruit tree (Crissa carandas) under saline
condition
11
LAYOUT OF THESIS
Chapter 1 Monoculture of Cajanus cajan (Vern Arhar) and Ziziphus mauritiana
(Varn Ber) under different range of salinities created by irrigation of
various sea salt concentrations
A Experiments on Cajanus cajan
Following experiments were performed under A
Experiment No 1 Effect of Pre-soaked seeds of C cajan in distilled water for
germination in water of different sea salt concentrations
Experiment No 2 Effect of Pre-soaked seeds of C cajan in various dilutions of sea salt
for germination in water of respective sea salt concentrations
Experiment No 3 Seedling establishment experiment of C cajan on soil irrigated with
sea salt of different concentrations
Experiment No 4 Growth and development of C cajan in Lysimeter (Drum pot culture)
being irrigated with water of different sea salt concentrations
Experiment No 5 Range of salt tolerance of nitrogen fixing symbiotic bacteria
associated with root of C cajan
B Experiments on Ziziphus mauritiana
Experiment No 6 Growth and development of Z mauritiana in large size clay pot being
irrigated with water of two different sea salt concentrations
Discussion (Chapter 1)
Chapter 2 Intercropping of Ziziphus mauritiana with Cajanus cajan
Experiment No 7 Physiological investigations on Growth of Ziziphus mauritiana and
Cajanus cajan intercropped in drum pot (Lysimeter) culture being
irrigated with water of sea salt concentration at two irrigation
intervals
Experiment No 8 Investigations of intercropping Ziziphus mauritiana with Cajanus
cajan on marginal land under field conditions
12
Discussion (Chapter 2)
Chapter 3 Investigations on rang of salt tolerance in Carissa carandas (varn
karonda) for determining possibility of growing at waste saline land
Experiment No 9 Investigation on the effect of higher range of salinities on growth of
Carissa carandas (varn karonda) created by irrigation of different
dilutions of sea salt
Discussion (Chapter 3)
13
1 Chapter 1
Monoculture of Cajanus cajan (Vern Arhar) and Ziziphus mauritiana
(Varn Ber) under different range of salinity created by irrigation of
various sea salt concentrations
11 Introduction
Scarcity of good quality water enforced the growers to irrigate the crops with
lowmoderately saline water at marginal lands which ultimately enhance soil salinity due
to high evapo-transpiration (Azeem and Ahmad 2011) To overcome this situation people
are now focusing on less-conventional plants which can grow on resource limited areas
and can produce edible biomass for human and animal consumption
Ziziphus mauritiana (varn grafted ber) is salt and drought tolerant plant which can
grow on marginal and degraded land (Morton 1987) It has wide spread crown and a short
bole fast growing tree with average bearing life of 25 years The ripe fruit (drupe) is juicy
hard or soft sweet-tasting pulp has high sugar content vitamins A amp C carotene
phosphorus and calcium (Nyanga et al 2013 2008 Pareek 2013) The leaves contain 6
digestible crude protein and an excellent source of ascorbic acid and carotenoids The
leaves are used as forage for cattlesheepgoats and also palatable for human consumption
(Sharma et al 1982 Bal and Mann 1978 Agrawal et al 2013) The timber is very hard
can be worked to make boats charcoal and poles for house building Roots bark leaves
wood seeds and fruits are reputed to have medicinal properties The tree also used as a
source of tannins dyes silk (via silkworm fodder) shellac and nectar (Dahiru et al 2006
Chrovatia et al 1993 Gupta 1993)
Some atmospherics nitrogen fixing bacterial associated deep rooted drought
tolerent leguminious plants like Cajanus cajan can fix up to 200 Kg nitrogen ha-1 year-1
due to symbiotic association of Rhizobium with its deep penetrating roots (Bhattacharyya
et al 1995) Total cultivated area of Pigeon pea is about 622 million hectare and global
annual crop production is around 474 million tonnes whereas total seed production of
this crop is about 015 million tonnes (FAOSTAT 2013) Its seeds are an excellent source
of good quality protein (up to 24) and foliage is used as animal fodder with high
nutritional value (Pandey et al 2014) Besides being used as food and fodder this plant
14
also have therapeutic value and it is used against diabetes fever dysentery hepatitis and
measles (Grover et al 2002) It also use traditionally as a laxative and was identified as
an anti-malarial remedy beside other medicinal species (Ajaiyeoba et al 2013 Qasim et
al 2010 2011 2014)
Following experiments were conducted to evaluate the seed germination seedling
establishment and growth of C cajan as well as grafted sapling of Z mauritiana under
various salinity regimes Investigations were also undertaken to find-out of their
intercropping has any beneficial effect on growth at marginal saline land saline
environment
15
12 Experiment No 1
Effect of Pre-soaked seeds of Cajanus cajan in distilled water for
germination in water of different sea salt concentrations
121 Materials and methods
1211 Seed collection
Seeds of C cajan were purchased from local seed market Mirpurkhas Sindh and were
tested to determine the effect of salinity on germination at the biosaline laboratory Botany
department Karachi University Karachi The best lot of healthy seeds having 100
germination was selected for further experiments
1212 Experimental Design
Seeds of C cajan were surface sterilized with 01 sodium hypochlorite solution for 2-3
minutes washed in running tap water then soaked in sterilized distilled water for one hour
(Saeed et al 2014) Sterilized glass petri plates (9cm) lined with filter paper were moist
with 10 ml of distilled water at different saline water of different sea salt concentrations
and their germination percentage was observed Their electrical conductivities on these
sea salt dilutions are mentioned in Table 11 Three replicates were used for each treatment
Ten seed were placed in each petri plate which were kept in temperature controlled
incubator (EYELA LTI-1000 Japan) at 28 plusmn 1ordmC in dark Experiment was continued for 7
days Data were recorded on daily bases Analyses of varience by using repeated measures
and the significant differences between treatment means were examined by least
significant difference (Zar 2010) All statistical analysis was performed using SPSS for
windows version 14 and graphs were plotted using Sigma plot 2000
Germination percentage of C cajan was recorded every 24 hours per seedling
evaluation procedure up to 07 days The final percent germination related with salinity in
accordance with Maas and Hoffman (1977) The percent germination was calculated using
the following formula (Cokkizgin and Cokkizgin 2010)
16
Germination index for C cajan was recorded according to AOSA (1990) by using
following formula
Where Gt is the number of germinated seed on day t and Dt is the total number of
days (1 - 7)
Coefficient of germination velocity of C cajan was calculated described by Maguire
(1962)
Where G represents the number of germinated seeds counted per day till the end of
experiment
Mean germination time of C cajan was calculated by Ellis and Roberts (1981) by
using following formula
Where lsquonrsquo is the number of germinated seeds in day d whereas Σn is the total
germinated seeds during experimental period
Germination rate was of C cajan determined according to following formula
(Shipley and Parent 1991)
Where numbers of germinated seeds were recorded from 1 to 7
17
122 Observations and Results
Cajanus cajan (imbibed in distilled water) grown at different salinity regimes showed 50
reduction at 16 salt concentration corresponding ECiw 168 dSm-1 (Table 1 2 Appendix
I)
Rate of germination was inversely correlated with sea salt concentration It was
significantly (p lt 0001) decreased from first day to final (day 7) of observation Higher
germination rate was recorded in control and at lower concentrations of sea salt in early
days of seed incubation with contrast to higher concentrations of sea salt which was
reduced with increasing day of incubation (Table 13 Appendix I)
A significant decrease (p lt 0001) in coefficient of germination velocity was
observed with increasing salinity (Table 14 Appendix I)
A significantly increase (p lt 0001) in mean germination time of seeds was observed
with increasing sea salt concentrations However the difference was insignificant at lower
salinities (Table 14 Appendix I)
A significant decrease (p lt 0001) in mean germination index was observed with
increasing salt concentrations except lower salinities More reduction was observed
byhond 16 and onward sea salt concentration (Table 14 Appendix I)
18
Table 11 Electrical conductivities of different sea salt solutions used in germination of C cajan
Sea salt () ECiw (dSm-1)
Non saline control 06
01 09
02 16
03 35
04 42
05 58
06 62
07 79
08 88
09 99
10 101
11 112
12 128
13 131
14 145
15 159
16 168
ECiw is the electrical conductivity of irrigation water measured in deci semen per meter
19
Table 12 Effect of irrigation water of different sea salt solutions on germination percentage (GP) per day
of C cajan seeds pre-soaked in non-saline water prior to germination with duration of time under
various salinity regimes
Sea Salt
(ECiw= dSm-1)
GP
1st day
GP
2nd day
GP
3rd day
GP
4th day
GP
5th day
GP
6th day
GP
7th day
Control 8333plusmn667 90plusmn00 9333plusmn333 9333plusmn333 9333plusmn333 9333plusmn333 9333plusmn333
09 8667plusmn333 9333plusmn333 9667plusmn333 9667plusmn333 100plusmn00 100plusmn00 100plusmn00
16 7667plusmn667 80plusmn10 8333plusmn882 8333plusmn882 8333plusmn882 8333plusmn882 8667plusmn667
35 6667plusmn333 9333plusmn333 9333plusmn333 9333plusmn333 9333plusmn333 9333plusmn333 9333plusmn333
42 70plusmn00 8667plusmn333 90 plusmn00 90 plusmn00 90 plusmn00 90 plusmn00 90 plusmn00
58 6333plusmn667 7333plusmn333 8333plusmn333 90 plusmn00 90 plusmn00 90 plusmn00 90 plusmn00
62 5667plusmn667 80plusmn577 90 plusmn00 90plusmn00 90 plusmn00 90 plusmn00 90plusmn00
79 5333plusmn333 70plusmn00 8333plusmn333 8333plusmn333 8333plusmn333 8333plusmn333 8333plusmn333
88 4000plusmn00 6667plusmn667 8333plusmn333 8333plusmn333 8333plusmn333 8333plusmn333 8333plusmn333
99 2667plusmn333 60 plusmn00 90 plusmn00 90plusmn00 90 plusmn00 90 plusmn00 90 plusmn00
101 2333plusmn333 70plusmn577 7333plusmn333 7333plusmn333 7333plusmn333 7333plusmn333 7333plusmn333
112 70plusmn577 7667plusmn333 80plusmn00 8333plusmn333 8333plusmn333 8333plusmn333 8333plusmn333
128 6667plusmn333 6667plusmn333 6667plusmn333 6667plusmn333 6667plusmn333 6667plusmn333 6667plusmn333
131 3333plusmn882 50plusmn00 5333plusmn333 5333plusmn333 5333plusmn333 5333plusmn333 5667plusmn333
145 3333plusmn667 40 plusmn00 50 plusmn577 50plusmn577 50 plusmn577 5333plusmn333 5333plusmn333
156 3667plusmn667 40plusmn577 4667plusmn882 4667plusmn882 50plusmn577 50plusmn577 5333plusmn667
168 1667plusmn882 3333plusmn333 3333plusmn333 3333plusmn333 3667plusmn333 3667plusmn333 4333plusmn333
LSD 005 Salinity 18496
Time (days) 13322
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and LSD among the treatments is recorded at p lt 005
20
Table 13 Effect of irrigation water of different sea salt solutions on germination rate (GR) per day
of seeds C cajan pre-soaked in non-saline water prior to germination with duration of
time under various salinity regimes
Sea Salt
(ECiw= dSm-1)
GR
1st day
GR
2nd day
GR
3rd day
GR
4th day
GR
5th day
GR
6th day
GR
7th day
Control 833plusmn067 450plusmn00 311plusmn011 233plusmn008 187plusmn007 156plusmn006 133plusmn005
09 867plusmn033 467plusmn017 322plusmn011 242plusmn008 200plusmn00 167plusmn00 143plusmn00
16 767plusmn067 400plusmn050 278plusmn029 208plusmn022 167plusmn018 139plusmn015 124plusmn010
35 667plusmn033 467plusmn017 311plusmn011 233plusmn008 187plusmn007 156plusmn006 133plusmn005
42 700plusmn00 433plusmn017 300plusmn00 975plusmn750 180plusmn00 150plusmn00 129plusmn00
58 633plusmn067 367plusmn017 278plusmn011 225plusmn00 180plusmn00 150plusmn00 129plusmn00
62 567plusmn067 400plusmn029 300plusmn00 225plusmn00 180plusmn00 150plusmn00 129plusmn00
79 533plusmn033 350plusmn00 278plusmn011 208plusmn008 167plusmn007 139plusmn006 119plusmn005
88 400plusmn00 333plusmn033 278plusmn011 208plusmn008 167plusmn007 139plusmn006 119plusmn005
99 267plusmn033 300plusmn00 300plusmn00 225plusmn00 180plusmn00 150plusmn00 129plusmn00
101 233plusmn033 350plusmn029 244plusmn011 183plusmn008 147plusmn007 122plusmn006 105plusmn005
112 700plusmn058 383plusmn017 267plusmn00 208plusmn008 167plusmn007 139plusmn006 119plusmn005
128 667plusmn033 333plusmn017 222plusmn011 167plusmn008 133plusmn007 111plusmn006 095plusmn005
131 333plusmn088 250plusmn00 178plusmn011 133plusmn008 107plusmn007 089plusmn006 081plusmn005
145 333plusmn067 200plusmn00 167plusmn019 125plusmn014 100plusmn012 089plusmn006 076plusmn005
156 367plusmn067 200plusmn029 156plusmn029 117plusmn022 100plusmn012 083plusmn010 076plusmn010
168 167plusmn088 167plusmn017 111plusmn011 083plusmn008 073plusmn007 061plusmn006 062plusmn005
LSD 005 Salinity 0481
Time (days) 0378
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and LSD among the treatments is recorded at p lt 005
21
Table 14 Effect of irrigation water of different sea salt solutions on mean germination rate (GR)
coefficient of germination velocity (GV) mean germination time (GT) mean
germination index (GI) and final germination (FG) of C cajan seeds pre-soaked in non-
saline water prior to germination under various salinity regimes
Sea Salt
(ECiw= dSm-1) GR GV GT GI FG
Control 2624plusmn100 369plusmn005 027plusmn00 2624plusmn100 9667plusmn333
09 2743plusmn063 365plusmn009 027plusmn001 2743plusmn063 100plusmn00
16 2398plusmn218 423plusmn036 024plusmn002 2398plusmn218 8333plusmn882
35 2467plusmn086 378plusmn005 026plusmn00 2467plusmn086 9333plusmn333
42 3169plusmn733 311plusmn058 035plusmn008 3169plusmn733 9333plusmn333
58 2264plusmn081 399plusmn015 025plusmn001 2264plusmn081 90plusmn00
62 2253plusmn073 400plusmn013 025plusmn001 2253plusmn073 9333plusmn333
79 2074plusmn081 402plusmn00 025plusmn00 2074plusmn081 8333plusmn333
88 1927plusmn043 449plusmn008 022plusmn00 1927plusmn043 90plusmn577
99 1853plusmn033 486plusmn009 021plusmn00 1853plusmn033 90plusmn00
101 1635plusmn056 470plusmn022 021plusmn001 1635plusmn056 8667plusmn882
112 2263plusmn042 369plusmn020 027plusmn001 2263plusmn042 9667plusmn333
128 1953plusmn098 341plusmn00 029plusmn00 1953plusmn098 9667plusmn333
131 1368plusmn059 440plusmn018 023plusmn001 1368plusmn059 6667plusmn333
145 1276plusmn099 446plusmn019 023plusmn001 1276plusmn099 60plusmn577
156 1289plusmn153 447plusmn030 023plusmn002 1289plusmn153 8000plusmn100
168 876plusmn104 589plusmn078 018plusmn002 876plusmn104 8667plusmn333
LSD005 5344 3312 0064 5344 1313
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and LSD among the treatments is recorded at p lt 005
22
13 Experiment No 2
Effect of Pre-soaked seeds of Cajanus cajan in various dilutions of sea
salt for germination in water of respective sea salt concentrations
131 Materials and methods
1311 Seed germination
Procedure of seed germination has been mentioned in Experiment No 1 earlier The seeds
were pre-soaked in various sea salt concentrations instead of non-saline water and
germinated in respective sea salt concentrations Their electrical conductivities mentioned
in Table 15 Data were calculated and analysed according to formulas given in Experiment
No 1
Since these pre-soaked seeds in different sea salt concentration showed 50
germination at 03 equivalent to ECiw= 42dSm-1 sea salt solution any further work
beyond ECiw= 42dSm-1was not continued
132 Observations and Results
The final percent germination related with salinity in accordance with Maas and
Hoffman (1977) linear relative threshold response model as follows
Relative Final Germination = 100-200 (Ke ndash 005)
Where threshold salt concentration was 005 and Ke is the concentration of salts
at which relative final germination may be predicted This model indicated 50
declined in final germination at 030 salt concentration corresponding to ECiw= 42
dSm-1 (Table 16 Appendix II)
Rate of germination was significantly decreased (p lt 0001) from first day to final
(day 07) of observation and it was inversely correlated with sea salt concentration High
germination rate was recorded in control and low sea salt concentrations in early days of
seed incubation compared to higher sea salt concentrations but the difference in rate was
reduced (Table 17 Appendix II)
23
A progressive decline (p lt 0001) in coefficient of germination velocity was
observed with increasing salinity and fifty percent reduction was observed at 021 sea
salt concentration (ECiw = 319 dSm-1 Figure 11 Appendix II)
Final germination percentage was decreased significantly with increasing sea salt
concentrations However the difference was insignificant at lower (ECiw = 16 dSm-1)
salinity (Figure 11 Appendix II)
Mean germination time of seeds was increased significantly (p lt 0001) with
increasing sea salt concentrations However the difference was insignificant at lowest
(ECiw = 09 dSm-1) salinity (Figure 11 Appendix II)
Mean germination index was also significantly decreased (plt0001) with
increasing salt concentrations except for ECiw = 09 dSm-1 salinity Fifty percent reduction
in mean germination index was observed at 0188 sea salt concentration (ECiw = 289
dSm-1 Figure 11 Appendix II)
24
Table 15 Electrical conductivities of different sea salt solutions used in germination of C cajan
Sea salt () ECiw (dSm-1)
0 04
005 09
01 16
015 24
02 32
025 39
03 42
ECiw is the electrical conductivity of irrigation water measured in deci semen per meter
25
Table 16 Effect of irrigation water of different sea salt solutions on germination percentage (GP) per day of C cajan seeds pre-soaked in respective sea salt concentrations
with duration of time
Sea salt
ECiw (dSm-1)
GP
1st day
GP
2nd day
GP
3rd day
GP
4th day
GP
5th day
GP
6th day
GP
7th day
Control 6667plusmn333 8667plusmn333 10000plusmn000 10000plusmn000 10000plusmn000 10000plusmn000 10000plusmn000
09 7000plusmn000 7667plusmn333 9000plusmn000 10000plusmn000 10000plusmn000 10000plusmn000 10000plusmn000
16 4667plusmn333 6000plusmn000 7333plusmn333 8000plusmn000 8667plusmn333 8667plusmn333 9000plusmn577
24 4333plusmn333 5000plusmn000 6000plusmn577 6667plusmn333 7333plusmn333 7333plusmn333 8000plusmn000
32 3000plusmn000 3333plusmn333 3667plusmn333 4333plusmn333 5000plusmn577 6000plusmn577 7000plusmn577
39 1667plusmn333 2333plusmn333 2333plusmn333 4000plusmn577 4333plusmn333 5000plusmn000 6000plusmn000
42 667plusmn333 1333plusmn333 2333plusmn333 2333plusmn333 3333plusmn333 3667plusmn333 5000plusmn000
LSD 005 Salinity 327 Time 327
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and LSD among the treatments was recorded at p lt 005
25
26
Table 17 Effect of irrigation water of different sea salt solutions on germination rate (GR) per day of Ccajan
seeds pre-soaked in respective sea salt concentrations with duration of time
Sea salt
(ECiw= dSm-1)
GR
1st day
GR
2nd day
GR
3rd day
GR
4th day
GR
5th day
GR
6th day
GR
7th day
Control 667plusmn033 433plusmn017 333plusmn000 250plusmn000 200plusmn000 167plusmn000 143plusmn000
09 700plusmn000 383plusmn017 300plusmn000 250plusmn000 200plusmn000 167plusmn000 143plusmn000
16 467plusmn033 300plusmn000 244plusmn011 200plusmn000 173plusmn007 144plusmn006 129plusmn008
24 433plusmn033 250plusmn000 200plusmn019 167plusmn008 147plusmn007 122plusmn006 114plusmn000
32 300plusmn000 167plusmn017 122plusmn011 108plusmn008 100plusmn012 100plusmn010 100plusmn008
39 167plusmn033 117plusmn017 078plusmn011 100plusmn014 087plusmn007 083plusmn000 086plusmn000
42 067plusmn033 067plusmn017 078plusmn011 058plusmn008 067plusmn007 061plusmn006 071plusmn000
LSD 005 Salinity 014
Time 014 Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and LSD among the treatments is recorded at p lt 005)
27
Sea salt (ECiw = dSm-1
)
Contr
ol
09
16
24
32
39
42
Germ
ination Index(s
eedd
ays
-1)
0
2
4
6
8
Fin
al germ
ination (
)
0
20
40
60
80
100
Coeff
icie
nt of
germ
ination v
elo
city
(seedd
ays
-1)
00
01
02
03
04
05
06
07
Sea salt (ECiw = dSm-1
)
Contr
ol
09
16
24
32
39
42G
erm
ination tim
e (
Days
)
0
1
2
3
4
LSD005 = 0086
a = 0664 b = 1572
R2 = 0905 n =21
LSD005 = 062
a = 1239
b = 9836
R2 = 0894 n=21
LSD005 = 053
a = 8560b = -2272
R2 = 0969 n=21
RGF = 100-200 (Ke -005) Ke = 030
Figure 11 Effect of irrigation water of different sea salt solutions on seed germination indices of C cajan
(Bars represent means plusmn standard error of each treatment and significance among the treatments
was recorded at p lt 005)
28
14 Experiment No 3
Seedling establishment experiment of Cajanus cajan on soil irrigated with
sea salt of different concentrations
141 Materials and methods
1411 Seedling establishment
Seedling establishment experiment was carried out in Biosaline research field Department
of Botany University of Karachi Surface sterilized seeds pre-soaked were sown in small
plastic pots filled with 15 Kg sandy loam soil provided with farm manure at 91 ratio (30
water holding capacity) Sea salt solutions of different concentrations mentioned above
were used for irrigation The electrical conductivity of soil saturated paste (ECe) was also
determined at the end of the experiment (Table 18) Data on seedlings emergence was
recorded and their height were measured after 14 days of salinity treatment EC of the soil
(ECe) was initially 054 dSm-1 Statistical analyses were done according to the procedures
given in Experiment No 1
Since germination percentage of seeds pre-soaked in non-saline water was found
better under different concentrations of sea salt the seeds sown in soil for taking for
seedling establishment were pre-soaked in distilled water
29
142 Observations and Results
1421 Seedling establishment
Seedling emergence from soil was reduced significantly (p lt 0001) with increasing salt
concentration of irrigation water Not a single seedling emerged from soil in ge ECiw= 39
dSm-1 saline water irrigation However lower salinities (ECiw= 09 16 dSm-1) showed
slight decrease in seedling emergence with respect to controls Seedling emergence related
with salinity in accordance with a quadratic model as follows
Equation for seedling emergence () = 977751+ 44344 salt ndash 22215238 (salt)2 plusmn
6578 r = 09810 F = 15358 (p lt 00001)
Fifty percent reduction in seedling emergence was noticed at 016 sea salt
concentration (ECiw = 241 dSm-1 Figure 12 Appendix III)
1422 Shoot height
Shoot height was measured after fourteen days of irrigation Shoot length was
significantly decreased (p lt 0001) with increasing salinity A lower decrease was
observed in low sea salt salinity (ECiw= 09 and 16 dSm-1) compared to controls while
higher decrease in shoot height was noticed from ECiw= 2 dSm-1sea salt concentration
Shoot height related with salinity as follows
Equation for shoot height (cm) = 9116714 ndash 3420286 salt plusmn 09221 r = 0968 F =
128893 (p lt 0001)
Fifty percent reduction in shoot height was estimated at 013 sea salt concentration
(ECiw = 210 dSm-1) (Figure 12 Appendix III)
30
Table 18 Electrical conductivities of different Sea salt concentrations and ECe of soil saturated paste at the
end of experiment (ECe = 0447 + 1204 (salt ) plusmn 02797 R = 0987 F = 72301 (p lt
000001)
Sea salt () ECiw (dSm-1) ECe (dSm-1)
0 04 05
005 09 161
01 16 278
015 24 354
02 32 433
025 39 483
03 42 552
Electrical conductivity of soil saturated paste determined after 14 days of saline water irrigation in pots
Figure 12 Effect of irrigating water of different sea salt solutions on seedling emergence (A) and shoot
length (B) of C cajan (Bars represent means plusmn standard error of each treatment where similar
letters are not significantly different at p lt 005)
e f
Sea salt (ECiw = dSm-1
)
Contr
ol
16
27
8
35
4
43
3
48
3
Shoot le
ngth
(cm
)
0
2
4
6
8
10ab
c
de
Contr
ol
16
27
8
35
4
43
3
48
3Seedlin
g e
merg
ence (
)
0
20
40
60
80
100a
bb
c
d
A B
31
15 Experiment No 4
Growth and development of Cajanus cajan in Lysimeter (Drum pot
culture) being irrigated with water of different sea salt concentrations
151 Materials and methods
1511 Drum pot culture
A modified drum pot culture (lysimeter) installed by Ahmad amp Abdullah (1982) at
Biosaline research field (Department of Botany University of Karachi) was used in
present experiment Each drum pot (60 cm diameter 90 cm depth) was filled with 200 kg
of sandy loam mixed with cow-dung manure (91) having 28 water holding capacity
They are fixed at cemented platform at slanting position with basal hole to ensure rapid
drain Over irrigation was practiced to avoid the accumulation of salt in the root zone
1511 Experimental design
Growth and development of C cajan in drum pots was carried out in six different drum
pot sets (each in triplicate) and irrigated with sea salt of following concentrations
Drum pot Sets Sea salt
()
ECiw ( dSm-1) of
irrigation water
Resultant ECe (dSm-1) after
end of experiment
Set I Non saline (C) 04 05
Set II 005 sea salt 09 16
Set III 001 sea salt 16 28
Set IV 015 sea salt 24 35
Set V 02 sea salt 28 38
Set VI 025 sea salt 34 43
Note ECiw is the electrical conductivity of irrigation water and ECe is the electrical conductivity of the saturated soil extract taken after
eighteen weeks at the end of experiment
Ten surface sterilized seeds with 01 sodium hypochlorite were sowed in each
drum pot and were thinned to three healthy and equal size seedlings after two weeks of
establishment in their respective sea salt concentration Each drum pot was irrigated with
15 liters non-saline or respective sea salt solution at weekly intervals Electrical
conductivity of soil was measured by EC meter (Jenway 4510) using saturated soil paste
32
at the end of experiment Experiment was conducted for a period of 18 weeks (July to
November 2009) during which environmental data which includes average humidity
(midnight 76 and noon 54) temperature (low 23oC and high 33oC) wind velocity (14
kmph) and rainfall (~4 cm) was recorded (Pakistan Metrological Department Karachi) is
given in Figure 13Statistics were analysed according to the procedures given in
Experiment No 1
1512 Vegetative and Reproductive growth
Shoot height was measured at every two week interval after seedling establishment Fresh
and dry weight of shoot was recorded at final harvest (18th week when pods were fully
matured) Leaf succulence (dry weight basis Abideen et al 2014) Specific shoot length
(SSL Panuccio et al 2014) and relative growth rate (RGR Moinuddin et al 2014) were
measured using following equations
Succulence (g H2O gminus1 DW) = (FW minus DW) DW
SSL = shoot length shoot dry weight
RGR (g gminus1 dayminus1) = (lnW2 - lnW1) (t2 - t1)
Whereas FW fresh weight DW dry weight W1 and W2 initial and final dry weights and
t1 and t2 initial and final time of harvest in days
Reproductive data in terms of number of flowers number of pods number of seeds
and seed weight per plants was recorded during reproductive period
1513 Analysis on some biochemical parameters
Biochemical analysis of leaves was carried out at grand period of growth Following
investigations was undertaken at different biochemical parameters
i Photosynthetic pigments
Fresh and fully expended leaves (at 2nd3rd nodal part) samples (01g) were crushed in 80
chilled acetone and were centrifuged at 3000rpm for 10 minutes Supernatant were
separated and adjusted to 5ml final volume The absorbance was recorded at 663nm and
645 nm on spectrophotometer (Janway 6305 UVVis) for chlorophyll content while 480
33
and 510 nm for carotenoids Chlorophyll ab ratio was calculated after the amount
estimated The chlorophyll and carotenoid contents were determined according to Strain
et al (1971) and Duxbury and Yentsch (1956) respectively
Chlorophyll a (microgml) = 1163 (A665) ndash 239 (A649)
Chlorophyll b (microgml) = 2011 (A649) ndash 518 (A665)
Total Chlorophylls (microgml) = 645 (A665) + 1772 (A649)
Carotenoids (microgml) = 76 (A480) ndash 263 (A510)
ii Total soluble sugars
Dry leaf samples (01g) were homogenized in 5mL of 80 ethanol and were centrifuged
at 4000 g for 10 minutes 10 mL diluted supernatant in 5mL Anthronrsquos reagent was kept
to boil in 100oC water bath for 30 minutes and were cooled in running tap water Optical
density was taken at 620nm for the determination of soluble carbohydrates according to
Fales (1951)Total soluble carbohydrates was estimated against glucose as standard and
was calculated from the equation mentioned and expressed in mgg-1 dry weight
Total carbohydrates (microgmL-1) = 228462 OD 097275 plusmn004455
iii Protein content
Fresh and fully expended leaves at 2nd3rd nodal part were taken for protein estimation
The protein contents were measured according to Bradford Assay reagent method against
Bovine Serum Albumin as standards (Bradford 1976) Dye stock was made to dissolved
50mg comassie blue in 25 ml methanol The solution is added to 50ml of 85 phosphoric
acid and diluted to 100 ml with distilled water 02g fresh leaf samples were mills in 5 ml
phosphate buffer pH7 5ml of assay reagent (diluting 1 volume of dye stock with 4 volume
distilled water) were added in 01 ml leaf extract used for enzyme assay Absorbance was
recorded at 590nm and was expressed in mgg-1 fresh weight Proteins were calculated
from the following best fit standard curve equation
Protein (microgml-1) = -329196 + 1142755 plusmn 53436
34
152 Observations and Results
1521 Vegetative and Reproductive growth
Effect of sea salt on vegetative growth including height fresh and dry weight of Cajanus
cajan is presented in (Figure 14 and 15 Appendix-VI) Comparative analysis showed
that plant growth (all three parameters) was significantly increased with time (plt 0001)
however it was linearly decreased (plt 0001) with increasing salinity (Figure 16
Appendix-VI) shows the water content succulence relative growth rate (RGR) and
specific shoot length (SSL) of Cajanus cajan Under saline conditions all parameters were
significantly reduced in comparison to control however SSL showed decline after ECe38
dSm-1 Salt induced growth reduction was more pronounced at ECe 38 and 43 dSm-1 in
which plants died before reaching the reproductive maturity after 12 and 14 weeks at sea
salt treatments respectively Therefore further analysis was carried out in plant grown up
to ECe= 35 dSm-1 sea salt concentrations
Salinity significantly reduced (plt 0001) reproductive parameters including
number of flowers pods seeds and seed weight (Figure 17 Appendix-VII) Among all
treatments highest reduction was observed in 315 dSm-1 in which number of flowers and
pods reduced up to 7187 and 70 respectively Similar trend was observed in total
number and weight of seeds which showed 80 and 8793 reduction respectively
1522 Study on some biochemical parameters
i Photosynthetic pigments
Figure 18 Appendix-VII shows the effect of salinity on pigments (chlorophyll a b ab
ratio and carotenoids) of C cajan leaves A slight increase in total chlorophyll contents
(1828) and chlorophyll ab ratio (1215) was observed at low salinity (ECe= 16 dSm-
1) however they were significantly reduced (4125 and 3630 respectively) in high salt
treatment (plt 0001) Chlorophyll a was higher than chlorophyll b in all treatments
however chlorophyll b was un-affected by salinity whereas total chlorophyll content and
ab ratio was disturbed due to change in chlorophyll a This reduction was more
pronounced at high salinity (ECe= 35 dSm-1) in which chlorophyll a total chlorophylls
and ab ratio was decreased by 505 412 and 3630 respectively Carotenoid content
was maintained at ECe= 16 dSm-1 and decreased with further increase in salinity
35
ii Total soluble sugars
Total soluble sugars in leaves of C cajan is presented in Figure 19 Appendix-VII Total
leaf sugars in C cajan were remained un-affected at 16 dSm-1 and subsequently decreased
with further increase in medium salinity Although total sugars were decreased at ECe 28
and 35 dSm-1 a significant increase (~25) of soluble sugars was observed at higher
salinities However this increment was accounted for decrease (504 ) in insoluble sugar
content at that salinity levels
iii Protein
Total protein in leaves of C cajan is presented in Figure 19 Appendix-VII An increase
in leaf protein content in C cajan was found at lower salinity regime (ECe= 16 dSm-1)
which was followed by significant reduction with further increase in salinity This decline
was 2040 at 28 which was more pronounced (5646 ) at high salinity level (ECe=
35dSm-1)
36
Months (2009)
Jun Jul Aug Sep Oct Nov Dec
Valu
es
0
10
20
30
40
50
60
70
80
90
Rainfall (cm)Low Temp (
oC)
High Temp (oC)
Humidity at noon () Wind (kmph)
Humidity at midnight ()
Figure 13 Environmental data of study area during experimental period (July-November 2009)
Time (Weeks)
2 4 6 8 10 12 14 16 18
Pla
nt heig
ht (c
m)
0
30
60
90
120
150
180
210
43 38 35 28 16 Control
Figure 14 Effect of salinity using irrigation water of different sea salt concentrations on height of C cajan
during 18 weeks treatment (Lines represent means plusmn standard error of each treatment represents
significant differences at p lt 005)
37
Sea salt (ECe= dSm
-1)
Cont 16 28 35 38 43
Sea salt (ECe= dSm
-1)
Cont 16 28 35 38 43
Fre
sh w
eig
ht (g
)
0
5
10
15
20
25
30
35Initial Final
a
b b
c c cab b
c c cC 16 28 35 38 43
Fre
sh w
eig
ht
(g)
012345 a
bb
bc ca a ab b c c
Dry weightMoisture
Figure 15 Effect of salinity using irrigation water of different sea salt concentrations on initial and final
biomass (fresh and dry) of C cajan (Bars represent means plusmn standard error of each treatment Different
letters represent significant differences at p lt 005)
Mo
istu
re (
)
0
20
40
60
80
100
Succu
lance
(
)
0
20
40
60
80
100
Sea salt (ECe= dSm
-1)
Co
nt
16
28
35
38
43
RG
R (
)
0
20
40
60
80
100
Co
nt
16
28
35
38
43
SS
L (
)
0
20
40
60
80
100
Sea salt (ECe= dSm
-1)
ab
b b
c c
a
b bc c c
a
b b
c c c
a a a ab
c
Figure 16 Percent change (to control) in moisture succulence relative growth rate (RGR) and specific
shoot length (SSL) of C cajan under increasing salinity using irrigating water of different sea
salt concentrations (Bars represent means plusmn standard error of each treatment Different letters
represent significant differences at p lt 005)
38
Sea salt (ECe= dSm-1)
Control 16 28 35
Tota
l seeds (
Pla
nt-1
)
0
20
40
60
80
100
120
140 Seed w
eig
ht (g
pla
nt -1
)
0
5
10
15
20
25
Num
ber
10
20
30
40
50
60
70 a
b
cc
a
a
b
b
b c
c
a
b
a
c c
Flowers
Pods
Seed weightTotal seeds
Figure 17 Effect of irrigating water of different sea salt solutions on reproductive growth parameters
including number of flowers pod seeds and seed weight of C cajan (Values represent means
plusmn standard error of each treatment Different letters represent significant differences at p lt
005)
39
Sea salt (ECe=dSm-1
)
Control 16 28 35
Caro
tinoid
s (
mg g
-1 F
W)
000
005
010
015
020
025
030
Chlo
rophyll
(mg g
-1 F
W)
00
02
04
06
08
ab
ratio
00
05
10
15
20
25
ab
ab
b
a
cd
b
a
c
d
a
b
c
d
a
a
ab
b
Figure 18 Effect of irrigating water of different sea salt solutions on leaf pigments including chlorophyll a
chlorophyll b total chlorophyll and carotenoids of C cajan (Bars represent means plusmn standard
error of each treatment Different letters represent significant differences at p lt 005)
40
Figure 19 Effect of irrigating water of different sea salt solutions on total proteins soluble insoluble and
total sugars in leaves of C cajan (Bars represent means plusmn standard error of each treatment
Different letters represent significant differences at p lt 005)
Sea salt (ECe= dSm
-1)
C 16 28 35
Pro
tein
(m
g g
-1 F
W)
00
01
02
03
04
05
06
Su
gar
s (m
g g
-1 F
W)
00
02
04
06
08
a ab b
a a
b b
a ab b
a
b
ab
c
SoluableInsoluable
41
16 Experiment No 5
Range of salt tolerance of nitrogen fixing symbiotic bacteria associated
with root of Cajanus cajan
161 Materials and methods
1611 Isolation Identification and purification of bacteria
Nodules of C cajan grow in large clay pots and irrigated with running tap water at
biosaline agriculture research field were collected from the lateral roots (about 15 cm soil
depth) Nodules were surface sterilized with sodium hypochloride (2) for 5 min and
vigorously washed with sterilized distilled water Each nodule was crushed with sterilized
rod in 5 ml distilled water The bacterial suspension was streaked on yeast extract mannitol
agar (YEM) (K2HPO4 05 g MgSO 4 025g Na Cl 01 g Manitol 10g Yeast Extract 1g
Agar 20 g in 1000 ml of Distilled water) with the help of sterilized wire lope Colonies
were identified by studying different phenotypic characters as Rhizobium fredii
(Cappuccino and Sherman 1992 Sawada et al 2003) Pure culture of Rhizobium species
was stored at -20oC temperature
1612 Preparation of bacterial cell suspension
Bacteria were multiplied by growing in YEM broth for 48 hrs on shaking incubator (140
rpm) at 37oC in dark The culture in broth was centrifuged at 4000 rpm for 10 min to
obtained bacterial cell pellet Pellet was washed and centrifuged twice with sterilized
distilled water Pellet then re-suspended in sterilized distilled water before use
1613 Study of salt tolerance of Rhizobium isolated from root nodules of
C cajan
Assessment for salinity tolerance of Rhizobium species was assessed on YEM agar
Salinity levels of 0 05 10 15 20 25 and 30 having electrical conductivity 06 90
188 242 306 366 and 423 dSm-1 respectively were maintained with NaCl Bacterial
cell suspension of 01 ml (5times 103 colony forming unitsml) was poured in each sterilized
Petri dish 10 ml of molten YEM agar was poured immediately and shake well before
solidification of agar Petri plates were incubated at 37deg C in dark Colonies were observed
and counted in colony counter after 48 h and photographed (Dubey et al 2012 Singh and
42
Lal 2015) There were three replicates of each treatment and data were transformed to
log10 before analysis
162 Observations and Results
Colonies of Rhizobium on YEM agar at different salinity levels is presented in Figure 110
and 111 Appendix-VIII A significant decrease (plt0001) in rhizobial colonies was
observed with increasing salinity However the difference between non saline control and
90 dSm-1 and as that of 242 dSm-1 and 302 dSm-1 salt (NaCl) concentration showed
nonsignificant difference in rizobial colonies Whereas drastic decreased was observed on
further salinity levels Rhizobial colonies were not found at 423 dSm-1salt concentration
NaCl (ECw= dSm
-1)
06 9 188 242 306 366 423
Rh
izo
bia
l co
lonie
s (l
og
10)
0
1
2
3
4 a a
b
c c
d
e
Figure 110 Growth of nitrogen fixing bacteria associated with root of C cajan under different NaCl
concentrations (Bars represent means plusmn standard error of each treatment among the treatments
is recorded at p lt 005)
43
Figure 111 Photographs showing growth of Rhizobium isolated from the nodules of C cajan invitro on
YEM agar supplemented with different concentrations of NaCl (ECw)
188
423 90
Control
366
306 242
44
17 Experiment No 6
Growth and development of Ziziphus mauritiana in large size clay pot
being irrigated with water of two different sea salt concentrations
171 Materials and methods
1711 Experimental design
The grafted plants obtained from the local nursery of Mirpurkhas Sindh were transported
to the Biosaline Agriculture Research field Department of Botany University of Karachi
and were transplanted carefully in large earthen pots containing 20 Kg sandy loam soil
mixed with cow dung manure at 91 ratio having about 5 liters of water holding capacity
with a basal hole for drainage of excess salts to avoid accumulation in the rhizosphere
Over irrigation with about 15 liters of non-saline saline water was kept weekly in summer
and biweekly in winter to avoid accumulation of salts in rhizosphere Plants were irrigated
to start with non-saline tap water for about two weeks for establishment All the older
leaves were fallen and new leaves were developed during establishment period Following
irrigation schedule of non-saline (control) and saline water was selected in view of Z
mauritiana being moderately salt tolerant plant which includes both low and as well as
higher concentrations of the salt in irrigation
Sea salt () ECiw (dSm-1)
of irrigation water
Average resultant ECe (dSm-1) of soil
with some fluctuation often over
irrigation
Non saline (Control) 06 12
04 63 72
06 101 111
ECiw = Electrical conductivity of irrigation water ECe = Electrical conductivity of saturated soil
Healthy and well established plants were selected of nearly equal height and
divided into three sets each contain three replicates (total nine pots) Salinity was provided
through irrigation water of different sea salt concentrations All pots except non-saline
control were initially irrigated with 01 sea salt solution and then sea salt concentration
45
in irrigation medium was increased gradually upto the required salinity level The salinity
level of soil was monitored by taken the electrical conductivity of saturated soil paste the
end of experiment The electrical conductivity of soil (ECe) maintained at the level of 12
72 and 111 dSm-1 respectively as described by Mass and Hoffman (1977)
1712 Vegetative and reproductive growth
Vegetative growth in terms of shoot height fresh and dry weight of shoot and number of
branches were noted at destructive harvesting at initial (establishment) 60 and 120 days
of growth For dry weight shoots were dried in oven at 70˚C for three days Shoot
succulence specific shoot length (SSL) moisture percentage and relative growth rate
(RGR) was calculated at final harvest by using formulas given in Experiment No 4
Whereas number of flowers in reproductive data were recorded at onset of reproductive
period
As regard of fruit formation the duration of experiment was not sufficient for fruit
setting and furthermore the amount of sol in pots was not sufficient for healthy growth of
this plant Secondly flowering and fruiting is reported to be poor at the time of 1st initiation
of reproductive period (Azam-Ali 2006) Furthermore statistical significance of flower
and fruit count also become far less due to their excess dropping at early stage Hence it
was decided to proceed with study of fruit formation in forthcoming field trials of their
intercropping culture
1713 Analysis on some biochemical parameters
Biochemical analyses were performed at the grand period (at the time of flower initiation)
in fully expended fresh leaves Chlorophyll contents soluble sugar contents and soluble
proteins were analyzed Leaves samples taken from 3rd 4th node below the apex according
to the procedures given in Experiment No 4
46
172 Observations and Results
1721 Vegetative and Reproductive growth
Effect of sea salt on vegetative growth of Z mauritiana including height fresh and dry
weight is presented in (Figure 112 Appendix-IX) Comparative analysis showed that
plant growth (all three parameters) was significantly increased with time (plt 0001)
however number of branches was decreased (plt 0001) with increasing salinity
Figure 113 shows the moisture content succulence relative growth rate (RGR)
and specific shoot length (SSL) of Z mauritiana A non-significant difference in shoot
succulence SSL and moisture content was observed with time salinity and interaction of
both factors However RGR showed decline Salt induced growth reduction was more
pronounced at higher salinities
In Z mauritiana plants number of flowers showed significant decrease (plt0001)
with increasing salinity treatment Flower initiation seems non-significant at early growth
(60 days) period in controls and salinity treatments However drastic decrease was
observed with increasing salinity in 120 days of observation (Figure 114 Appendix-IX)
1722 Study on some biochemical parameters
i Photosynthetic pigments
The effect of Z mauritiana leaves pigments (chlorophyll a b ab ratio) on salinity shower
a slight difference in chlorophyll lsquoarsquo over control However chlorophyll lsquobrsquo contents
showed increase over control in both salinity treatments due to which the total chlorophylls
were also enhanced compared to controls Chlorophyll ab ratio was significantly
(plt0001) decreased in both salinities as compared to control (Figure 115 Appendix-IX)
ii Sugars and protein
In Z mauritiana plant soluble sugars were significantly decreased (plt0001) over controls
whereas proteins showed little decrease under salinity treatments compared to controls
(Figure 116 Appendix-IX)
47
Control 72 111
Fre
sh w
eig
ht (g
)
0
150
300
450
600
750
900
Sea salt (ECe= dSm
-1)
Control 72 111
Dry
weig
ht (g
)
0
150
300
450
600
750
900
Num
ber
of bra
nches
3
6
9
12
15
18
Heig
ht (c
m)
20
40
60
80
100
120
140
160
Initial 60 days 120 days
AcBb
Ba
AcBb Ba
AcBb Ba
Ac
BbBa
Figure 112 Effect of salinity using irrigation water of different sea salt concentrations on height number of
branches fresh weight and dry weight of shoot of Zmauritiana after 60 and 120 days of
treatment (Bars represent means plusmn standard error of each treatment Different letters represent
significant differences at p lt 005)
48
120 days 60 days InitialS
uccula
nce (
g g
-1 D
W)
00
03
06
09
12
Sea salt (ECe= dSm
-1)
SS
L (
cm
g-1
)
00
01
02
03
04
05
Control 72 111
Mois
ture
(
)
0
10
20
30
40
50
60
Control 72 111
RG
R (
mg g
-1 d
ay
-1)
0
5
10
15
20
a a aa a a a a a a
a aa a a a a a
a a aa a a a a a a a
b
b b
c
Figure 113 Effect of salinity using irrigation water of different sea salt concentrations on succulence
specific shoot length (SSL) moisture and relative growth rate (RGR) of Z maritiana (Bars
represent means plusmn standard error of each treatment Different letters represent significant
differences at p lt 005)
49
Sea salt (ECe= dSm
-1)
Control 72 111
Num
ber
of flow
ers
0
20
40
60
80
100
120
140 60 days120 days
Ac
BbBa
Figure 114 Effect of salinity using irrigation water of different sea salt concentrations on number of flowers
of Z mauritiana (Bars represent means plusmn standard error of each treatment Different letters
represent significant differences at p lt 005)
Sea salt (ECe= dSm
-1)
Control 72 111
Ch
loro
ph
yll
(mg g
-1)
00
03
06
09
12
15
18
bba
bba
bb
a
chl b chl a ab
ab
ra
tio
00
05
10
15
20
Figure 115 Effect of salinity using irrigation water of different sea salt concentrations on leaf pigments
including chlorophyll a chlorophyll b total chlorophyll and chlorophyll ab ratio of Z mauritiana (Values
represent means plusmn standard error of each treatment Different letters represent significant differences at p lt
005)
50
Figure 116 Effect of salinity using irrigation water of different sea salt concentrations on total sugars and
protein in leaves of Z mauritiana (Bars represent means plusmn standard error of each treatment
Different letters represent significant differences at p lt 005)
Sea salt (ECe= dSm
-1)
C 04 06
Pro
tein
s (m
g g
-1)
0
10
20
30
40
50
60
70
80
Solu
ble
sugar
s (m
g g
-1)
0
3
6
9
12
15
18a
a
bb
b b
Control 72 111
51
18 Discussion
Seed germination is the protrusion of radicle from the seed which is adversely affected by
salinity stress (Kaymakanova 2009) Salinity imposes the osmotic stress by accumulation
of Na+ and Cl- which decrease soil water potential that ultimately inhibits the imbibition
process (Othman 2005) Effect of seed germination against salinity is reported in linear
threshold response model of Maas and Hoffman (1977) The germination of a salt tolerant
desert legume Indigofera oblongifolia and a desert graminoid Pennisetum divisum are
also reported to behave to salinity in similar manner (Khan and Ahmad 1998 2007) Many
workers used chemical (organic inorganic) salt temperature biological and soil matrix
priming techniques to enhance seed germination percentage and especially germination
rate in saline medium (Ashraf et al 2008 Ashraf and Foolad 2005)Encouraging results
in most of the species of glycophytes and hydrophytes were found by presoaking in pure
water prior to germinating under saline condition Our study supports this finding and
seeds soaked in distilled water prior to germination performed better than those which
were presoaked in sea salt solutions Salinity adversely affects at all germination
parameters (germination percentage germination rate coefficient of germination velocity
and germination index) directly proportional with increasing salinity (Tayyab et al 2015)
With increase in time a delayed germination at higher salinity was found Higher sea salt
(168 dSm-1 for pure water presoaking and 35 dSm-1 for presoaking in respective
salinities) showed 50 or more reduction in all germination indices as compared to control
(Table 13-16 Figure 11)Our results are parallel with the finding of other workers such
as Kafi and Goldani (2001) who found the same trend in chickpea at higher salinities Pujol
et al (2000) reported that increased salinity inhibit the seed germination as well as delays
germination initiation in various halophyte species as well Similar response was also
found in some other crops such as pepper (Khan et al 2009) sunflower (Vashisth and
Nagarjan 2010) and eggplant (Saeed et al 2014) Salt tolerance within species may vary
at germination and other growth phases (Khan and Ahmad 1998)
According to our results C cajan appeared to be a salt sensitive in initial growth
phase specially when presoaked in saline medium (Figure 12) however at later growth
stages it proved relatively salt tolerant Salt stress delays or either seize the metabolic
activities during seed germination in salt sensitive and even in salt tolerant plants (Khan
and Ahmad 1998 Ali et al 2013b) Salinity also imposes the oxidative stress due to
52
overproduction of reactive oxygen species which may alter metabolic activities during
germination growth and developmental stages (Zhu 2001 Munns 2005
Lauchli and Grattan 2007)
In our study seeds of pigeon pea were unable to emerge beyond ECe39 dSm-1 sea
salt concentration Height of seedling was significantly affected by increasing salinity
(Figure 12) Similar results are also reported in Indian mustered (B juncea Almansouri
et al 2001) some Brassica species (Sharma et al 2013) and tomato cultivars (Jamil et
al 2005) Growth retardation with increasing salinity may be due to reduced
photosynthetic efficiency and inhibition of enzymatic and non-enzymatic proteins
(Tavakkoli et al 2011) Furthermore salt stress also limit the DNA and RNA synthesis
leads to reduced cell division and elongation during germination growth and
developmental stage
Khan and Sahito (2014) found variation in salt tolerance within species subspecies
and provenance level Furthermore the salt tolerance of a species may also vary at
germination and growth phases (Khan and Ahmad 1998 Ali et al 2013a) Srivastava et
al (2006) suggested that the genetic variability influences salinity tolerance eg wild
species like Cajanus platycarpus C scaraboides and C sericea showed better salt
tolerance than C cajan In this connection Wardill et al (2006) has also reported genetic
diversity in Acacia nilotica C cajan in this study appeared to be a salt sensitive at
germination in compression with later stages of growth Seedling establishment at saline
solution faces adverse effects when emerging radicle and plumule come in contact with
salt effected soil particle or saline water hence percent seedling establishment remains
less than germination percentage observed at petri plate Ashraf (1994) found that salinity
tolerance of different varieties of C cajan do not much differ at germination and early
growth stages whereas at adult growth stage show improvement in salt tolerance
Soil salinity is a major limiting factor for plant growth and yield production
particularly in leguminous plants (Guasch-Vidal et al 2013 Tayyab et al 2016) In
present study Plant height RGR fresh and dry biomass were severely reduced with
increasing salinity and plant was unable to grow after ECe= 43 dSm-1(Figure 14-16)
This growth inhibition of C cajan may be accounted for individual and synergistic effect
of water stress nutrient imbalances and specific ions toxicities (Hasegawa et al 2000
Silvera et al 2001) Salt induced ion imbalance results in lower osmotic potential which
53
alter physiological biochemical and other metabolic processes leading to overall growth
reduction (Del-Amor et al 2001) Excessive amount of salt in cytoplasm challenge the
compartmentalization capacity of vacuole and disrupts cell division cell elongation and
other cellular processes (Munns 2005 Munns et al 2006) Our results are parallel with
some other studies in which significant growth inhibition of peas chickpea and faba beans
have been reported against salt stress (El-Sheikh and Wood 1990 Delgado et al 1994)
Singla and Garg (2005) also observed a similar salt sensitive growth response in Cicer
arietinum In our study the fresh and dry biomass of C cajan also showed inhibitory
behavior to salt stress (Figure 15) Hernandez et al (1999) also found significant reduction
in dry biomass of pea plant and common bean (40 and 84 respectively) when grown
in saline medium Mehmood et al (2008) also found similar results in Susbania sasban
Salinity also has imposed deleterious effects on reproductive growth of C cajan
Production of flowers and pods are significantly decreased in response to salinity (Figure
19) Increase in flower shedding leads to decreased number of pods indicating salt
sensitivity of plant at reproductive phase which was more pronounced at high salinity
(Vadez et al 2007) Furthermore seed production and weight of seed per plant was also
linearly decreased Salt induced reduction of reproductive growth has also been found in
mung bean in which 60 and 12 less pods and seeds were produced respectively at 06
saline solution (Qados 2010) Similar results are reported in faba bean (De-Pascale and
Barbieri 1997) tomato (Scholberg and Locascio 1999) maiz sunflower (Katerji et al
1996) and watermelon (Colla et al 2006) Salinity reduces reproductive growth by
inhibiting growth of flowers pollen grains and embryo which leads to inappropriate ovule
fertilization and less number of seeds and fruits (Torabi et al 2013)
On biochemical parameters total chlorophyll and chlorophyll ab ratio has
increased in low salinity in contrast the adverse effect at higher salinity could be due to
high Na+ dependent breakdown of these pigments (Li et al 2010 Yang et al 2011)
Chlorophyll a is usually more prone to Na+ concentration and decrease in total chlorophyll
is mainly attributed to the destruction of chlorophyll a (Fang et al 1998 Eckardt 2009)
This diminution could be due to the destruction of enzymes responsible for green pigments
synthesis (Strogonov et al 1973) and increased chlorophyllase activity (Sudhakar et al
1997) Thus insipid of leaf was a visible indicator of salt induced chlorophyll damage
which was well correlated with quantified values as reported in other legume species
54
(Soussi et al 1998 Al-Khanjari et al 2002) In this study chlorophyll a was found to be
more sensitive than chlorophyll b (Figure 18) Garg (2004) also found similar reduction
in chlorophyll pigments (a b and total chlorophyll) in chickpea cultivars under salinity
stress
At low salinity (16 dSm-1) total carotenoids remained unaffected along with
increased total chlorophyll (Figure 18) which may suggest a role of carotenoids in
protection of photosynthetic machinery (Sharma et al 2012) Similar response was found
in Cajanus indicus and Sesamum indicum (Rao and Rao 1981) however
Sivasankaramoorthy (2013) and Ramanjulu et al (1993) reported slight increase of leaf
carotenoids in Zea maiz and mulberry when exposed to NaCl High salinity was destructive
for both leaf pigments (chlorophyll and carotenoids) of C cajan which was in accordance
with Reddy and Vora (1985) who found similar decrease in some other salt sensitive crops
Salinity led to the conversion of beta-carotene to Zeaxanthin which protect plants against
photo-inhibition (Sharma and Hall 1991)
In present study with increasing salinity water content and succulence of C cajan
were significantly reduced which indicated loss of turgor (Figure 16) Our data suggest
that decreased succulence by lowering water content may help in lowering leaf osmotic
potential when exposed to increasing salinity which is in agreement with findings of Parida
and Das (2005) and Abideen et al (2014) In addition increased production and
accumulation of organic substances is also necessary to sustain osmotic pressure which
provide osmotic gradient to absorb water from saline medium (Hasegawa et al 2000
Cha-um et al 2004) Compatible solutes including carbohydrates amino acids proteins
and ammonium compounds play important roles in water relations and cell stabilization
(Ashraf and Harris 2004) In this study C cajan produce more soluble sugars (Figure 18)
which is considered as a typical plant response under saline conditions (Murakeozy et al
2003) Sugars serve as organic osmotica and their available concentration is related to the
degree of salt stress and plantrsquos tolerance (Ashraf 1994 Murakeozy et al 2003) Sugars
are involved in osmoprotection osmoregulations carbon storage and radical scavenging
activities (Pervaiz and Satyawati 2008) On the other hand insoluble and total sugars were
reduced in higher salinity which is also supported by Parida et al (2002) and Gadallah
(1999) who found similar results in Bruguiera parviflora and Vicia faba
55
Total soluble proteins of C cajan were reduced due to deleterious effects of salinity
(Figure 18) The accumulation of Na+ in cytosol disrupts the protein and nucleic acid
synthesis (Bewley and Black 1985) Gill and Sharma (1993) and Muthukumarasamy and
Panneerselvam (1997) also reported decreased protein content with increasing salinity in
Cajanus cajan seedlings Similar results were found when tomato (Azeem and Ahmad
2011) Zingiber officinale (Ahmad et al 2009) and Sorghum bicolor (Ali et al 2013a)
were grown under variable salt concentrations (Figure 19)
Nodule formation of Rhizobium in Legume depends upon interaction between soil
chemistry of salt composition and osmotic regimes of salt and water (Velagaleti et al
1990 Zahran 1991 Zahran and Sprent 1986) Salinity reduces plant growth directly
through ion and osmotic effects and indirectly by inhibiting Legume-Rhizobium
association (El-Shinnawi et al 1989) Studies demonstrated a more sensitive response of
rhizobial N-fixing mechanism than growth of plant to abiotic stresses including salinity
(Mhadhbi et al 2004) In nodules metabolic disturbance initiated with the production of
ROS leading to tissues injury and loss of nodule function (Becana et al 2000) In general
it slow down the nitrogenase activity and decrease nodule protein and leghemoglobin
content which decreased becteroid development (Mhadhbi et al 2008) In consequence
plant suffer directly by salt induced ion toxicity low water uptake and photosynthetic
damage and indirectly through weak association of symbionts due to high energy demand
for nodule function (Pimratch et al 2008) In our study the isolated rhizobial strain from
nodules of C cajan was found to be tolerant to salinity even up to 2 (ECw= 306 dSm-1)
NaCl (Figure 110 and 111) Some of the other species of Rhizobium such as Brady
Rhizobium have been shown salt tolerant even at higher concentration than their
leguminous hosts (Zahran 1999) For instance a number of rhizobial species can tolerate
up to 06 NaCl (Yelton et al 1983) while Rhizobium meliloti can tolerate 175 to
40 NaCl and R leguminosarum can tolerate can tolerate upto 2 NaCl (Abdel-Wahab
and Zahran 1979 Sauvage et al 1983 Breedveld et al 1991 Helemish 1991
Mohammad et al 1991 Embalomatis et al 1994 Mhadhbi et al 2011) Rhizobia
isolated from soybean and chickpea can tolerate up to 2 NaCl with a difference of fast-
growing and slow growing strains (El-Sheikh and Wood 1990 Ghittoni and Bueno 1996)
Similarly Rhizobium from Vigna unguiculata can survive up to up to 55 NaCl
(Mpepereki et al 1997)
56
Present study shows an increase in vegetative growth in terms of plant height and
fresh and dry weight of shoot with increasing time under non-saline and saline conditions
but the increase was rapid at early period of growth (Figure 112) All the vegetative
growth parameters determined were reduced under salinity stress compared to non-saline
control Measurements of shoot moisture succulence specific shoot length and RGR
(Figure 113) indicate that Z mauritiana adjusted in its water relation over coming
negative water and osmotic potential with increase in salinity levels increased There is
evidence that water and osmotic potentials of salt tolerant plants become more negative in
higher salinities (Khan et al 2000) These altered water relations and other physiological
mechanisms help plants to get by adverse abiotic stress like that of drought and salinity
(Harb et al 2010) However the results clearly showed that salinity had an inhibitory
effect on growth but the decline was less at early sixty days and more during later 60-120
days in compression to controls Growth inhibition in shoot has been observed in number
of plants including different species of halophytes (Keiffer and Ungar 1997) chickpea
(Cicer arietinum Kaya et al 2008) and different wheat cultivars (Triticum aestivum
Moud and Maghsoudo 2008)
Salinity also caused reduction in the number of branches and the number of flowers
in Z mauritiana however reduction in the number of flowers is non-significant in ECe=
72 dSm-1 salinity treatment in comparison with non-saline control (Figure 114) The main
reason for this reduction could be attributed to suppression of growth under salinity stress
during the early developmental stages (shooting stage) of the plants These results are
similar to those reported by Ahmad et al (1991) and Khan et al (1998) As affirmed by
Munns and Tester (2008) suppression of plant growth under saline conditions may either
be due to osmotic effect of saline solution which decreases the availability of water for
plants or the ionic effect due to the toxicity of sodium chloride High salt concentration in
rooting medium also reduced the uptake of soil nutrients a phenomenon which affected
the plant growth thus resulting in less number of branches per plant Various abiotic
stresses such as temperature drought salinity light and heavy metals altered plant
metabolism which ultimately affects plant growth and productivity Amongst these
salinity stress is a major problem in arid and semiarid regions of the world (Kumar et al
2010) Salinity has an adverse effect on several plant processes including seed
germination seedling establishment flowering and fruit formation and ripening (Sairam
and Tyagi 2004) Salinity stress also imposes additional energy requirements on plant
57
cells and less carbon is available for growth and flower primordial initiation (Cheesman
1988) The lesser decrease in number of flowers at lower salinity (ECe= 72 dSm-1) has
been attributed to the fact that the cells of apex are un-vacuolated and the incoming salts
accumulated in the cytoplasm Munns (2002) further suggested a well-controlled phloem
transport of toxic ions from these cells prevented any change in reproductive development
Our findings showed an increase in total chlorophyll contents particularly
chlorophyll b contents were enhanced more than chlorophyll a contents under salinity
stress (Figure 115) In general the total chlorophyll contents decreased under high salinity
stress and this may be due to accumulation of toxic ions in photosynthetic tissues and
functional disorder of stomatal opening and closing (Khan et al 2009) The increase in
total chlorophylls appearing at salinity levels is considered as an important indicator of
salinity tolerance in plants (Katsuhara et al 1990 Demiroglu et al 2001) In another
study on Z mauritiana (cv Banara sikarka) the chlorophyll contents has shown decrease
with increasing salinity and sodicity but the seedlings treated with low salinity (ECe of 5
mmhoscm-1) shows slightly higher values than controls (Pandey et al 1991) Our study
also suggests that increase in total chlorophylls adapted this plant increased its tolerance
to salt stress
Slight decrease in protein has been shown under salinity treatments compared to
controls (Figure 16) Proteins play diverse roles in plants including involvement in
metabolic pathways as enzyme catalyst source of reserve energy and regulation of osmotic
potential under salt stress (Pessarakli and Huber 1991 Mansour 2000) Salts may
accumulate in cell cytoplasm and alter their viscosity depending on the response of plant
to salinity stress (Hasegawa et al 2000 Paravaiz and Satyawati 2008) The decrease in
protein contents under increasing salinity has also been documented in several plants
including Lentil lines (Ashraf and Waheed 1993) sorghum (Ali et al 2013a) and sugar
beet (Jamil et al 2014)
Soluble sugars were also decreased with increasing salinity treatments in our study
(Figure 16) Decrease in soluble sugars due to salinity has also been reported in Viciafaba
(Gadallah 1999) some rice genotypes (Alamgir and Ali 1999) Bruguiera parviflora
(Parida et al 2002) and Lentil (Sidari et al 2008) However the accumulation of soluble
sugars under salinity stress is considered as strategy to tolerate stress condition due to their
58
involvement in osmoprotection osmotic adjustment and carbon storage (Parida et al
2002 Parvaiz and Satyawati 2008)
From these experiments it is evident that C cajan is a salt sensitive plant at every
level of its life cycle starting from germination to growth phases Germination capacity
and salt tolerance ability of this species can be enhanced by water presoaking treatment
Growth reduction with increasing salinity could be attributed to physiological and
biochemical disturbances which ultimately affect vegetative and plant reproductive
growth Its roots are well associated with nitrogen fixing rhizobia and these
microorganisms were salt tolerant in in-vitro cultures Another fruit baring species of
marginal lands Z mauritiana showed growth improvement in lower salinity and its growth
was not much affected in high saline mediums owing to its controlled biochemical
responses
59
2 Chapter 2
Intercropping of Z mauritiana with C cajan
21 Introduction
Increasing soil salinity fresh water scarcity and agricultural malpractice creating shortage
of food crops for human and animal consumption (Bhandari et al 2014) and making
prices high Traditional agriculture which has been practiced since centuries using multi
species at a time in a given space could be a potential solution to narrow down the growing
edges of this supply demand scenario Plant species with innate resilience to abiotic
stresses like salinity and drought could be considered suitable to serve this purpose
especially for arid regions where marginal lands can be utilized to generate economy
Presence of such type of local systems in the region highlight their potential advantage in
crop production income generation as well as sustainability (Somashekar et al 2015)
For instance reports are available on successful intercropping of multipurpose trees
shrubs and grasses like millets pulses and some oil seed and fodder crops Green part of
these species usually mixed and used for cattle feed especially during the lean period The
utilization of the inter-row spaces of fruit trees like Ziziphus mauritiana for growing edible
legumes can generate further income by similar input (Dayal et al 2015) As an option
to this Cajanus cajan could serve as better intercropped as it provides protein rich food
nutritious fodder and wood for fuel which helped to uplift the socio-economic condition
of poor farmers Integrated agricultural practices improve the productivity of each crop by
keeping cost of production under sustainable limits (Arabhanvi and Pujar 2015)
Keeping in mind the above mentioned scenario in present study the possibility to
increase production of a non-conventional salt tolerant fruit tree (Z mauritiana) by
intercropping with a leguminous plant (C cajan) was investigated to produce edible fruits
and fodder simultaneously from salt effected waste lands
60
22 Experiment No 7
Physiological investigations on Growth of Ziziphus mauritiana and
Cajanus cajan intercropped in drum pot (Lysimeter) culture being
irrigated with water of sea salt concentration at two irrigation intervals
221 Materials and Methods
2211 Growth and Development
Experiment was designed to investigate the effect of intercropping on growth and
development of Z mauritiana (a fruit tree) and C cajan (a leguminous fodder) in drum
pot culture irrigated with water of 03 sea salt concentrations at two irrigation intervals
2212 Drum pot culture
Drum pot culture as recommended by Boyko (1966) and modified by Ahmed and
Abdullah (1982) was used for the present investigation as described in chapter 1
2213 Experimental Design
Three sets of 18 plastic drums (lysimeter) were used in this experiment One plant of Z
mauritiana were grown in each lysimeter Three replicates were kept for each treatment
comprising of 06 drums in each set which was further divided in two sub-sets First sub-
set was irrigated at every 4th and second subset at every 8th day
Set ldquoArdquo =Ziziphus mauritiana (Sole crop)
Set ldquoBrdquo = Cajanus cajan (Sole crop)
Set ldquoCrdquo = Ziziphus mauritiana + Cajanus cajan (intercropped)
The effect of salinity on sole crops of C cajan and Z mauritiana on salinity created
by various dilutions of sea salt has been investigated in chapter 1 Concentration of 03
sea salt considered equal level to its 50 reduction has been selected in present
experiment In addition irrigation was given in sub-sets in two intervals to investigate to
have some idea of its water conservation
61
2214 Irrigation Intervals
Sub-set 1 Irrigation was given every 4th day
Sub-set 2 Irrigation was given every 8th day
In set lsquoArsquo and lsquoCrsquo six month old saplings of Ziziphus mauritiana (vern grafted
ber) plants of nearly equal height and good health were transplanted in drum pots Plants
were irrigated to start with non-saline tape water for about two weeks for purpose of
establishment All the older leaves fell down and new leaves immerged during
establishment period
In set lsquoBrsquo and lsquoCrsquo Ten healthy sterilized seeds of Cajanus cajan imbibed in distill
water were sown in each drum pot and irrigated to start with tap water and after
establishment of seedlings only six seedlings of equal size with eqal distance (about one
feet) between C cajan and that of Z mauritiana were kept for further study The sowing
time of cajanus cajan seeds in both sets (B and C) was the same In drum pot lsquoCrsquo it was
sown when sapling of Z mauritiana have undergone two weeks of their establishment
period in tap water
When seedlings of C cajan reached at two leaves stage irrigation in all the sets
(ABC ) was started with gradual increase sea salt concentration till it reached to the
salinity level of treatment (03) in which they were kept up to end of experiment Each
drum was irrigated with enough water sea salt solution which retains 15 liters in soil at
field capacity Rest of water drain down with leaching of accumulated salt in root
rhizosphere
Vegetative growth of Z mauritiana plant was noted monthly in terms of height
volume of canopy while in C cajan height and number of branches was noted Shoot
length root length number of leaves fresh and dry weight of leaf stem and root leaf
weight ratio root weight ratio stem weight ratio specific shoot and root length plant
moisture leaves succulence and relative growth rate was observed and calculated at final
harvest in both the plant species growing individually (sole) or as intercropping at two
irrigation intervals
Investigations were undertaken on nitrate content relative water content and
electrolyte leakage at grand period of growth Amount of photosynthetic pigments soluble
62
carbohydrates proline content soluble phenols and Protein contents were also investigated
in fully expended leaves
Activity of catalase (CAT) ascorbate peroxidase (APX) guaiacol peroxidase
(GPX) superoxide dismutase (SOD) (Anti-oxidant enzymes) and nitrate reductase (NR)
activity was also observed in on both the Z mauritiana and C cajan leaves growing as
sole and as intercropped at two different irrigation intervals
The procedures of above mentioned analysis as follows
Leaf succulence (dry weight basis) Specific shoot length (SSL) and relative
growth rate (RGR) were measured according to the equations given in chapter 1
2215 Estimation of Nitrate content
NO3 was estimated through Cataldo et al (1975) 01g fresh leaf samples were boiled in
50 mL distilled water for 10 min 01mL of sample were added to mixed in 04 mL 50
salicylic acid (wv dissolved in 96 H2SO4 ) and allowed to stand for 20 min at room
temperature 95 mL of 2N NaOH was slowly mixed at last The samples were permissible
to cool NO3 concentration was observed at 410 nm and was calculated according to the
standard curve expressed in mg g-1 fresh weight
2216 Relative Water content (RWC)
Young and fully expended leaf was excise from each plant removing dust particles
preceding to Relative water content (RWC) Fresh weights (FW) were taken to all leaf
samples and were immersed in distilled water at 4 degC for 10 hours The soaked leaf samples
were taken out and surfeit water was removed by tissue paper Weighted again these leaf
samples for turgid weight (TW) and were oven dried at 70 degC Dry weight (DW) was
recorded after 24 hrs The RWC of leaf was calculated by the following formula
RWC () = [FW ndash DW] [TW ndash DW] x 100
2217 Electrolyte leakage percentage (EL)
EL was measured according to Sullivon and Ross (1979) Young and fully expended
leaves removing dust particles were taken 20 disc of 6mm diameter were made through
63
porer and were placed in the test tube containing 10ml de-ionized water First electrical
conductivity (EC lsquoarsquo) was record after shaken the tubes These test tubes now were placed
at 45-50oC warmed water bath for 30 min and observed second Electrical conductivity (EC
lsquobrsquo) Finally tubes were placed at 100oC water bath for ten min and obtained third and final
Electrical conductivity (EC lsquocrsquo) The electrolyte leakage was calculated in percentage by
using following formula
EL () = (EC b ndash EC a) EC b x 100
2218 Photosynthetic pigments
Photosynthetic pigments including chlorophyll a chlorophyll b total chlorophyll
chlorophyll ab ratio and carotinoids were estimated according to the procedure given in
chapter 1
2219 Total soluble sugars
Dry leaf samples (01g) were milled in 5mL of 80 ethanol and were centrifuged at 4000
g for 10 minutes and were estimated according to the procedure described in chapter 1
22110 Proline content
The proline contents were determined through Bates et al (1973) Each dried leaf powder
sample (01 g) was grinded and homogenized in 5 ml of 3 (wv) sulphosalicylic acid and
were centrifuged at 5000 g for 20 minutes 2ml supernatant was boiled by adding 2 ml
glacial acetic acid and 2 ml ninhydrin reagent (prepared by dissolving 125 g ninhydrin in
30 ml of glacial acetic acid and 20 ml 6 M phosphoric acid) in caped test tube The tubs
were kept in boiling water bath (100oC) for 1 hour After cooling 4 ml of toluene was
added to each tube and vortex Two layers were appeared the chromophore layer of
toluene was removed and their absorbance was recorded at 590nm against reference blank
of pure toluene The proline concentrations in leaves were determined from a standard
curve prepared from extra pure proline of (Sigma Aldrich) and were calculated from the
equation and were expressed in mgg-1 of leaf dry weight
Proline (microgmL-1) = -074092 + 1660767 (OD) plusmn054031
64
22111 Soluble phenols
The dried leaf powder (01g) was milled in 3ml of 80 methanol and was centrifuged at
10000g for 15 min (Abideen et al 2015) Final volume (5ml) were adjusted by adding
80 methanol Soluble phenols were determined by using Singleton and Rossi (1965) ie
5 ml of Folin-Ciocalteu reagent (19 ratio in distilled water) and 4 ml of 75 Na2CO3
were added to 01 ml supernatant The absorbance was recorded at 765 nm after incubation
of 30 minutes at room temperature The soluble phenols concentration in leaf tissues was
determined from a standard curved prepared from Gallic acid
22112 Total soluble proteins
The protein contents were measured according to Bradford Assay reagent method against
Bovine Serum Albumin as standards (Bradford 1976) Procedure was followed as given
in chapter 1
22113 Enzymes Assay
Enzyme extract prepared as given below was used for study of enzymes mentioned in text
The juvenile and expended leaf excised was frozen in liquid nitrogen and were stored at -
20 degC These leaf samples (100mg) was firmed in liquid nitrogen and were mills in 3 ml
of ice chilled potassium phosphate buffer (pH = 7 01 M) with 1mM EDTA and 1 PVP
(wv) The homogenate was filtered through a four layers of cheesecloth and were
centrifuged at 21000 g using refrigeration centrifuge (Micro 17 TR Hanil Science
Industrial Co Ltd South Korea) at 4 degC for 20 min The supernatant was separated and
stored at -20 degC and used for investigation on following enzymes
i Superoxide dismutase (SOD)
SOD (EC 11511) antioxidant enzymeactivity was measured through Beauchamp and
Fridovich (1971) derived on the inhibition of nitroblue tetrazolium (NBT) reduction by
produced O2minus using riboflavin photo-reduction 50 mM of pH 78 phosphate buffer (with
01mM EDTA 13 mM methionine) 75 microM nitroblue tetrazolium (NBT) 2 microM riboflavin
and 100 microl of enzyme extract was added to 3ml reaction mixture Riboflavin was added at
the last before the reaction was initiated under fluorescent lamps for 10 min Exposed and
un-exposed to florescence lamp without enzyme extract were used to serve as calibration
65
standards Activity was measured at 560nm Unit of SOD activity was defined as the
amount of enzyme required for 50 inhibition of NBT conversion
ii Catalase (CAT)
CAT (EC 11116) antioxidant enzyme activity was precise according to Aebi (1984)
derived on H2O2 reduction at 240nm for 30 s (ε = 36 M-1 cm-1)100mM potassium
phosphate buffer (pH=7) with 30mM H2O2 and 50 microl of diluted enzyme extract (adding in
last) was added to 3ml reaction mixture The decrease in absorbance due to H2O2 reduction
was measured at 240 nm and expressed in micromol of H2O2 reduced m-1g-1 fresh weight at 25
degC
iii Ascorbate peroxidase (APX)
Nakano and Asada (1981) method was used for APX (EC 111111) antioxidant
enzymeactivity by measuring the decrease in ascorbate oxidation by H2O2 The reaction
mixture (3ml) contained potassium phosphate buffer (50mM pH=7) 01mM H2O2 050
mM Ascorbate and 100 microl of enzyme extract and were observed 290 nm for 1 min 25 degC
(extinction coefficient 28 mM-1cm-1)
iv Guaiacol peroxidase (GPX)
GPX (EC 11117) antioxidant enzymeactivity was estimated through Anderson et al
(1995) 3ml of 50 mM potassium phosphate buffer (pH 7) guaiacol 75 mM H2O2 10 mM
reaction mixture with 20 microl of enzyme extract adding at last Increase in absorbance was
observed due to the formation of tetra-guaiacol at 470 nm for 2 min (extinction coefficient
266 mM-1cm-1)
v Nitrate reductase (NR)
The NR activity in leaves was observed through Long and Oaks 1990 Fresh leaf samples
(01g) were placed in 5ml of 100mM potassium phosphate pH 75 (added to 10
Isopropanol and 25mM KNO3) Tubes were vacuumed for 10 min to remove air from the
mixture and were placed in water bath shaker at 33oC for 60 min in dark The tubes were
placed in hot water (100oC) for 5 min 15 mL from the reaction mixture were added in 05
mL 20 sulphanilamide (wv dissolve in 5N HCl) and 025 mL 008 N-1-Napthylene-
66
diamine dihydrochloride Final volume up to 60 ml was made by adding distilled water
Color developed over the next 20 min Absorbance was measured at 540 nm using
spectrophotometer
67
222 Observations and Results
Sole and intercropped Ziziphus mauritiana
2221 Vegetative growth
Growth of Z mauritiana in terms of shoot root and plant length and number of leaves in
two different cropping system (sole and intercrop with C cajan) in two different irrigation
intervals has been presented in Figure 21 Appendix-XII A significant increase (plt0001)
in plant length was observed in 8th day irrigation in both the cropping systems in Z
mauritiana At 4th day of irrigation interval a non-significant increase in length was
observed in intercropped plants compared to sole crop Similarly at 8th day of irrigation
plants attain almost same heights in both the cropping systems
A significant increase (plt001) in root length was observed in sole Z mauritiana
at 8th day of irrigation compared to other treatments Smallest root length revealed in plants
that were irrigated at 4th day under sole crop system
The shoot length was significantly increase (plt0001) in plants which were
irrigated at 8th day under intercropped system However shoot length remains unaffected
when comparing the different cropping system at both the irrigation intervals
A significant increase (plt0001) in number of leaves was observed in intercropped
Z mauritiana plants compared to plants cultivated according to sole system However
more increase was observed in 4th day irrigated intercropped plant as compared to 8th day
The difference in number of leaves in sole crop at both irrigating intervals remains same
i Fresh weight
Figure 22 Appendix-XII showed fresh and dry weight of stem root and leaf of Z
mauritiana plant in two different cropping system (sole and intercrop with C cajan) in
two different irrigation intervals A significant increase (plt0001) in fresh weights of leaf
stem and root was observed in intercropping (with C cajan) 4th and 8th day of irrigation
interval compared to individual cropping of Z mauritiana In 4th day of irrigation the
increment was more pronounced in fresh weights of root (7848) leaves (4130) and
stem (4047) respectively with comparison to the crop growing alone Similarly
intercropping in 8th day of irrigation showed better growth of leaves (28) stem (12)
68
and root (31) against sole crop Whereas decrease in leaves 33 (plt005) stem 70
(plt0001) and root 60 (plt0001) fresh weights were observed in 8th day of irrigation
compared to 4th day intercropping However the difference was non-significant between
two sole crops irrigated at 4th and 8th day interval
ii Dry weight
Intercropping with comparison to the sole crop showed significant (plt0001) increase in
dry weights of leaves root and stem of Z mauritiana at 4th and 8th day of irrigation (Figure
22 Appendix-XII) At 4th day of irrigation intercropping showed an increment in dry
weights of Leaves (4366) stem (4109) and root (754) compared to the sole crop
Similar increase was observed in leaves (plt0001) stem (plt0001) and root (plt0001)
weights after 8th day of irrigation However intercropping at 8th day irrigation showed an
increment in root (19) stem (11) whereas a slight decrease (1) in leaves dry weight
When comparing irrigation time an increase in stem dry weight at 4th day whereas decline
in leaves dry weight was observed Root dry weights were more or less similar at both
irrigation intervals
iii Leaf weight ratio (LWR) root weight ratio (RWR) stem weight
ratio (SWR)
Leaf weight ratio (LWR) root weight ratio (RWR) stem weight ratio (SWR) of Z
mauritiana plant grown in two different cropping system (sole and intercrop with C cajan)
in two different irrigation intervals has been presented in Figure 23 Appendix-XII An
increased in LWR and SWR was recorded at 8th day of irrigation compared to 4th day of
irrigation in both cropping systems whereas decrease in RWR was observed LWR and
SWR remained un-change in sole and inter crop system However RWR increased in
intercrop system compared to sole crop system
iv Specific shoot length (SSL) specific root length (SRL)
Specific shoot length (SSL) specific root length (SRL) of Z mauritiana plant grown in
two different cropping system (sole and intercrop with C cajan) in two different irrigation
intervals has been presented in Figure 23 Appendix-XII SSL was observed higher in 8th
day of irrigation compared to 4th day in both the cropping systems However the increase
69
in SSL was lesser in sole crop compared to intercropping Similarly SRL was recorded
lesser in 4th day of irrigation compared to 8th day of irrigation in both cropping systems
Intercropped plants showed decline in SRL compared to sole crop plants Greatest SRL
revealed in plants that were irrigated after 8th day and planted according to sole crop
system
v Plant moisture
The moisture content of Z mauritiana plant grown in two different cropping system (sole
and intercrop with C cajan) in two different irrigation intervals has been presented in
Figure 23 Appendix-XII The moisture content of plants was significantly decreased
(plt005) in sole crop while increased (plt005) in intercropping at 8th day of irrigation
compared to 4th day At 4th day moisture remained same in both cropping system
However significant increase in moisture contents was observed in inter-crop system
compared to sole crop system after 8th day of irrigation
vi Plant Succulence
Succulence of Z mauritiana plant grown in two different cropping system (sole and
intercrop with C cajan) in two different irrigation intervals has been presented in Figure
23 Appendix-XII Plant succulence in 8th day was significantly reduced in sole crop
whereas increased in intercropping system In 4th day irrigated plants decrease in
succulence was noticed compared to plants that were irrigated at 8th day under sole crop
system However significant increase (plt0001) was observed in intercropped plants
irrigated at 4th day compared to 8th day
vii Relative growth rate (RGR)
Relative growth rate (RGR) of Z mauritiana plant grown in two different cropping system
(sole and intercrop with C cajan) in two different irrigation intervals has been presented
in Figure 23 Appendix-XII Relative growth rate remains unchanged at both irrigation
times under sole crop system However decline in 8th day was observed compared to 4th
day of irrigation under intercrop system Greatest RGR was recorded in plants that were
irrigated at 4th day under intercrop system
70
2222 Photosynthetic pigments
Photosynthetic pigments including Chlorophyll a chlorophyll b total chlorophyll
Chlorophyll ab ratio and carotinoids of Z mauritiana plant grown in two different
cropping system (sole and intercrop with C cajan) in two different irrigation intervals has
been presented in Figure 24 Appendix-XII
i Chlorophyll contents
A significant increase (plt0001) in chlorophyll a b and total chlorophyll was observed in
plants growing as sole crop compared to intercropped system at both the irrigation
intervals Higher chlorophyll contents were also recorded in plants that were irrigated at
8th day compared to 4th day of irrigation The chlorophyll ab ratio increased in 4th day
while decline in 8th day in intercropped system compared to sole crop However overall
results showed non-significant changes
ii Carotinoids
A significant increase (p lt 0001) in leaf carotinoids was observed in sole crop compare
to intercropped system at both irrigation times in Z mauritiana Least carotene content
was estimated in plants that were irrigated at 4th day under intercrop system
2223 Electrolyte leakage percentage (EL)
Electrolyte leakage percentage (EL) of Z mauritiana plant grown in two different
cropping system (sole and intercrop with C cajan) in two different irrigation intervals has
been presented in Figure 25 Appendix-XII A non-significant result was observed in
electrolyte leakage in plant growing at varying cropping system and irrigating intervals
2224 Phenols
Total phenolic contents in leaves of Z mauritiana plant grown in two different cropping
system (sole and intercrop with C cajan) in two different irrigation intervals has been
presented in Figure II25 Appendix-XII A significant increase (plt001) in total phenolic
contents was observed in intercropped growing at both irrigation interval compared to sole
crop However the increase was more pronounced at 8th day of irrigation Maximum
phenolic contents were measured in plants irrigated at 8th day under intercropped plants
71
2225 Proline
Total proline contents in leaves of Z mauritiana plant grown in two different cropping
system (sole and intercrop with C cajan) in two different irrigation intervals has been
presented in Figure 25 Appendix-XII A significant decreased (plt0001) was observed
in Z mauritiana cultivated according to intercropped system in both irrigation intervals
Maximum decrease was observed in intercropped plants irrigated at 8th day whereas
highest phenolic contents were observed in plants irrigated at 4th day under sole crop
system
2226 Protein and sugars
Protein and sugar contents in leaves of Z mauritiana plant grown in two different cropping
system (sole and intercrop with C cajan) in two different irrigation intervals has been
presented in Figure 26 Appendix-XII A nonsignificant difference in total protein and
sugar contents in Z mauritiana plants was observed in two different (4th and 8th day)
irrigation intervals However the interaction with time and irrigation interval also showed
nonsignificant result
2227 Enzyme essays
Antioxidant enzymes like Catalase (CAT) Ascorbate peroxidase (APX) Guaiacol
peroxidase (GPX) Superoxide dismutase (SOD) and Nitrate reductase activity in leaf of
Z mauritiana plant grown in two different cropping system (sole and intercrop with C
cajan) in two different irrigation intervals has been presented in Figure 27 and 28
Appendix-XII
i Catalase (CAT)
A significant decreased (plt0001) in catalase activities was observed in Z mauritiana
leaves in intercropped system in both time interval with compare to sole crop at 4th day
irrigated plant However maximum decline was in sole plants irrigated at 8th day interval
However their interaction with time was nonsignificant
72
ii Ascorbate peroxidase (APX)
A significant increase (plt0001) in APX activity was observed in 8th day irrigation in both
sole and intercropped plants with compare to sole and intercropped at 4th day irrigation
interval More increase (plt0001) was observed in intercropped Z mauritiana at 8th day
Whereas nonsignificant decrease was observed in two different cropping system in 4th day
irrigation interval However interaction between time and the treatments shows significant
values
iii Guaiacol peroxidase (GPX)
A significant (plt0001) increase in GPX was observed in 8th day intercropped Z
mauritiana plant with compare to irrigation intervals as well as cropping system However
at 4th day both cropping system showed nonsignificant difference Whereas more decline
was observed in 8th day sole crop The ANOVA reflects significant (plt005) interaction
between time and the cropped system
iv Superoxide dismutase (SOD)
A nonsignificant increase in SOD was observed in intercropped at 8th day irrigation
interval Whereas there was nonsignificant differences in 4th day intercropped and at both
time intervals of sole crop However interaction between time interval and the two
cropping system shows nonsignificant result
v Nitrate and Nitrate reductase
A significant increase (plt0001) in nitrate content and activity of nitrate reductase was
observed in intercropped plants of both irrigation intervals Increase in activity was
observed (plt0001) in intercropped Z mauritiana at 4th day
73
Sole and intercropped Cajanus cajan
2228 Vegetative growth
Growth of C cajan in terms of shoot root and plant length and number of leaves was
observed in two different cropping system (sole and intercrop with Z mauritiana) in two
different irrigation intervals has been presented in Figure 21 Appendix-XIII XIV A
significant increase (plt001) in plant length was observed in intercropped C cajan
compared to sole crop at both irrigation interval Whereas sole crop at 8th day interval
showed better results as compare to sole of 4th day Similarly root length remains
unaffected and showed non-significant change in both cropping systems and even at two
different irrigation intervals While shoot length was significantly (Plt001) decreased in
sole crop compared to intercropped at 4th day irrigation Whereas non-significant
difference be observed in rest of cropping systems growing at different irrigation interval
A significant increase (plt001) in leaves number was observed in intercropped
plants compared to sole crop at 4th and 8th day irrigation interval However most
significant decrease (plt0001) was observed in sole crop at 4th day
i Fresh weight
Figure 22 Appendix-XIV showed fresh and dry weight of stem root and leaf of C cajan
plant in two different cropping system (sole and intercrop with C cajan) in two different
irrigation intervals A significant increase (plt001) in fresh weight of leaf was observed in
intercropping (with Z mauritiana) at 4th and 8th day of irrigation interval compared to
individual cropping of C cajan The increase in intercropped system compared to sole
crop was more pronounced at 4th day (42) of irrigation than the 8th day (1701) Plants
showed higher leaves fresh weights in 8th day of irrigation compared to 4th day Similarly
the interaction between cropping system and the irrigation interval was significant
(Plt005)
An insignificant difference was observed in stem at 4th (15) and 8th (12) days
fresh weights in both intercropping system at two different irrigation intervals The
interaction between cropping system and the irrigation interval also showed non-
significant result
74
A non-significant difference in root fresh weight was observed in two different
cropping systems (sole and intercropped) in 4th and 8th day of irrigation intervals However
fresh weight of crop at 8th day irrigation interval was significantly increase (plt0001) over
4th day irrigation interval Similar pattern was observed in 4th day irrigated sole and
intercropped C cajan
ii Dry weight
A significant increase in leaves (42) stem (24) and root (18) dry weights were
observed in 4th day irrigation under intercropped system compared to sole However in 8th
day of irrigation this increase of dry weights was not much prominent Under sole crop
system dry weights of leaves stem and root was increased markedly in 8th day compared
to 4th day However in intercrop system the difference in dry weights was insignificant
between 8th and 4th day of irrigation
iii Leaf weight ratio (LWR) root weight ratio (RWR) stem weight
ratio (SWR)
Leaf weight ratio (LWR) root weight ratio (RWR) stem weight ratio (SWR) of C cajan
grown in two different cropping system (sole and intercrop with Z mauritiana) in two
different irrigation intervals has been presented in Figure 23 Appendix-XIV A
significant increase (plt0001) in LWR was observed at 8th day of irrigation compared to
4th day intercropped Similar pattern was noticed in RWR however SWR showed
insignificant difference between 4th and 8th day of irrigation A slight increase in LWR was
noticed in intercropped plants compared to sole Whereas RWR declined in intercrop
compared to sole and SWR remains un-changed
iv Specific shoot (SSL) root length (SRL)
Specific shoot length (SSL) specific root length (SRL) of C cajan grown in two different
cropping system (sole and intercrop with Z mauritiana) in two different irrigation
intervals has been presented in Figure 23 Appendix-XIV SSL and SRL were observed
to increase in sole crop compared to intercrop at 4th day of irrigation However increase
SSL and SRL was recorded in intercropped compared to sole at 8th day of irrigation A
general decline in SSL and SRL was noticed in 8th day of irrigation compared to 4th day
75
v Plant moisture
The moisture content of C cajan plant grown in two different cropping system (sole and
intercrop with Z mauritiana) in two different irrigation intervals has been presented in
Figure 23 Appendix-XIV The moisture content of plants was decreased significantly
(plt005) at 8th day irrigation interval compared to 4th day in sole crop Whereas non-
significant increase was observe in intercrop plants at 8th day of water irrigation
vi Plant succulence
Succulence of C cajan plant grown in two different cropping system (sole and intercrop
with Z mauritiana) in two different irrigation intervals has been presented in Figure 23
Appendix-XIV A significant increase (plt001) was observed in intercropped plants of C
cajan compared to sole crop at both irrigation interval However succulence increased in
sole crop and decreased in intercrop plants at 8th day of irrigation compared to 4th day
vii Relative growth rate (RGR)
Relative growth rate (RGR) of C cajan plant grown in two different cropping system (sole
and intercrop with Z mauritiana) in two different irrigation intervals has been presented
in Figure 23 Appendix-XIV A significant increase in RGR was observed in 8th day
compared to 4th day in both the cropping systems Highest increase was observed in
intercropped at 8th day irrigation At 4th day irrigation intervals intercropped plants
showed better RGR compared to Sole crop
2229 Photosynthetic pigments
Photosynthetic pigments including Chlorophyll a chlorophyll b total chlorophyll
Chlorophyll ab ratio and carotinoids of C cajan plant grown in two different cropping
system (sole and intercrop with Z mauritiana) in two different irrigation intervals has been
presented in Figure 24 Appendix-XIV
i Chlorophyll contents
A significant increase (plt005) in Chlorophyll a b and total chlorophyll was observed in
intercrop plants at 8th day irrigation interval Whereas at 4th day irrigation interval Sole
76
plants showed better results as compare to intercrop plants Plants at 8th day significantly
increase chlorophyll a b and total chlorophyll compared to 4th day of irrigation
Interactions between cropping systems and irrigation intervals were found significant
(chlorophyll a (plt001) chlorophyll b (plt001) and total chlorophyll (plt0001)
respectively) However the ratio of chlorophyll ab showed non-significant values in
cropping irrigation interval and their interaction
ii Carotenoids
A significant increase (plt001) in carotinoids was observed in intercropped C cajan at 8th
day of irrigation Whereas non-significant increase was observed in sole crop at 4th day
irrigation interval with compare to intercrop However the irrigation intervals showed
significant (plt0001) difference Whereas interaction of cropping system with irrigation
time also showed significant correlation (plt0001)
22210 Electrolyte leakage percentage (EL)
Electrolyte leakage percentage (EL) of C cajan plant grown in two different cropping
system (sole and intercrop with Z mauritiana) in two different irrigation intervals has been
presented in Figure 25 Appendix-XIV A non-significant increase in EL percentage was
observed in sole crop compared to intercrop plants growing at 4th and 8th day of irrigation
No significant change was noticed between the irrigation times to C cajan The interaction
between cropping system (sole and intercropped) and irrigation interval (4th and 8th day)
also showed non-significant
22211 Phenols
Total phenolic contents in leaves of C cajan plant grown in two different cropping system
(sole and intercrop with Z mauritiana) in two different irrigation intervals has been
presented in Figure 25 Appendix-XIV A nonsignificant result was observed in total
phenolic contents of C cajan growing as sole and intercropped system at two different
irrigation intervals However the interaction between irrigation intervals with crop system
showed significant (p lt 005) results
77
22212 Proline
Total proline contents in leaves of C cajan plant grown in two different cropping system
(sole and intercrop with Z mauritiana) in two different irrigation intervals has been
presented in Figure 25 Appendix-XIV Proline contents in leaves of C cajan showed
nonsignificant increase at 4th day of irrigation interval in both sole and intercropped
system Whereas the interaction between irrigation intervals showed significant (Plt001)
results
22213 Protein and Sugars
Protein and sugar contents in leaves of C cajan plant grown in two different cropping
system (sole and intercrop with Z mauritiana) in two different irrigation intervals has been
presented in Figure 26 Appendix-XIV A less significant difference (plt005) was
observed in two different (4th and 8th day) irrigation intervals However there was
nonsignificant difference in two cropped system More decrease was observed at 4th day
intercropped plants Whereas nonsignificant increase in 8th day intercropped and 4th day
sole plants were observed However interaction between crop and time of irrigation
showed significant results (plt0001)
22214 Enzyme assay
Antioxidant enzymes like Catalase (CAT) Ascorbate peroxidase (APX) Guaiacol
peroxidase (GPX) Superoxide dismutase (SOD) and Nitrate reductase activity in leaf of
C Cajan plant grown in two different cropping system (sole and intercrop with Z
mauritiana) in two different irrigation intervals has been presented in Figure II27
Appendix-XIV
i Catalase (CAT)
A significant increase (plt001) in catalase activity was observed in intercropped C cajan
at 8th day of irrigation with compare to other irrigation time and cropped system Whereas
increase was observed in sole crop at 4th day irrigation interval with compare to 8th day
However the irrigation intervals and the interaction between cropping system with
irrigation interval also showed nonsignificant correlation
78
ii Ascorbate peroxidase (APX)
A non-significant increase in APX was observed in intercropped plant in 4th and 8th day
irrigation interval with compare to sole crops Sole crop at 8th day showed maximum
decline However the difference between cropping system and their interaction with
irrigation interval also showed nonsignificant results
iii Guaiacol peroxidase (GPX)
A significant increase (plt005) in GPX activity was observed in 8th day sole crop
However there was nonsignificant difference among intercropped at two time interval and
sole crop at 4th day irrigation Whereas interaction with time to irrigation interval also
showed less significant results
iv Superoxide dismutase (SOD)
A significant decrease (plt0001) in SOD activity was observed in intercropped at 8th day
irrigation interval with compare to 4th day Maximum decrease was observed in 8th day
intercropped Whereas sole crop at 8th day also showed better result to 4th day sole crop
However ANOVA showed significant correlation among crop system at two time interval
and 4th day irrigation
v Nitrate and Nitrate reductase
Nitrate content and activity of nitrate reductase was nonsignificant in both cropping
system using both irrigation intervals However nonsignificant increase was observed in
nitrate content and activity of nitrate reductase in intercropped Z mauritiana at 8th day
79
Sole IntercropSole Intercrop
No o
f le
aves
0
20
40
60
Len
gth
(cm
)
0
40
80
120
160
200
2404
th day
Cajanus cajan
a
RootShoot
ab
a
a
b
a
a
8th
day
Figure 21 Vegetative parameters of Z mauritiana and C cajan at grand period of growth under sole and
intercropping system at 4th and 8th day irrigation intervals (Bars represent means plusmn standard error
of each treatment and significance among the treatments was recorded at p lt 005)
Sole IntercropSole Intercrop
No of
leav
es
0
200
400
600
Len
gth
(cm
)
0
40
80
120
160
200
240
Ziziphus mauritiana
RootShoot
4th
day 8th
days
b b
a a
a
b
cc
80
Sole Intercrop
Dry
wei
ght
(g)
50
100
150
200
250
300
Fre
sh w
eight
(g)
100
200
300
400
500
Sole Intercrop
4th
day 8th
day
a
b
c
a
b b aa
b
b
c c
a
bc
a
c
ba
b
c
a
b
c
Leaf Stem Root
Ziziphus mauritiana
Sole Intercrop
Dry
wei
ght
(g)
2
4
6
8
10
12
Fre
ah w
eight
(g)
5
10
15
20
25
30
35
40
Sole Intercrop
4th
day 8th
day
aa
b
a
a
b
a
b
c
a
b
c
a
c
b
a a
b
a
b
c
a
b
c
Leaf Stem Root
Cajanus cajan
Figure 22 Fresh and dry weight of Z mauritiana and C cajan plants under sole and intercropping system
at 4th and 8th day irrigation intervals (Bars represent means plusmn standard error of each treatment
and significance among the treatments was recorded at p lt 005)
81
Figure 23 Leaf weight ratio (LWR) root weight ratio(RWR) shoot weight ratio(SWR)specific shoot
length (SSL) specific root length (SRL) plant moisture Succulence and relative growth rate (RGR) of
Zmauritiana and C cajan grow plants under sole and intercropping system at 4th and 8th
day irrigation
intervals (Bars represent means plusmn standard error of each treatment and significance among the treatments
was recorded at p lt 005)
Sole Intercrop
Mo
istu
re (
)
0
20
40
60
80
SS
L (
cm g
-1)
01
02
03
04
05
06
RW
R (
g g
-1 D
W)
005
010
015
020
LW
R (
g g
-1 D
W)
01
02
03
04
05
06
07
Sole Intercrop
Su
ccu
lan
ce
(g H
2O
g-1
DW
)00
05
10
15
20
25
RG
R
(g g
-1 d
ay-1
)
001
002
003
004
005
SR
L (
cm g
-1)
05
10
15
20
25
SW
R (
g g
-1 D
W)
02
04
06
08
10
Ziziphus mauritiana
a a
bb
b
a
bb
a
b
aa
a aa
b
a
bb
c
b
a
bb
b
aa a
ba
bc
4th day
8th day
82
(Figure 23 continuedhellip)
Sole Intercrop
Mo
istu
re (
)
0
20
40
60
80
SS
L (
cm g
-1)
2
4
6
8
10
12
RW
R (
g g
-1 D
W)
002
004
006
008
010
012
014
LW
R (
g g
-1 D
W)
01
02
03
04
05
06
07
08
Sole Intercrop
Su
ccu
lan
ce
(g H
2O
g-1
DW
)
00
05
10
15
20
25
RG
R
(g g
-1 d
ay-1
)
001
002
003
004
005
SR
L (
cm g
-1)
5
10
15
20
25
SW
R (
g g
-1 D
W)
02
04
06
08
10
Cajanus cajan
a aab
a aaa
a
bba
a
b b
c
a aab
a
bbb
abbb
aa
bc
8th day
4th day
83
Sole Intercrop
Car
oti
noid
s (m
g g
-1 F
W)
00
01
02
03
04
05
Ch
loro
phyll
(m
g g
-1 F
W)
00
03
06
09
12
15
Sole Intercrop
4th
day 8th
day
Ch
loro
phyll
ab
rat
io
00
05
10
15
20
25Chl ab
Ziziphus mauritiana
a a
bb
a
b
a
b
a ab
b
Chl aChl b
Figure 24 Leaf pigments of Zmauritiana and C cajan grow plants under sole and intercropping system at
4th and 8th
day irrigation intervals (Bars represent means plusmn standard error of each treatment and
significance among the treatments was recorded at p lt 005)
Sole Intercrop
Car
oti
noid
s (m
g g
-1 F
W)
00
01
02
03
04
05
Ch
loro
phyll
(m
g g
-1 F
W)
00
03
06
09
12
15
18
Sole Intercrop
4th
day 8th
day
ab r
atio
00
05
10
15ab
ab
Cajanus cajan
bb b
a
a
b
cc
bb b
a
84
Ele
ctro
lyte
lea
kag
e(
)
0
5
10
15
4th
day 8th
dayP
hen
ols
(m
g g
-1)
0
5
10
15
20
25
30
Sole Intercrop
Pro
line
( g g
-1)
0
10
20
30
40
Sole Intercrop
Ziziphus mauritiana
a a a
a
b b ba
a
b
c
d
Figure 25 Electrolyte leakage phenols and prolein of Z mauritiana and C cajan at grand period of growth
plants under sole and intercropping system at 4th and 8
th day irrigation intervals (Bars represent
means plusmn standard error of each treatment and significance among the treatments was recorded at
p lt 005)
85
(Figure 25 continuedhellip)
E
lect
roly
te l
eakag
e(
)
0
20
40
60
80
4th
day 8th
day
Phen
ols
(m
g g
-1)
0
2
4
6
8
10
12
Sole Intercrop
Pro
line
( g g
-1)
000
003
006
009
012
015
018
Sole Intercrop
Cajanus cajan
a aa
a
a a aa
aa a
a
86
Sole Intercrop
Sugar
s (m
g g
-1)
0
20
40
60
Sole Intercrop
Pro
tein
(m
g g
-1)
00
02
04
06
4th
day 8th
day
Ziziphus mauritiana
a aa a
a
a a a
Sole Intercrop
Sugar
s (m
g g
-1)
0
10
20
30
Sole Intercrop
Pro
tein
(m
g g
-1)
00
02
04
06
08
10
4th
day 8th
dayCajanus cajan
ab
a
c
a
b
cc
Figure 26 Total protein and sugars in leaves of Z mauritiana and C cajan plants under sole and
intercropping system at 4th and 8th
day irrigation intervals (Bars represent means plusmn standard
error of each treatment and significance among the treatments was recorded at p lt 005)
87
Sole Intercrop
SO
D (
Unit
s m
g-1
)
0
2
4
6
8
10
12
14
Sole Intercrop
Cat
alas
e (U
nit
s m
g-1
)
0
5
10
15
20
25
AP
X (
Unit
s m
g-1
)
0
20
40
60
80
GP
X (
Unit
s m
g-1
)
00
01
02
03
04
05
4th
day 8th
day
Ziziphus mauritiana
a
bc
c
a
b
cc
a
c
b
b
b bb
a
Figure 27 Enzymes activities in leaves of Z mauritiana and C cajan plants under sole and intercropping
system at 4th and 8th
day irrigation intervals (Bars represent means plusmn standard error of each
treatment and significance among the treatments was recorded at p lt 005)
88
(Figure 27 continuedhellip)
Sole Intercrop
SO
D (
Unit
s m
g-1
)
0
1
2
3
4
5
Sole Intercrop
Cat
alas
e (U
nit
s m
g-1
)
0
2
4
6
8
4th
day 8th
dayG
PX
(U
nit
s m
g-1
)
00
05
10
15
20
25
Cajanus cajan
aA
PX
(U
nit
s m
g-1
)
0
20
40
60
80
100
bb
b
aaa
b
a
bbb
a
c
a
b
89
Sole Intercrop
NO
3 (
mM
ol
g-1
)
00
02
04
06
08
10
12
14
8th
day
Sole Intercrop
Nit
rate
Red
uct
ase
(mM
ol
g-1
)
0
1
2
3
4
4th
day
Nitrate reductaseNO
3
Ziziphus mauritiana
a
b
c
cb
b
b
a
Sole Intercrop
NO
3 (
mM
ol
g-1
)
00
02
04
06
08
10
12
8th
day
Sole Intercrop
Nit
rate
Red
uct
ase
(mM
ol
g-1
)
0
2
4
6
8
10
12
4th
dayCajanas cajan
a
bb
b
aa
aa
Nitrate reductase NO3
Figure 28 Nitrate reductase activity and nitrate concentration in leaves of Z mauritiana and C cajan plants
under sole and intercropping system at 4th and 8th
dayirrigation intervals (Values represent means
plusmn standard error of each treatment and significance among the treatments was recorded at p lt
005)
90
23 Experiment No 8
Investigations of intercropping Ziziphus mauritiana with Cajanus cajan
on marginal land under field conditions
231 Materials and Methods
2311 Selection of plants
Ziziphus mautitiana and Cajanus cajan were selected for this study as described in chapter
1
2312 Experimental field
Field of Fiesta Water Park was selected to investigate intercropping of Z mauritiana with
Ccajan It is situated about 50 km from University of Karachi at super highway toward
HyderabadThe area of study has subtropical desert climate with average annual rain fall
is ~20 cmmost of which is received during the monsoon or summer seasonSince summer
temperature (April to October) are approx 30-35 degC and the winter months (November to
March) are ~20 degC Wind velocity is generally high all the year Topography of the area
was uneven with clay- loam soil having gravels Xerophytic plants are pre-dominantly
present in the area including Prosopis spp Acacia spp Euphorbia spp Caparus
deciduas etc
2313 Soil analysis
Before conducting experiment soil of Fiesta Water Park field was randomly sampled at
three locationsatone feet of depthusing soil augerThese soil samples were analyzed in
Biosaline Research Laboratory Department of Botany University of Karachi to
determine its physical and chemical properties
i Bulk density
Bulk density was determinedin accordance with Blake and Hartge (1986) by using the
following formula
Bulk density = Oven dried soil (g) volume of soil (cm3)
91
ii Soil porosity
Soil porosity was calculated in accordance with Brady and Weil (1996) by using the
following formula
Soil porosity = 1- (bulk density Particle density) times 100
Where particle density = 265 gcm3
iii Soil texture and particle size
Soil particle size was determined by Bouyoucos hydrometric method in accordance with
Gee and Or (1986)On the basis of clay silt and sand percentages soil texture was
determined by using soil texture triangle presented in Figure 31
iv Water holding capacity
Water holding capacity in percentages was calculatedaccording to George et al (2013)
v pH and Electrical conductivity of soil (ECe)
Soil saturated paste was made with de-ionized water and leave for 24 hours Soil solution
was extracted through Buckner funnel and suction pump (Rocker 300) pH of soil
solution was taken on Adwa AD1000 pHMV meter and ECe was taken on electrical
conductivity meter (4510 Jenway)
2314 Experimental design
Six months old grafted Ziziphus mauritiana saplings were carefully transported in field of
Fiesta Water Park
Three equal size plots of 100times10 sq ft were prepared for this experiment
Plot ldquoArdquo = Ziziphus mauritiana (Sole crop)
Plot ldquoBrdquo = Cajanus cajan (Sole crop)
Plot ldquoCrdquo = Ziziphus mauritiana + Cajanus cajan (intercropped)
In plot lsquoArsquo and lsquoCrsquo pits of two cubic feet depth were prepared in two parallel rows
at a distance of 10 feet (Yaragattikar amp Itnal 2003)so that the distance of pits within the
row and the distance of pits between the rows were same Each row bears nine pits
Eighteen healthy saplings of nearly equal height and vigor of Z mauritiana were
92
transplanted in the pits and were fertilized with cow-dong manure Plants were irrigated
with underground (pumped) water initially on alternate day for two weeks older leaves
fall down completely and new leaves appeared in this establishment period Later the
irrigation interval was kept fortnightly Electrical conductivity of irrigated water (ECiw)
was 24 plusmn 05 dSm-1
After establishment of Z mauritiana water soaked seeds of intercropping plant (C
cajan) were sown in plot lsquoCrsquo Three vertical lines (strips design) of equal distance were
made between the rows of Z mauritiana The distance between the line was one feet
Eleven C cajan were maintained in each line at a distance of one feet which constitute a
total of 33 C cajan in 3 lines There were 264 plants of C cajan arranged in strip pattern
as intercrop for eighteen Z mauritiana A sole crop of C cajan in plot lsquoBrsquo was arranged
with the same manner to serve as control Similarly plot lsquoArsquo was served as control of Z
mauritianaThe experiment was observed up to reproductive yield of each plant
Field diagram Theoritical model of intercropping system used in this study showing sole crop in Plot lsquoArsquo
(Z Mauritiana) and Plot lsquoBrsquo (C cajan) while Plot lsquoCrsquo represents intercropping of both
species at marginal land
Six Z mauritiana plants were randomly selected from their two rows of block lsquoCrsquo
which were facing two rows of C cajan on either sides Similarly ten plants of C cajan
facing Z mauritiana were randomly selected for further study At the same manner six Z
mauritiana from block lsquoArsquo and ten C cajan from block lsquoBrsquo grown as sole crop were
selected as control for further study
93
2315 Vegetative and reproductive growth
Vegetative growth of Z mauritiana plant was noted in terms of height volume of canopy
while height and number of branches in Ccajan bimonthly after establishment Fresh and
dry weightsof leaves stem and root were observed at final harvest in both plant species
growing as sole or intercropping
Reproductive growth of Z mauritiana such as number length and diameter fruit
weight per ten plant and average fruit yield was measured at termination of the experiment
Whereas reproductive growth in C cajan was monitored in terms of number of pods
number of seeds weight of pods and weight of seed
2316 Analyses on some biochemical parameters
Following biochemical analysis was conducted in Fully expended leavesof Z mauritiana
and C cajan growing as sole and as intercropped at grand period of growth Additionally
fruits of Z mauritiana were also analyzed for their protein soluble and insoluble sugars
and total phenolic contents
i Photosynthetic pigments
Photosynthetic pigments including chlorophyll a chlorophyll b and total chlorophyll were
estimated in leaves of Z mauritiana and C cajan according to procedure described in
chapter 1
ii Protein in leaves
Protein contents were estimated in leaves of Z mauritiana and C cajan according to
procedure described in chapter 1
iii Total soluble sugars in leaves
Total soluble sugars were estimated in leaves of Z mauritiana and C cajanaccording to
procedure described in chapter 1
94
iv Phenolic contents in leaves
Phenolic content were estimated in leaves of Z mauritiana and C cajan according to
procedure described in chapter 1
2317 Fruit analysis
i Protein in fruit
Protein content in fruit of Z mauritiana was estimated according to procedure described
in chapter 1
ii Total soluble sugars in fruits
Total soluble sugars in ripe fruits of Z mauritiana were estimated according to procedure
described in chapter 1
iii Phenolic contents in fruits
Phenolic contents in fruits of Z mauritiana were estimated according to procedure
described in chapter 1
2318 Nitrogen estimation
Nitrogen was also estimated in root zone soil as well as in fully expended leaves of Z
mauritiana and C cajan plants
Total nitrogen in leaves and soil was estimated through AOAC method 95504
(2005) One g of dried powdered sample in round bottle flask was digested in presence of
20 mL H2SO4 15 mL K2SO4 and 07g CuSO4 at 400oC heating mental After digestion 80
ml distilled water was added in digest Then distillation was done at 100oC by adding 100
mL of 45 NaOH (drop wise) in digested solution Steam was collected in 35 mL of 01M
HCl in a flask Three samples of 10 mL each steam collected solution were taken and 2-3
drops of methyl orange was added as indicator Titration was made with 01M NaOH
Changeappearance of color indicates the completion of reactionPercent nitrogen was
calculated through following equation
N = (mL of acid times molarity) ndash (mL of base times molarity) times 14007
95
2319 Land equivalent ratio and Land equivalent coefficient
The LER defined the total land area needed for sole crop system to give yield obtained
mixed crop It is mainly used to evaluate the performance of intercropping (Willey 1979)
Land equivalent ratio (LER) of two crops was estimated according to (Willey 1979) by
using formula
Whereas partial LER of Z mauritiana calculated according to
Similarly Partial LER of Ccajan were calculated as
Land equivalent coefficient (LEC) an assess of dealings the effectiveness of relationship
of two crops (Alhassan et al 2012) was calculated by using (Adetiloye et al 1983)
equation as
Yield was calculated in gram fresh weight LER and LEC of height and total chlorophyll
were also calculated by using above formula by substituting their values with yield (fruits
of Z mauritiana and seeds of C cajan) to height fruits and chlorophyll respectively
23110 Statistical analysis
Data were analyzed by using (ANOVA) and the significant differences between treatment
means wereexamined by least significant difference (Zar 2010) All statistical analysis
was performed using SPSS for windows version 14 and graphs were plotted using Sigma
plot 2000
LER= Yield of Z mauritiana + Yield of C cajan (in intercropped) + Yield of C cajan + Yield of Z mauritiana (in intercropped)
Yield of Z mauritiana (sole) Yield of C cajan (sole)
Partial LER = Yield of Z mauritiana + Yield of C cajan (in intercropped)
Yield of Z mauritiana (sole)
Partial LER = Yield of C cajan + Yield of Z mauritiana (in intercropped)
Yield of C cajan (sole)
LEC = Partial LER of Z mauritiana times Partial LER of C cajan
96
232 Observations and Results
2321 Vegetative parameters
Vegetative growth parameters of Z mauritiana include plant height volume of canopy
grown individually as well as intercropped with C cajan is presented in Figure 29
Appendix-XV A significant increase in height and canopy volume of Z mauritiana with
time (p lt 0001) and cropping system (p lt 005) was observed However the interaction
between time and cropping system showed non-significant results In general the
intercropped plants were showed higher values in all vegetative parameters than sole crop
and this increase was more pronounced after 60 days
Figure 29 Appendix-XVII showed the vegetative growth parameters of C cajan
including height and number of branches Height of C cajan was significantly increased
(plt0001) with increasing time in plants growing sole and as intercropped with Z
mauritiana The interaction with time to crop height also showed significant (plt0001)
results in both cropping systems However slight decline in height of intercropped C
cajan was noticed at 120 days compared to sole crop Number of branches was significant
increased (plt0001) in both crops with increasing time The interaction of time with
branches also showed significant (plt0001) results in both cropping systems However
number of branches was slightly increased in intercropped plants at 120 days compared to
sole crop
2322 Reproductive parameters
i Fruit number and weight (fresh and dry)
Reproductive parameters of Z mauritiana and C cajan at grand period of growth under
sole and intercropping system has been presented in Figure 210 Appendix-XVI XVIII
Individual and interactive effect of time (p lt0001) and treatment (plt001) on number and
fresh weight of fruits of Z mauritiana was showed significant results Similarly plants
grown with C cajan showed significant increase (p lt0001) in fresh weight of fruits (p
lt005) whereas fruit dry weight and circumference was non-significant in comparison to
sole crop
97
In C cajan flowers were appeared only at blooming phase (during 60 days of treatment)
and no difference in number of flowers was observed in both cropping systems (sole and
with Z mauritiana (Figure 210 XVII)
Leguminous pods were initiated soon after flowering period (during 60 days) and
last till end of the experiment (120 days) A significant increase (plt0001) in pod numbers
was observed with increasing time in both sole and intercropped system But non-
significant differences in number of pods of both cropping system and their interaction
with time were observed
Similarly number and weight of C cajan seeds were showed non-significant difference
in both cropping systems
2323 Study on some biochemical parameters
i Photosynthetic pigments
Leaf pigments of Zmauritiana and C cajan grow plants under sole and intercropping has
been presented in Figure 211 Appendix-XVI XVIII In Z muritiana leaves A significant
increase (plt005) in chlorophyll a chlorophyll b total chlorophyll and carotinoids was
observed when grown as intercrop whereas the effect on chlorophyll ab ratio was non-
significant as that of sole one
In C cajan a slight decrease (plt005) in chlorophyll lsquobrsquo and total chlorophyll
(plt001) was observed in intercropped plants compare to sole one Whereas chlorophyll
lsquoarsquo chlorophyll ab ratio and carotinoids showed nonsignificant difference between sole
and intercropped C cajan
ii Total proteins sugar phenols
Sugars protein and phenols in leaves of Z mauritianaand C cajan at grand period of
growth under sole and intercropping system is presented in Figure 212 Appendix-XVI
XVIII Total proteins and soluble and insoluble sugar content of Z mauritiana leaves was
unaffected throughout the experiment However an increase in total phenolic content
(plt001) was observed in intercropped Z mauritiana plants than grown individually
98
In C cajan total soluble sugars protein and phenols in leaves showed non-
significant differences between sole to intercropped plants
Sugars protein and phenols in fruits of Z mauritiana grown under sole and
intercropping system is presented in Figure 213 Appendix-XVI A non-significant
increase was observed in phenolic as well as in soluble insoluble and total sugar contents
in fruits of Z mauritiana plants grown with C cajan (intercrop) as compare to the fruits
of sole crop
2324 Nitrogen Contents
Nitrogen in leaves and in soil of Z mauritiana and C cajan growing under sole and
intercrop system is presented in Figure 214 Appendix-XVI XVIII ANOVA showed a
non significant effect on nitrogen content of leaf as well as root zone soil of Z mauritiana
and C cajan grown individually or as intercropping system
2225 Land equivalent ratio (LER) and land equivalent coefficient
(LEC)
Land equivalent ratio (LER) Land equivalent coefficient (LEC) of height chlorophyll and
yield of of Z 98auritiana and C cajan growing as sole and intercropping system in has
been presented in Table 22 The LER using height of both species was nearly 2 in which
PLER of Z mutitania was 48 and PLER of C cajan was 519 Whereas the calculated
values of the land equivalent coefficient (LEC) of Z mauritiana and C cajan remained
9994
The LER using yield of both species was above 2 in which PLER of Z mauritiana
was 46 Whereas PLER of C cajan was 543 However the calculated values of LEC
of both species were 100
The LER using total chlorophylls of both species were more than 25 in which
PLER of Z mauritiana was 344 and as that of PLER of C cajan was 655 Whereas
the calculated values of LEC was 999 of both the species
99
Table 21 Soil analysis data of Fiesta Water Park experimental field
Serial number Parameters Values
1 ECe (dSm-1) 4266plusmn0536
2 pH 8666plusmn0136
3 Bulk density (gcm3) 123plusmn0035
4 Porosity () 53666plusmn1333
5 Water holding capacity () 398plusmn2811
6 Soil texture Clay loam
7 Sand () 385plusmn426
8 Silt () 3096plusmn415
9 Clay () 305plusmn1
Ece is the electrical conductivity of saturated paste of soil sample
Figure 29 Soil texture triangle (Source USDA soil classification)
100
Ziziphus mauritiana
Days
0 60 120
Volu
me
(m3)
0
10
20
30
Days
0 60 120
Hei
ght
(cm
)
0
50
100
150
200
250
Sole Intercrop
a
a
bb
c c
aa
bb
c c
Cajanus cajan
Days
0 60 120
Bra
nch
es (
)
0
10
20
30
Days
0 60 120
Hei
ght
(cm
)
0
50
100
150
200
250
300
Sole Intercrop
aa
bb
c c
aa
bb
c c
Figure 210 Vegetative growth of Z mauritiana and C cajan growing under sole and intercropping
system (Bars represent means plusmn standard error of each treatment and significance among the
treatments was recorded at p lt 005)
101
Ziziphus mauritiana
Fresh Dry
Fru
it w
eig
ht
(g)
0
50
100
150
200
Days
0 60 120 180
Nu
mb
er o
f F
ruit
s
0
100
200
300
Sole Intercrop
a
b
a
b
c
c
dd
Cajanus cajan
0 60 120
Num
ber
of
Pods
0
50
100
150
200
Days
0 60 120
Num
ber
of
Flo
wer
s
0
50
100
150
Sole Intercrop
Days
aa
bb
c c
Sole Intercrop
Num
ber
of
See
ds
0
100
200
300
400
500
See
d W
eight
(g)
0
10
20
30
40
50
60Number of seedsSeed weight
Figure 211 Reproductive growth of Z mauritiana and C cajan growing under sole and intercropping
system (Bars represent means plusmn standard error of each treatment and significance among the
treatments was recorded at p lt 005)
102
Ziziphus mauritiana
Cajanus cajan
Figure 212 Leaf pigments of Zmauritiana and C cajan growing under sole and intercropping (Bars
represent means plusmn standard error of each treatment and significance among the treatments was
recorded at p lt 005)
Sole Intercrop
Car
ote
noid
s (m
g g
-1)
00
01
02
03C
hlo
rophyl
l (m
g g
-1)
00
02
04
06
08
ab r
atio
00
05
10
15
20
25
ab
ab
Sole Intercrop
Car
ote
no
ids
(mg
g-1
)
00
01
02
03
Ch
loro
ph
yll
(m
g g
-1)
00
02
04
06
08
10
ab
rat
io
0
1
2
3
4ab
ab
103
Ziziphus mauritiana
Sole Intercrop
Lea
f P
hen
ols
(m
g g
-1)
0
2
4
6
8
10
12
Lea
f P
rote
ins
(mg
g-1
)
0
2
4
6
8
Lea
f S
ug
ars
(mg
g-1
)
0
5
10
15
20
25
30
35SoluableInsoluable
Figure 213 Sugars protein and phenols in leaves of Z mauritiana and C cajan at grand period of growth under
sole and intercropping system (Bars represent means plusmn standard error of each treatment and
significance among the treatments was recorded at p lt 005)
104
(Figure 212 continuedhellip)
Cajanus cajan
Sole Intercrop
Lea
f P
hen
ols
(m
g g
-1)
0
2
4
6
8
Lea
f P
rote
ins
(mg g
-1)
00
05
10
15
20
Lea
f S
ugar
s (m
g g
-1)
0
2
4
6
8
105
Ziziphus mauritiana
Sole Intercrop
Fru
it P
hen
ols
(m
g g
-1)
0
2
4
6
8
10
12
14
Fru
it P
rote
ins
(mg g
-1)
00
02
04
06
08
10
Fru
it S
ugar
s (m
g g
-1)
0
5
10
15
20
25
30
35 SoluableInsoluable
Figure 214 Sugars protein and phenols in fruits of Z mauritiana grown under sole and intercropping
system (Bars represent means plusmn standard error of each treatment and significance among the
treatments was recorded at p lt 005)
106
Z mauritiana
Sole Intercrop
Nit
rogen
(
)
0
1
2
3
4
5
6
7 LeafSoil
Cajanus cajan
Sole Intercrop
Nit
rogen
(
)
0
1
2
3
4
5
6
7 LeafSoil
Figure 215 Nitrogen in leaves and in soil of Z mauritiana and C cajan growing under sole and intercrop
system (Bars represent means plusmn standard error of each treatment and significance among the
treatments was recorded at p lt 005)
107
Table 22 Land equivalent ratio (LER) and Land equivalent coefficient (LEC) with reference to height chlorophyll and yield of of Z mauritiana and C cajan growing
under sole and intercropping system
Plant species Parameters Formulated with
reference to Height
Formulated with
reference to Total
Chlorophyll
Formulated with reference to Yield
(fresh weight of Z mauritiana fruit
and seed of C cajan)
Z mauritiana Partial LER 1027 1666 1159
C cajan Partial LER 0950 0877 0993
Intercropped
Total LER 1977 2543 2152
Z mauritiana amp C cajan
(Sole and intercropped) LEC 0975 1461 1151
107
108
24 Discussion
Intercropping is a common practice used to obtain better yield on a limited area through
efficient utilization of given resources which may not be achieved by growing each crop
independently (Mucheru-Muna et al 2010) In this system selection of appropriate crops
planting rates and their spatial arrangement can reduce competition for light water and
nutrients (Olowe and Adeyemo 2009) In general increased growth (biomass height
volume circumference biomass succulence SSL SRL SSR LWR SWR RWR and
RGR) of each species is a good indicator of successful intercropping The SRL and SSL
measure the ratio between the lengths of root or shoot per unit dry weight of respective
tissues (Wright and Westoby 1999) The weight ratio of leaf stem and root to total plant
weight (LWR SWR and RWR) describes the allocation of biomass towards each organ to
maximize overall relative growth rate (RGR) which explains how plant responds to certain
type of condition (Reynolds and Antonio 1996) In this study height and canopy volume
of Z mauritiana and height and branches of C cajan were increased when grown together
in comparison to sole crop in field experiment (Figure 29) Whereas in drum pot culture
biomass generally the length of plant canopy volume number of leaves RGR LWR
SWR RWR SSL and SRL were either higher or unaffected in both species growing in
intercropping at 4th and 8th days intervals (Figure 21-23) Similar beneficial effects on
growth of other intercrops have also been reported under different conditions (Yamoah
1986 Atta-Krah 1990 Kass et al 1992 Singh et al 1997) Dhyani and Tripathi (1998)
observed increased height stem diameter crown width and timber volume of three
intercropped species than sole crop Bhat et al (2013) also revealed significant
improvement in annual extension height and spread in apple plants intercropped with
leguminous plants
The increased growth of both intercropped plants of this study was well reflected
by their biochemical parameters Leaf pigments like chlorophyll a chlorophyll b and total
chlorophyll were either higher or remained unaffected (Figure 211) in both intercropped
plants than sole crops of field experiments Whereas in drum pot culture chlorophyll
content (Figure 24) was higher only in intercropped C cajan (specially in 8th days) Bhatt
et al(2008) and Massimo and Mucciarelli (2003) also reported the increased accumulation
of chlorophyll a b and total chlorophylls in leaves of soybean and peppermint when
109
grown with their respective intercrops Our results are also in agreement with Liu et al
(2014) and Otusanya et al (2008) reported similar results in Lycopersican esculentum and
later in Capsicum annum as well Some other reports are also available which shows non-
significant effect on leaf pigments in both cropping systems (Shi-dan 2012 Luiz-Neto-
Neto et al 2014)The synthesis and activity of chlorophyll depends on severity and type
of applied stress it generally increase in low saline mediums (Locy et al 1996) or
remained unaffected however sometimes stimulated (Kurban et al 1999 Parida et al
2004 Rajesh et al 1998)
Proteins and carbohydrates (sugars) perform vast array of functions which are
necessary for plant growth and reproduction (Copeland and McDonald 2012) Variation
in their contents helps to predict plant health which is usually decreased with applied stress
(Arbona et al 2013) Both are also the compulsory factors of animals diet since they
cannot manufacture sugars and some of the components of proteins which must be
obtained from food (Bailey 2012) In our experiment protein content was either remained
unchanged or increased which indicated a good coordination of both intercrops in field
and drum pot experiments (Figure 26 and 212) Liu et al (2014) also found that protein
and sugars were not affected in tomatogarlic intercrops In another experiment similar
results were found when corn was grown with and without intercropping (Borghi et al
2013)
Reactive oxygen species (ROS) are produced as a spinoff of regular metabolism
however under stress the overproduction of ROS may lead to oxidative damage (Baxter et
al 2014) In low concentrations ROS worked as messengers to regulate several plant
processes and also helps to improve tolerance to various biotic and abiotic stresses (Miller
et al 2009 Nishimura and Dangl 2010 Suzuki et al 2011) but when the concentration
goes beyond the critical limit ROS would become self-threatening at every level of
organization (Foreman et al 2003) To maintain a proper workable redox state an
efficient scavenging system of enzymatic (SOD CAT GPX and APX) andor non-
enzymatic (polyphenols sugars glutathione and ascorbic acid) antioxidants is required
which would be of critical importance when plant undergoes stress (Sharma et al 2012)
Among these enzymes SOD is a first line of defense which converts dangerous superoxide
radicals into less toxic product (H2O2) In further CAT APX and GPX worked in
association to get rid off from the excessive load of other oxygen radicals or ions (H2O2
110
OH- ROO etc) In this study antioxidant enzymes (SOD CAT GPX and APX) were
found to work in harmony which was not affected during 4th day treatment in both species
in comparison to sole crop (Fig 27) showing strong antioxidant defense which was not
compromised by cropping system When comparing in 8th day treatment a significant
general increase in all enzyme activities were observed in both species except for SOD
and GPX of C cajan (Fig 27) These results displayed relatively better performance and
tight control over the excessive generation of ROS which would be predicted in this case
due to less availability of water than in 4th day treatment (Karatas et al 2014 Doupis et
al 2013) Similarly by coping oxidative burst and maintaining cellular redox equilibrium
plants were able to improve growth performance especially in Z mauritiana (Fig 21)
Water deficit affect stomatal conductance which could bring about changes in
photosynthetic performance hence overproduction of ROS is usually found among
different crops (Moriana et al 2002 Miller et al 2010) As a response tolerant plants
overcome this situation by increased activity of antioxidant enzymes which was evident in
Wheat Rice olive etc (Zhang and Kirkham 1994 Sharma and Dubey 2005 Guo et al
2006 Sofo et al 2005)
Phenolic compounds despite their role in physiological plant processes are
involved in adsorbing and neutralizing reactive oxygen species (ROS Ashraf and Harris
2004) The overproduction of ROS may cause several plant disorders Plants produce
secondary compounds like polyphenols to maintain balance between ROS generation and
detoxification (Posmyk et al 2009) Increased synthesis and accumulation of phenolic
compounds is reported to safeguard cellular structures and molecules especially under
biotic abiotic constraints (Ksouri et al 2007 Oueslati et al 2010) In this study
intercropped Z mauritiana of field and both species in drum pot culture showed higher
phenolic content than individual crop (Figure 25 and 212) which may be attributed to
adaptive mechanism for scavenging free radicals to prevent cellular damage (Rice-Evans
1996)
In terms of fruit yield we observed that Z mauritiana is suitable for intercropping
as suggested by Yang et al (1992) Number of flowers fruits and fruit fresh weight of
both species either increased considerably or no-affected in intercropped plants compared
to individual ones (Figure 210) Moreover fruit quality of Z mauritiana includes proteins
phenols and soluble extractable and total sugars were also higher in intercropped plants
111
(Figure 213) Results of this study are better than other experiments reported by
Sharma (2004) Kumar and Chaubey (2008) and Kumar et al (2013) who did not find
influence of other understory forage crops (like Aonla) on the yield of Z mauritiana
However in other case the yield of intercropped ber was some time higher (Liu 2002)
Singh et al 2013 found no adverse effects on the yield of pigeonpea when intercropped
with mungbean however it improved the grain yield of associated species
A leguminous plant C cajan is used in this experiment as secondary crop which
can supplement Z mauritiana by improving soil fertility Results of both experiments
showed that the nitrogen was higheror un-affected (Figure 214) in soils of intercropped
plants which supports our hypothesis that leguminous intercrop increase N supply This
can be achieved by acquisition of limited resources to manage rootrhizosphere
interactions which can improve resource-use efficiency (Zhang et al 2010
Shen et al 2013 White et al 2013b Ehrmann and Ritz 2014 Li et al 2014) As a
consequence it impact on overall plant performance which starts from high photosynthetic
activity by increasing chlorophyll results in more availability of photoassimilate for
growth and reproductive allocation (Eghball and Power 1999) Use of C cajan in tree
intercropping proved beneficial for producing high yield crops and for the environment
(Gilbert 2012 Glover et al 2012)
Land equivalent ratio (LER) is commonly used to evaluate the effectiveness of
intercropping by using the resources of same environment compared with sole crop
(Vandermeer 1992 Rao et al 1990 1991 Cao et al 2012) It is the ratio of area for sole
crop to intercrop required to produce the equal amount of yield at the same management
level (Mead and Willey 1980 Dhima et al 2007) On the other hand land equivalent
coefficient (LEC) describe an association that concern with the strength of relationship It
is the proportion of biomassyield of one crop explained by the presence of the other crop
The LER 1 or more indicate a beneficial effect of both species on each other which increase
the yield of both crops as compare to single one (Zada et al 1988) In this experiment all
LER values were about 2 or more than 2 while LEC values were around 1 or more than
one in ZizyphusCajnus intercropping Both LER and LEC values were in descending
order of chlorophylls gt yield gt height (Table 22) However the partial LER was higher in
Zizyphus than Cajanus in all cases These results describe the superiority of intercropping
over sole cropping where LER values are even gt2 Some other studies reported LER from
112
09-14 (Bests 1976) 12-15 (Cunard 1976) and up to 2 (Andrews and Kassam 1976)
Similar results were reported in poplarsoybean system (Rivest et al 2010) black
locustMedicago sativa (Gruenewald et al 2007) wheatjujube (Zhang et al 2013)
Acacia salignasorghum (Droppelmann et al 2000 Raddad and Luukkanen 2007) The
high LER values in our system indicating a harmony in resource utilization in both species
which was also corroborated with their respective LEC values The greater LEC values (gt
025) suggesting an inbuilt tendency of studied crops to give yield advantage (Kheroar and
Patra 2013) Experiments based on traditional practices of growing legumes with cereals
demonstrated greater and continuous cash returns than individual-crops (Baker 1978) In
addition the same authors found further increase in cash returns by increasing the
proportion of cereal and incorporating maize with sorghum and millet In agreement with
our findings similar reports are also available from different intercropping systems
including sesamegreengram (Mandal and Pramanick 2014) maizeurdbean (Naveena et
al 2014) and pegionpeasorghum (Egbe and Bar-Anyam 2010)
After detailed investigations of both species using two different experiment designs
(drum pot and field) it is evident that intercropping had beneficial effects on growth
physiology biochemisty and yield of both species Furthermore by using this system
higher outcome interms of edible biomass and green fodder using marginal lands can be
obtained in a same time using same land and water resources which can help to eliminate
poverty and uplift socio-economic conditions
113
3 Chapter 3
Investigations on rang of salt tolerance in Carissa carandas
(varn karonda) for determining possibility of growing at waste
saline land
31 Introduction
Carissa carandas commonly known as Karonda or lsquoChrist thornrsquo belonging to family
Apocynaceae shows capability of growing under haloxeric conditions It is an important
plant which has established well at tropical and subtropical arid zone under high
temperatures It is large evergreen shrub and having short stem It has fork thorn and hence
used as hedges or fence around fields The leaves are oval or elliptic 25 to 75 cm long
dark green leathery and secrete white milk if detached The fruits are oblong broad- ovoid
or round 125- 25 cm long It has thin but tough epicarp Fruits are in clusters of 3-10
Young fruits are pinkish white and become red or dark purple on maturation
The plant is propagated through seed in August and September Budding and cutting
could also be undertaken Planting is started after first shower of monsoon Plants raised
from seeds are able to flower within two years Flowering starts in March and fruit ripen
from July to September (Kumar et al 2007) The fruit possess good amount of pectin and
acidity hence used in prickle jelly jam squash syrup and in chutney by the commercial
name lsquoNakal cherryrsquo (Mandal et al 1992) They are rich in vitamin C and good source
of Anthocyanin (Lindsey et al 2000) Its fruits also are one of the richest source of iron
(391 mg 100gm) (Tyagi et al 1999) Juice of its root is also used to treat various
microbial diseases such as diarrhea dysentery and skin disease (Taylor et al 1996)
Hence its range of salt and suitability for cultivation at waste saline land or with saline
water irrigation is being undertaken for commercial exploitation by preparing jams jellies
and prickles (Kumar 2014) Investigations on its growth and development at higher range
of salinities are being undertaken with an interest to cultivate it if profitable at highly saline
waste land
114
32 Experiment No 9
Investigation on the effect of higher range of salinities on growth of
Carissa carandas (varn karonda) created by irrigation of different
dilutions of sea salt
321 Materials and methods
3211 Drum Pot Culture
Drum pot culture as recommended by Boyko (1966) and modified by Ahmed and
Abdullah (1982) was used for the present investigation which was been already described
in Chapter 1 earlier
3212 Plant material
About six months old sapling of Carissa carandas (varn Karonda) having almost equal
height and volume poted in polythene bag in 3kg of soil fertilized with cow-dong manure
were purchased from the Noor nursery Gulshan-e-Iqbal Karachi Sindh and were
transported to the Biosaline research field department of Botany University of Karachi
3213 Experimental setup
Plants were transplanted in drum pot (Homemade lysimeter) filled with sandy loam mixed
with cow dung manure (91) Each drum pot was irrigated weekly during summer and
fortnightly during winter months with 20 liters tap water (Eciw= 0 6 dSm-1) or water of
sea salt concentrations of various ie 03 (Eciw = 42 dSm-1) 04 (Eciw =61 dSm-1)
06 (Eciw = 99 dSm-1) and 08 (Eciw = 129 dSm-1) The plants were established initially
by irrigation with tap water for two weeks and later salinity was gradually increased till
desired percentage is achieved for different treatments by dessolving of sea salt in
irrigation water Three replicates were maintained for each treatment Urea DAP and
KNO3 were the source of NPK provided in the ratio 312 50g granules Osmocot (Scotts-
Sierra Horticulture Products) and 50g Mericle-Gro (Scotts Miracle-Gro Products Inc)
were dissolved in irrigation water per drum after six months at six monthly intervals
Height and volume of canopy of these plants were recorded prior to the starting the
experiment and then after every six months interval
115
Since the vegetative growth performance in plants irrigated with 03 sea salt (Eciw = 42
dSm-1) was found comparatively better than control and only 26 decrease was noticed
in volume of canopy at plant irrigated with 04 sea salt (Eciw = 61 dSm-1) (Table III41)
the onward investigations were focused at higher salinity levels and plants were irrigated
with 06 (Eciw = 99 dSm-1) and 08 (Eciw = 129 dSm-1) sea salt in rest of experiment
3214 Vegetative parameters
Vegetative growth on the basis of plant height and volume were recorded while
reproductive growth was observed on the basis of number of flowers and number and
weight of fruits per plant Length and diameter of fruit were also recorded in ten randomly
selected fruits
3215 Analysis on some biochemical parameters
Following biochemical analysis of leaves was performed at grand period of growth (onset
of flowers)
i Photosynthetic pigments
Fresh fully expended leaves (01g) was crushed in 80 chilled acetone Further procedure
was followed described in chapter 1
ii Soluble sugars
Dry leaf samples (01g) were milled in 5mL of 80 ethanol and were centrifuged at 4000
g for 10 minutes Same procedure was followed as described in chapter 1
iii Protein content
The protein contents were measured according to Bradford Assay reagent method against
Bovine Serum Albumin which was taken for standard (Bradford 1976) as described in
chapter 1
iv Soluble phenols
The dried leaf powder (01g) was milled in 3ml of 80 methanol and was centrifuged at
10000g for 15 min Further procedure has been described in chapter 2
116
3216 Mineral Analysis
Estimation of Na+ and K+ were made according to Chapman and Pratt (1961) Oven dried
grinded Leaves (1g) furnace at 550ordmC for 6 hours and were digested in 5 ml of 2N HCl
Diluted and filtered solution was used to estimated Na+ and K+ in flame photometer
(Petracourt PFP I) The concentration of these ions was calculated against the following
standard curve equations
Na+ (ppm) = 0016135x1879824
K+ (ppm) = 0244346x1314603
117
322 Observations and Result
3221 Vegetative parameters
Vegetative growth in terms of height and volume of canopy of C carandas growing under
salinities created by irrigation of different dilutions of sea salt is presented in Table 32
Appendix-XIX A significant increase (plt0001) in plant height and volume of canopy
was observed with increasing time but the increase was rapid at early period of growth
However there was significant (plt0001) reduction under salinity stress The interaction
of time and salinity also showed significant (plt001) effect on plant parameters but the
increase in height and volume of canopy at Eciw= 42dSm-1of sea salt salinity was more
than control Plants irrigated with Eciw= 61 dSm-1 and Eciw= 99 dSm-1sea salt solution
showed decrease in height with respect to control but the difference between their
treatments was insignificantly higher decrease was observed in Eciw= 129 dSm-1 sea salt
irrigated plants
3222 Reproductive parameters
Reproductive growth in terms of flowers and fruits numbers flower shedding percentage
fresh and dry weight of ten fruit their length and diameter under salinities created by
irrigation of different dilutions of sea salt is presented in Table 33 Appendix-XX Number
of flowers and fruits significantly (plt0001) decreased with increasing salinity treatment
Difference in flower initiation seems non-significant at early growth period in controls and
salinity treatments However drastic decrease was observed in plants irrigated beyond
Eciw= 99 dSm-1 with increase in salinity
Flowers shedding percentage (Table 33 Appendix-XX) show an increase directly
proportional with increase in salinity however the difference in number of flowers
between the plants irrigated with Eciw= 99 dSm-1 and Eciw= 129 dSm-1 sea salt solution
is of little significance level (plt001)
Fresh and dry weight of average fruits (plt001) and their diameter (plt001) showed
decrease with increasing salinity whereas diameter and length of fruits showed non-
significant difference
118
3224 Study on some biochemical parameters
i Photosynthetic Pigments
Photosynthetic Pigments including Chlorophyll a chlorophyll b total chlorophyll
chlorophyll a b ratio and carotenoids of C carandas growing under salinities created by
irrigation of different dilutions of sea salt is presented in Figure 31 Appendix-XX The
chlorophyll contents of leaves significantly decreased (plt0001) over control with
increasing salinity however Chlorophyll rsquobrsquo at Eciw= 99 dSm-1salinity shows significant
increase (plt0001) over control Similarly Carotenoids at Eciw= 99 dSm-1 salinity show a
bit less significant increase (plt001) compare to control while at higher salinity (Eciw=
129 dSm-1) the decline is observed at all above mentioned parameters
iii Protein Sugars and phenols
Some biochemical parameters including Protein sugars and phenolic contents of C
carandas growing under salinities created by irrigation of different dilutions of sea salt is
presented in Figure 31 Appendix-XX Soluble proteins in leaves show non-significant
decrease at Eciw= 99 dSm-1salinity as compared with controls but a significant decrease
(plt005) was noted at Eciw= 129 dSm-1 salinity Sugars also showed non-significant
decrease at both the salinity whereas on contrary soluble phenols showed significant
increase (plt0001) with increasing salinity
3225 Mineral analysis
Mineral analysis including Na and K ions performed in leaves of C carandas growing
under salinities created by irrigation of different dilutions of sea salt is presented in Figure
32 Appendix-XX Sodium significantly increased (plt0001) all the way with increasing
salinity of growth medium Whereas significant decrease (plt0001) was observed in
Potassium with increasing salinity K+Na+ ratio show continuous increase with increasing
salinity
119
Table 31 Electrical conductivities of different sea salt concentration used for determining
their effect on growth of C carandas
Treatment
Sea salt ()
ECiw of irrigation water (dSm-1) ECe of soil saturated paste
(dSm-1)
Non-saline control 06 09
03 42 48
04 61 68
06 99 112
08 129 142
Whereas ECiw and ECe are the electrical conductivities of irrigation water and soil saturated past measured in deci semen per meter
120
Table 32Vegetative growth in terms of height and volume of canopy of C carandas growing under salinities created by irrigation of different dilutions of
sea salt
Treatment
Sea salt
(ECiw dSm-1)
Initial values prior to
starting saline water
irrigation
Growth at different salinities after 06 months
Height Volume Height Volume of canopy
cm m3 cm
increase
over initial
values
increase
decrease over
control
m3 increase over
initial values
increase
decrease
over control
Control 3734plusmn455 0029plusmn0001 8227plusmn4919 5363plusmn830 - 014plusmn0015 7952plusmn269 -
42 3674plusmn1415 0026plusmn0003 9930plusmn6142 6280plusmn205 +1710 019plusmn0017 8593plusmn098 +806
61 3752plusmn1243 0026plusmn0001 6490plusmn5799 4132plusmn485 -2305 012plusmn0010 7740plusmn117 -282
99 3819plusmn4499 0028plusmn0005 5793plusmn5821 3123plusmn1446 -4185 009plusmn0008 6759plusmn377 -1499
129 3676plusmn3114 0026plusmn0008 5250plusmn4849 2775plusmn1276 -4836 006plusmn0005 5690plusmn1110 -2844
LSD0 05
Salinity
Time Fisherrsquos least significant difference
91
172
002
0005
Values represent means plusmn standard error of each treatment and significance among the treatments was recorded at p lt 005
120
121
Table 33 Vegetative growth in terms of height and volume of canopy of C carandas growing under salinities
created by irrigation of different dilutions of sea salt
Treatment
Sea salt
(ECiw dSm-1)
Growth at different salinities after 12 months
Height Volume of canopy
cm
increase
over initial
values
increase
decrease over
control
m3
increase
over initial
values
increase
decrease over
control
Control 16214 plusmn633 7674plusmn307 - 077plusmn012 9689plusmn449 -
99 9736plusmn1048 6056plusmn561 -2109 034plusmn006 9367plusmn412 -333
129 6942plusmn565 4741plusmn480 -3822 022plusmn002 9064plusmn623 -645
Table 33 continuedhellip
Treatment
Sea salt
(ECiw= dSm-1)
Growth at different salinities after 18 months
Height Volume of canopy
Cm
increase
over initial
values
increase
decrease over
control
m3
increase
over initial
values
increase
decrease over
control
Control 1676plusmn1135 7776plusmn756 - 094plusmn011 9701plusmn578 -
99 10547plusmn842 6351plusmn666 -1833 045plusmn010 9445plusmn1024 -264
129 7581plusmn593 5154plusmn716 -3372 030plusmn003 9318plusmn580 -395
Table 33 continuedhellip
122
Table 33 continuedhellip
Treatment
Sea salt
(ECiw= dSm-1)
Growth at different salinities after 24 months
Height Volume of canopy
Cm
increase
over initial
values
increase
decrease over
control
m3
increase
over initial
values
increase
decrease over
control
Control 1911plusmn6
05 8055plusmn941 - 121plusmn015 9837plusmn522 -
99 1110plusmn5
31 6557plusmn543 -1859 053plusmn002 9509plusmn1032 -334
129 8754plusmn10
67 5990plusmn801 -2564 040plusmn008 9287plusmn745 -560
Table 33 continuedhellip
Treatment
Sea salt
(ECiw= dSm-1)
Growth at different salinities after 30 months
Height Volume of canopy
Cm
increase
over initial
values
increase
decrease over
control
m3
increase
over initial
values
increase
decrease over
control
Control 2052plusmn1126 8182plusmn676 - 146plusmn029 9873plusmn729 -
99 11700plusmn816 6743plusmn610 -1759 070plusmn011 9565plusmn850 -312
129 9628plusmn552 6189plusmn573 -2436 050plusmn004 9417plusmn1011 -462
LSD0 05 Salinity 77 007
Time 168 016
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and significance among the treatments was recorded at p lt 005
123
Table 34 Reproductive growth in terms of flowers and fruits numbers flower shedding percentage fresh and dry weight of ten fruit and their totals
perplant fruit length and diameter of C carandas growing under salinities created by irrigation of different dilutions of sea salt
Treatment
Sea salt
(ECiw= dSm-1)
Flower Fruits Flower
shedding
Weight of
Ten
fruit(fresh)
Weight of
Ten
fruit(dry)
Weight of
total fruitplant
(fresh)
Weight of
total fruitplant
(dry)
length
fruit
diameter
fruit
Numbers Numbers g g g g mm mm
Control 19467plusmn203 16600plusmn231 1468plusmn208 2282plusmn022 605plusmn009 37891plusmn891 10047plusmn283 1800plusmn003 1423plusmn006
99 12050plusmn202 7267plusmn491 3980plusmn307 1880plusmn035 530plusmn029 13695plusmn1174 3880plusmn469 1732plusmn037 1297plusmn011
129 12567plusmn549 6967plusmn203 4449plusmn082 1541plusmn023 435plusmn026 10742plusmn470 3041plusmn268 1711plusmn015 1233plusmn038
LSD0 05 Salinity 1514 1417 929 115 097 3785 1494 0971 097
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and significance among the treatments was recorded at p lt 005
123
124
Sea Salt (ECiw
= dSm-1
)
Cont 99 129
Car
ote
nio
ds
(mg
g-1
)
00
01
02
03
04
Ch
loro
ph
yll
(m
g g
-1)
00
01
02
03
04
05
06
ab
rat
io
00
05
10
15
20
25
30
35
ab
Chl a Chl b
a
a
a a
b
bcbc
a
b
c
a a
b
Figure 31 Chlorophyll a chlorophyll b total chlorophyll chlorophyll a b ratio carotenoids contents of C
carandas growing under salinities created by irrigation of different dilutions of sea salt (Bars
represent means plusmn standard error of each treatment and significance among the treatments was
recorded at p lt 005)
125
Sea Salt (ECiw
= dSm-1
)
Cont 99 129
Ph
eno
ls (
mg
g-1
)
0
5
10
15
20
Pro
tein
s (m
g g
-1)
0
1
2
3
4
Su
gar
s (m
g g
-1)
0
30
60
90
120
150Soluble Insoluble
a
a
a
a
a
a
b
b
b
c
ab
a
a
b
Figure 32 Total protein sugars and phenolic contents of C carandas growing under salinities created by
irrigation of different dilutions of sea salt (Bars represent means plusmn standard error of each treatment
and significance among the treatments was recorded at p lt 005)
126
Sea Salt (ECiw
= dSm-1
)
Cont 99 129
Ions
(mg
g-1
DW
)
0
20
40
60
80
100
120
KN
a ra
tio
00
01
02
03
04
05
06
07
Na K KNa
c
a
b
b
a
c
a
b
c
Figure 33 Mineral analysis including Na and K ions was done on leaves of C carandas growing under salinities
created by irrigation of different dilutions of sea salt (Bars represent means plusmn standard error of each
treatment and significance among the treatments was recorded at p lt 005)
127
33 Discussion
The volume and height of plants were increased per unit time under saline conditions This
increase was observed after six months in 03 sea salt (ECiw = 42 dSm-1) treated plants in
comparison to control (Table 32) Slight decrease was observed at 04 sea salt
(ECiw=61dSm-1) irrigation after which (Eciw= 99 dSm-1 and Eciw = 129 dSm-1sea salt) the
growth was significantly inhibited (Table 33) Noble and Rogers (1994) also noticed a general
decrease in growth of some of the glycophytes Humaira and Ahmad (2004) and Rivelli et al
(2004) also reported a proportional decrease in height of canola with increasing salinity
Cotton plants irrigated with saline water or those grown at saline soil are reported to increase
Na+ content in leaves accompanied by significant reduction in vegetative biomass (Meloni et
al 2001) Bayuelo-Jimenez et al (2003) observed salt induced growth inhibition of tomato
plant which was higher in shoot than root
Reproductive growth in terms of number of flowers number of fruits fruit length and
diameter were decreased and percent flower shedding increased with increasing salinity
(Table 34) These effects were higher at Eciw= 99 dSm-1and then maintained with further
salinity increment However weight of fruits (fresh and dry) and total fruits per plant were
linearly decreased with increasing medium salt concentrations A decrease in different phases
of reproductive growth like flowering fertilization fruit setting yield and quality of seeds etc
are reported to be seriously affected at different level of salinity by various workers (Lumis et
al 1973 Waisel 1991 Shannon et al 1994 Tayyab et al 2016) Cole and Mclead (1985)
and Howie and Lloyd (1989) reported severe effects of different salinity treatments on
flowering intensity fruit setting and number of fruits of Citrus senensis Walker et al (1979)
also reported reduction in the fruit weight during early ripening stage of Psidium guajava
Decrease in fruit diameter of strawberries (Fragaria times ananassa) has been reported with
salinity (Ehlig and Bernstein 1958)
In this study photosynthetic pigments of C carandas were decreased with salinity and
this decrease was more sever at Eciw = 129 dSm-1sea salt salinity (Figure 31) Such a decline
in amount of leaf pigments across different salinity regimes was also reported in cotton
(Ahmed and Abdullah 1979) Pea (Hernandez et al 1995 and Hernandez et al 1999) Vicia
128
faba (Gadallah 1999) Mulberry genotype (Agastian et al 2000) and B parviflora (Parida et
al 2004)
Leaf sugars and protein were decreased in both salinity levels (Figure 32) which could
be attributed to inhibition in transport of photosynthetic product (Levit 1980) Decrease
synthesis and mobilization of glucose fructose and sucrose has been demonstrated in number
of plants growing under salt stress (Kerepesi and Galiba 2000) Inhibition in the protein and
nucleic acid synthesis in Pisum sativum and Tamarix tetragyna plants were also reported by
Bar-Nun and Poljahoff-Mayber (1977) Melander and Harvath (1977) suggested that salt
induced reduction in protein is due to increase in protein hydrolysis
A significant increase in leaves phenol with increase in salinity (Figure 32) was
observed in present investigation was also demonstrated previously in Achilleacollina (Giorgi
et al 2009) Lactuca sativa (Kim et al 2008) and B parviflora (Parida et al 2004)
Inspite of over irrigation of saline water and maintaining leaching fraction of about
40 in drum pots accumulation of salts in rhizosphere soil was not completely avoided which
was evident in the differences between ECiw and ECe values (Table 31) Deposition of salts
in rhizosphere soil interferer absorption of minerals in plants For instance leaf Na+ content
of C carandas was significantly increased while K+ decreased with increasing soil salinity
(Figure 33) Over accumulation of toxic ions disturbed plant water status which directly
affects plant growth (Flowers et al 1977 Greenway and Munns 1980) A negative
relationship between Na+ and K+ concentration in roots and leaves of guava was also reported
by Ferreira et al (2001) Increase in Na+ content decreased K+ availability and K+Na+ ratio
in Vicia taba (Gadallah 1999) and also affect the uptake of other essential minerals in
Casurina equsetifolia (Dutt et al 1991)
Carissa carandas found to be a good tolerant to salinity and drought and it can produce
edible fruits from marginal lands of arid areas Fruits of this species can be consumed in a raw
form as well as in industrial products like pickles jams jellies and marmalades
129
4 Conclusions
In the light of above mentioned investigations it appears that pre-soaking treatment of Cajanus
cajan seeds has initiated metabolic processes at faster rate earlier which has helped seeds to
start germinative metabolism prior to be effected by toxic Na+ ions at higher salinities Cajanus
cajan and Ziziphus mauritiana were found to be the good companions for intercropping These
species synergistically enhanced the growth and biochemical performance of each other by
improving fertility of marginal land and maintaining harmony among different physiological
parameters which was missing in their sole crop Their intercropping could produce fodder
and delicious fruits even from under moderately saline substrate up to profitable extant
Carissa carandas also tolerated low and moderately salinities well by adjusting proper
regulation of physiological and biochemical parameters of growth It can provide protein rich
edible fruits jams jellies and pickles of commercial importance for benefit of poor farmer
from moderately saline barren land
130
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Sharma P HS Gujral and B Singh (2012) Antioxidant activity of barley as affected by
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Sharma P V Sardana and S Banga (2013) Salt tolerance of Indian mustard (Brassica
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Sharma PK and DO Hall (1991) Interaction of salt stress and photoinhibition on
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168
6 THESIS APENDECES
Appendix-I One way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution of seed germination of pre-soaked seeds of C cajan in non-saline water prior to germination under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Mean
germination rate
(GR)
Salinity treatment 4422 20 221133 21015 0000
Error 441949 42 10522
Total 4864 62
Mean germination
velocity (GV)
Salinity treatment 418813 20 20941 51836 0000
Error 169671 42 40398
Total 588484 62
Mean
germination
time (GT)
Salinity treatment 0271 20 0013 8922 0000
Error 0064 42 0002
Total 0335 62
Mean germination
Index (GI)
Salinity treatment 4422 20 221133 21015 0000
Error 441949 42 10523
Total 4864607 62
Final
germination
(FG)
Salinity treatment 32107 20 1605397 25285 0000
Error 2666 42 63492
Total 34774 62
Appendix-II Two way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution of seed germination of pre-soaked seeds of C cajan in non-saline water prior to germination under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Germination percentage per
day
Salinity treatment 509583 20 25479 19187 0000
Time 53156 9 5906 4663 0002
Salinity treatment times time 251743 180 1398576 1053 ns
Error 531130 400 1327825
Total 1375283 629
Germination
rate per day
Salinity treatment
Time 761502 9 84611 83129 0000
Salinity treatment times time 442265 20 22113 24630 0000
Error 359117 400 0898
Total 2108622 629
Appendix-III One way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution of seed
germination of pre-soaked seeds of C cajan in respective saline water prior to germination under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Final mean germination
velocity (GV)
Salinity treatment 0538 6 0089 35585 0000
Error 0035 14 0003
Total 0573
Final mean
germination time (GT)
Salinity treatment 20862 6 3477 26256 0000
Error 1854 14 0132
Total 22716 20
Final mean germination
index (GI)
Salinity treatment 110514 6 18419 190215 0000
Error 1356 14 0097
Total 111869 20
Final
germination percentage (GP)
Salinity treatment 6857 6 1142857 40 0000
Error 400 14 28571
Total 7257 20
Appendix-IV Two way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution of seed
germination of pre-soaked seeds of C cajan in respective saline water prior to germination under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Germination percentage per
day
Salinity treatment 86644 6 14440816 505428 0000
Time 23378 6 3896 136373 0000
Salinity treatment times time 2717 36 75472 2641 0001
Error 2800 98 28571
Total 115540 146
Germination rate
per day
Salinity treatment 117386 6 19564 360762 0000
Time 128408 6 21401 394636 0000
Salinity treatment times time 58747 36 1632 30091 0000
Error 5314 98 0054
Total 309855 146
169
Appendix-V One way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution on seedling
emergence and height of germinating seeds of C cajan under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Seedling height of C cajan
Salinity treatment 200822 5 40056 169666 0000
Error 2833 12 0236
Total 203115 17
Seedling
emergence of C cajan
Salinity treatment 24805 6 4134 6381 000
Error 9070 14 647867
Total 33875 20
Appendix-VI Two way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution on growth and
development of C cajan in lysemeter (Drum pot) under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Plant height of
C cajan
Salinity treatment 261079 5 52215 720259 0000
Time 126015 8 15751 132488 0000
Salinity treatment times time 76778 40 1919 16144 0000
Error 11413 96 118893
Total 477028 161
Appendix-VII One way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution on growth
and development of C cajan in lysemeter (Drum pot) under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Number of
Flowers of C
cajan
Salinity treatment 3932 3 131075 39719 0000
Error 264 8 33
Total 419625 11
Number of pods
of C cajan
Salinity treatment 1473 3 491 23105 0000
Error 170 8 2125
Total 1643 11
Number of
seedspod of C cajan
Salinity treatment 3 3 1
Error 0 8 0
Total 3 11
Number of seeds plant of
C cajan
Salinity treatment 19332 3 6444 45621 0000
Error 1130 8 14125
Total 20462 11
Weight of
seeds plant of C cajan
Salinity treatment 592976 3 197658 85572 0000
Error 18478 8 2309
Total 611455 11
Chlorophyll a
of C cajan
Salinity treatment 0117 3 0039 81241 0000
Error 0004 8 0000
Total 0121 11
Chlorophyll b
of C cajan
Salinity treatment 0004 3 0001 15222 0001
Error 0001 8 0000
Total 0005 11
Total chlorophyll of
C cajan
Salinity treatment 0160 3 0053 164401 0000
Error 0002 8 0000
Total 0162 11
Chlorophyll a b
ratio of C cajan
Salinity treatment 242 3 0806 9327 0005
Error 0692 8 0086
Total 3112 11
Carotenoids of
C cajan
Salinity treatment 0015 3 0005 4510 0039
Error 0009 8 0001
Total 0025 11
Soluble sugars
of C cajan
Salinity treatment 0043 3 0014 6515 0015
Error 00178 8 0002
Total 0061 11
Insoluble
sugars of C
cajan
Salinity treatment 0118 3 0039 36262 0000
Error 0008 8 0001
Total 0127 11
Total sugars of
C cajan
Salinity treatment 0019 3 0006 4239 0045
Error 0012 8 0001
Total 0031 11
Protein of C cajan
Salinity treatment 0212 3 0070 15735 0001
Error 0036 8 0004
Total 0248 11
170
Appendix-VIII One way ANOVA for completely randomized design for range of salt tolerance of nitrogen fixing symbiotic bacteria
associated with root of C cajan
Variables Source Sum of Squares df Mean Square F-value P
Nodule
associated
Rhizobial colonies of C
cajan
Salinity treatment 35927 2 17963 229402 0000
Error 1409 18 0078
Total 37337 20
Appendix-IX Two way ANOVA for completely randomized design for growth and development of Z mauritiana in large size clay pot being irrigated with water of two different sea salt concentration
Variables Source Sum of Squares df Mean Square F-value P
Height of
Z mauritiana
Time 91030 2 45515 839 0000
Salinity treatment 3268 2 1634 10 0000
Time times Salinity treatment 1533 4 383 238 ns
Error 6751 42 161
Total 104554 71
Number of
branches of
Z mauritiana
Time 25525 2 127625 25333 0000
Salinity treatment 86333 2 43166 11038 0000
Time times Salinity treatment 27416 4 6854 1752 ns
Error 16425 42 3910
Total 6575 71
Number of
flowers of
Z mauritiana
Time 73506 2 36753 167777 0000
Salinity treatment 12133 2 6066 25061 0000
Time times Salinity treatment 27824 4 6956 28736 0000
Error 10166 42 242063
Total 127759 71
Fresh weight of
Shoot of
Z mauritiana
Time 3056862 2 1528431 340777 0000
Salinity treatment 107829 2 53914 12020 0000
Time times Salinity treatment 51303 4 12825 2859 0031
Error 251167 56 4485
Total 3515820 71
Dry weight of Shoot of
Z mauritiana
Time 784079 2 392039 338932 0000
Salinity treatment 26344 2 13172 11387 0000
Time times Salinity treatment 13042 4 3260 2818 0033
Error 64774 56 1156690
Total 913855 71
Succulence of
Z mauritiana
Time 0002 2 0001 0214 ns
Salinity treatment 0006 2 0003 0682 ns
Time times Salinity treatment 0007 4 0002 0406 ns
Error 0199 45 0004
Total 51705 54
Spacific shoot
length of Z mauritiana
Time 0000 2 914 0176 0000
Salinity treatment 0002 2 0001 2096 ns
Time times Salinity treatment 0003 4 0001 1445 ns
Error 0023 45 0001
Total 6413 54
Moisture
contents of Z mauritiana
Time 1264 2 0632 0243 ns
Salinity treatment 3603 2 1801 0691 ns
Time times Salinity treatment 4172 4 1043 0400 ns
Error 117146 45 2603
Total 131675 54
Relative growth
rate of Z mauritiana
Time 1584206 1 1584206 532968 ns
Salinity treatment 18921 2 9460 3183 ns
Time times Salinity treatment 61624 2 30812 10366 0000
Error 89172 30 2972
Total 4034 36
Appendix-X One way ANOVA for completely randomized design for growth and development of Z mauritiana in large size clay pot
being irrigated with water of two different sea salt concentration
Variables Source Sum of Squares df Mean Square F-value P
Chlorophyll a
of Z mauritiana
Salinity treatment 0004 2 0002 7546 0003
Error 0006 21 0000
Total 0010 23
Chlorophyll b of Z mauritiana
Salinity treatment 0037 2 0018 4892 0018
Error 0080 21 0003
Total 0117 23
171
Total
chlorophyll of
Z mauritiana
Salinity treatment 0144 2 0072 39317 0000
Error 0038 21 0002
Total 0182 23
Chlorophyll ab ratio of
Z mauritiana
Salinity treatment 1499 2 0749 33416 0000
Error 0471 21 0022
Total 1969 23
Total soluble
sugars of
Z mauritiana
Salinity treatment 378271 2 189135 36792 0000
Error 107952 21 5140
Total 486223 23
Total protein contents of
Z mauritiana
Salinity treatment 133006 2 66502 5861 0009
Error 238268 21 11346
Total 371274 23
Appendix-XI Three way ANOVA for split-split plot design for physiological investigations on growth of Z mauritiana and C cajan in
drum pot being irrigated with water of sea salt concentration at two irrigation intervals
Variables Source Sum of Squares df Mean Square F-value P
Height of
Z mauritiana
Time 4499 2 2249 28888 0004
Crop 448028 1 448028 2208 ns
Irrigation intervals 2523 1 2523 2774 ns
Time times Crop 928088 2 464044 2288 ns
Time times irrigation interval 1120400 2 560200 0615 ns
Crop times irrigation interval 2690151 1 2690 2957 ns
Time times Crop times irrigation interval 171927 2 85963 0094 ns
Error 10916 12 909732
Total 35
Canopy volume of Z mauritiana
Time 7943 2 3971 6554 ns
Crop 0382 1 0382 0579 ns
Irrigation intervals 0068 1 0069 0103 ns
Time times Crop 0265 2 0133 0201 ns
Time times irrigation interval 1142 2 0571 0852 ns
Crop times irrigation interval 0722 1 0722 1077 ns
Time times Crop times irrigation interval 1998 2 0999 1491 ns
Error 8043 12 0670
Total 29439 35
Appendix-XII Two way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C
cajan in drum pot being irrigated with water of sea salt concentration at two irrigation intervals
Variables Source Sum of Squares df Mean Square F-value P
Plant length of
Z mauritiana
Crop 2986 1 2986 75322 0000
Irrigation interval 2986 1 2986 75322 0000
Crop times Irrigation interval 15336 1 153367 3868 ns
Error 317166 8 39645
Total 292428 12
Shoot length of
Z mauritiana
Crop 1069741 1 1069741 30890 0000
Irrigation interval 1069741 1 1069741 30890 0000
Crop times Irrigation interval 253001 1 253001 73058 0026
Error 27704 8 3463
Total 103376 12
Root length of
Z mauritiana
Crop 19763 1 19763 2671 ns
Irrigation interval 481333 1 481333 65059 0000
Crop times Irrigation interval 800333 1 800333 108177 0000
Error 59186 8 7398
Total 49165 12
Main branches
of Z mauritiana
Crop 33333 1 33333 5797 0042
Irrigation interval 48 1 48 8347 0020
Crop times Irrigation interval 0333 1 0333 0057 ns
Error 46 8 575
Total 2888 12
Lateral
branches of Z mauritiana
Crop 1344083 1 1344083 41356 0000
Irrigation interval 54675 1 54675 16823 0000
Crop times Irrigation interval 784083 1 784083 24125 0000
Error 26 8 325
Total 22465 12
Leaf numbers of
Z mauritiana
Crop 22465 12 98283 96482 0000
Irrigation interval 25025 1 25025 24566 0001
Crop times Irrigation interval 11907 1 11907 11688 0009
Error 8149 8 1018667
172
Total 2037850 12
Shootroot ratio
of Z mauritiana
Crop 0027 1 0027 1842 ns
Irrigation interval 0001 1 0001 0097 ns
Crop times Irrigation interval 0825 1 0825 54909 0000
Error 0120 8 0015
Total 27776 12
Plant fresh
weight of Z mauritiana
Crop 398107 1 398107 577818 0000
Irrigation interval 139514 1 139514 20249 0000
Crop times Irrigation interval 146898 1 146898 21321 0000
Error 5511 8 688982
Total 7248659 12
Plant dry weight of Z mauritiana
Crop 87808 1 87808 471436 0000
Irrigation interval 57893 1 57893 31082 0000
Crop times Irrigation interval 61132 1 61132 32821 0000
Error 14900 8 186257
Total 1875710 12
Stem fresh
weight of
Z mauritiana
Crop 46687 1 46687 227539 0000
Irrigation interval 17933 1 17933 87402 0000
Crop times Irrigation interval 20180 1 20180 98351 0000
Error 16414 8 205185
Total 1718530 12
Root fresh weight of
Z mauritiana
Crop 58450 1 58450 2295 0000
Irrigation interval 42186 1 42186 165641 0000
Crop times Irrigation interval 37307 1 37307 146487 0000
Error 203746 8 25468
Total 357145 12
Leaf fresh weight of
Z mauritiana
Crop 29970 1 29970 19089 0000
Irrigation interval 117018 1 1170187 7453 0025
Crop times Irrigation interval 2310 1 2310 14714 0004
Error 125596 8 15699
Total 699711 12
Stem dry weight
of Z mauritiana
Crop 13587 1 13587 216591 0000
Irrigation interval 11856 1 11856 18899 0000
Crop times Irrigation interval 6787763 1 6787 108197 0000
Error 50188 8 62735
Total 4689795 12
Root dry weight
of Z mauritiana
Crop 1358787 1 13587 216591 0000
Irrigation interval 1497427 1 14974 118615 0000
Crop times Irrigation interval 128773 1 12877 1020052 0000
Error 100993 8 12624
Total 124421 12
Leaf dry weight
of Z mauritiana
Crop 2374 1 2374 135380 0000
Irrigation interval 8748 1 8748 4987 ns
Crop times Irrigation interval 26403 1 2640 150539 0000
Error 140313 8 17539
Total 127170 12
Plant moisture of Z mauritiana
Crop 22082 1 22082 5608 0045
Irrigation interval 38702 1 38702 9830 0013
Crop times Irrigation interval 44406 1 44406 11279 0009
Error 31496 8 3937
Total 29872 12
Stem moisture of Z mauritiana
Crop 0005 1 0005 0000 ns
Irrigation interval 110663 1 110663 12023 0008
Crop times Irrigation interval 0897 1 0897 0097 ns
Error 73633 8 9204
Total 28532 12
Root moisture of Z mauritiana
Crop 235266 1 235266 16502 0003
Irrigation interval 3923 1 3923 0275 ns
Crop times Irrigation interval 0856 1 0856 0060 ns
Error 114051 8 14256
Total 17572 12
Leaf moisture
of Z mauritiana
Crop 130413 1 130413 47746 0000
Irrigation interval 22256 1 22256 8148 0021
Crop times Irrigation interval 210662 1 210662 77127 0000
Error 21850 8 2731
Total 38888 12
173
Relative growth
rate of Z mauritiana
Crop 0000 1 0000 287467 0000
Irrigation interval 0000 1 0000 164217 0000
Crop times Irrigation interval 0000 1 0000 179626 0000
Error 0000 8 0000
Total 0009 12
Relative water
contents of Z
mauritiana
Crop 37381 1 37381 1380 ns
Irrigation interval 49871 1 49871 1841 ns
Crop times Irrigation interval 13496 1 13496 0498 ns
Error 216649 8 27081
Total 50855 12
Chlorophyll a of Z mauritiana
Crop 0103 1 0103 32466 0000
Irrigation interval 0003 1 0003 1075 ns
Crop times Irrigation interval 0000 1 0000 0187 ns
Error 0025 8 0003
Total 1498 12
Chlorophyll b
of Z mauritiana
Crop 0027 1 0027 196164 0000
Irrigation interval 0002 1 0002 15656 0004
Crop times Irrigation interval 0006 1 0006 45063 0000
Error 0001 8 0000
Total 0456 12
Total chlorophyll
of Z mauritiana
Crop 0257 1 0257 53469 0000
Irrigation interval 0001 1 0001 0315 ns
Crop times Irrigation interval 0002 1 0002 0442 ns
Error 0038 8 0004
Total 3736 12
Chlorophyll a b ratio of
Z mauritiana
Crop 0002 1 0002 0028 ns
Irrigation interval 0169 1 0169 1696 ns
Crop times Irrigation interval 1064 1 1064 10643 0011
Error 0799 8 0099
Total 43067 12
Carotenoids of
Z mauritiana
Crop 0018 1 0018 42747 0000
Irrigation interval 0002 1 0002 5298 0050
Crop times Irrigation interval 0003 1 0003 8118 0021
Error 0003 8 0000
Total 0451 12
Phenol of
Z mauritiana
Crop 24641 1 24641 13168 000
Irrigation interval 5078 1 5078 2714 ns
Crop times Irrigation interval 10339 1 10339 5525 0046
Error 14969 8 1871
Total 6289 12
Proline of Z mauritiana
Crop 0001 1 0001 52288 0000
Irrigation interval 0000 1 0000 6972 0029
Crop times Irrigation interval 0000 1 0000 0358 ns
Error 0000 8 0000
Total 0005 12
Protein of Z mauritiana
Crop 200001 1 200001 296 ns
Irrigation interval 69264 1 69264 102 ns
Crop times Irrigation interval 4453 1 4453 006 ns
Error 540367 8 67545
Total 814086 11
CAT enzyme of
Z mauritiana
Crop 74171 1 74171 11404 0009
Irrigation interval 299930 1 299930 46117 0000
Crop times Irrigation interval 15336 1 15336 2358 ns
Error 52029 8 65036
Total 441467 11
APX enzyme of
Z mauritiana
Crop 191918 1 191918 6693 0032
Irrigation interval 4665 1 4665 162723 0000
Crop times Irrigation interval 336912 1 336912 11750 0009
Error 229383 8 28672
Total 5423 11
GPX enzyme of
Z mauritiana
Crop 0000 1 0000 0020 ns
Irrigation interval 0103 1 0103 5893 0041
Crop times Irrigation interval 0109 1 0109 6220 0037
Error 0140 8 0017
Total 0353 11
SOD enzyme Crop 8471 1 8471 1364 ns
174
of
Z mauritiana
Irrigation interval 6220 1 6220 1001 ns
Crop times Irrigation interval 21142 1 21142 3405 ns
Error 49664 8 6208
Total 85498 11
NR enzyme of
Z mauritiana
Crop 7520 1 75208333333 37253364154 0003
Irrigation interval 1360 1 1360 6737 0318
Crop times Irrigation interval 0016 1 0016 0079 ns
Error 1615 8 0201
Total 10512 11
Nitrate of
Z mauritiana
Crop 003 1 003 3028 ns
Irrigation interval 0018 1 0018 1831 ns
Crop times Irrigation interval 0003 1 0003 0336 ns
Error 0079 8 0009
Total 0130 11
Appendix-XIII Three way ANOVA for split-split design for physiological investigations on growth of Z mauritiana and C cajan in drum
pot being irrigated with water of sea salt concentration at two irrigation intervals
Variables Source Sum of Squares df Mean Square F-value P
Height of
C cajan
Time 14990 2 7495 235059 0000
Crop 7848 1 7848 42235 0000
Irrigation intervals 749056 1 749056 9676 0009
Time times Crop 2638 2 1319140 7098 00262
Time times irrigation interval 309932 2 154966 2001 ns
Crop times irrigation interval 9127 1 9127 0117 ns
Time times Crop times irrigation interval 31974 2 15987 0206 ns
Error 928935 12 77411
Total 29065 35
Apendix-XIV Two way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C
cajan in drum pot being irrigated with water of sea salt concentration at two irrigation intervals
Variables Source Sum of Squares df Mean Square F-value P
Plant length of C cajan
Crop 1056563 1 1056563 12331 0007
Irrigation interval 21675 1 21675 2529 ns
Crop times Irrigation interval 137363 1 137363 1603 ns
Error 68544 8 8568
Total 334030 12
Shoot length of C cajan
Crop 808520 1 808520 36580 0000
Irrigation interval 165020 1 165020 7466 0025
Crop times Irrigation interval 285187 1 285187 12902 0007
Error 17682 8 22102
Total 224013 12
Root length of C cajan
Crop 16567 1 16567 0674 ns
Irrigation interval 3520 1 3520 0143 ns
Crop times Irrigation interval 26700 1 26700 1087 ns
Error 196453 8 24556
Total 11133 12
Main branches
of C cajan
Crop 80083 1 80083 64066 0000
Irrigation interval 10083 1 10083 8066 0021
Crop times Irrigation interval 075 1 075 06 ns
Error 10 8 125
Total 335 12
Letral branches
of C cajan
Crop 0 1 0
Irrigation interval 0 1 0
Crop times Irrigation interval 0 1 0
Error 0 8 0
Total 0 12
Leaf numbers
of C cajan
Crop 1776333 1 1776333 16679 0003
Irrigation interval 972 1 972 9126 0016
Crop times Irrigation interval 176333 1 17633 1655 0234
Error 852 8 1065
Total 22342 12
Shootroot ratio of C cajan
Crop 0385 1 0385 0638 0447
Irrigation interval 0007 1 0007 0011 0916
Crop times Irrigation interval 2669 1 2669 4424 0068
Error 4825 8 0603
Total 264061 12
Crop 76816 1 76816 7494853 0025
175
Plant fresh
weight of
C cajan
Irrigation interval 730236 1 730236 7124832 0028
Crop times Irrigation interval 266869 1 266869 2603812 0145
Error 81993 8 102491
Total 25941 12
Plant dry weight of C cajan
Crop 38270 1 38270 1150145 0009
Irrigation interval 53046 1 53046 15942 0003
Crop times Irrigation interval 20202 1 20202 6071 0039
Error 26619 8 3327
Total 4150 12
Stem fresh weight of
C cajan
Crop 16100 1 16100 1462 ns
Irrigation interval 9900 1 9900 0899 ns
Crop times Irrigation interval 00675 1 0067 0006 ns
Error 8806 8 11007
Total 3318 12
Root fresh weight of
C cajan
Crop 0190 1 0190 0248 ns
Irrigation interval 27331 1 27331 35753 0000
Crop times Irrigation interval 2698 1 2698 3529 0097
Error 6115 8 0764
Total 432050 12
Leaf fresh
weight of C cajan
Crop 541363 1 541363 13825 0005
Irrigation interval 347763 1 347763 8881 0017
Crop times Irrigation interval 208333 1 208333 5320 0049
Error 313246 8 39155
Total 7236 12
Stem dry weight
of C cajan
Crop 10323 1 10323 11530 0009
Irrigation interval 0452 1 0452 0505 ns
Crop times Irrigation interval 0232 1 0232 0259 ns
Error 7162 8 0895
Total 125151 12
Root dry weight
of C cajan
Crop 0007 1 0007 012 ns
Irrigation interval 0607 1 0607 972 0014
Crop times Irrigation interval 0367 1 0367 588 0041
Error 05 8 0062
Total 3515 12
Leaf dry weight
of C cajan
Crop 9363 1 9363 15649 0004
Irrigation interval 34003 1 3400 5683 0000
Crop times Irrigation interval 11603 1 11603 19392 0002
Error 4786 8 0598
Total 95072 12
Plant moisture of C cajan
Crop 199182 1 19918 6011 0039
Irrigation interval 272215 1 27221 8215 0020
Crop times Irrigation interval 76654 1 76654 2313 0166755
Error 265079 8 33134
Total 38272 12
Stem moisture
of C cajan
Crop 100814 1 10081 3290 0107246
Irrigation interval 53460 1 53460 1744 0223065
Crop times Irrigation interval 19778 1 1977 0645 0444938
Error 245119 8 30639
Total 31036 12
Root moisture
of C cajan
Crop 26266 1 26266 1389 ns
Irrigation interval 223809 1 223809 11836 0008
Crop times Irrigation interval 0097 1 0097 0005 ns
Error 151272 8 18909
Total 58346 12
Leaf moisture
of C cajan
Crop 2623 1 2623 39350 0000
Irrigation interval 1765 1 1765 26477 0000
Crop times Irrigation interval 1425 1 1425452 21378 0001
Error 533411 8 66676
Total 36263 12
Relative growth
rate of C cajan
Crop 0000 1 0000 17924 0002
Irrigation interval 0000 1 0000 21296 0001
Crop times Irrigation interval 0000 1 0000 88141 0017
Error 0000 8 0000
Total
Crop 256935 1 256935 1560 ns
Irrigation interval 268827 1 26882 1633 ns
176
Electrolyte
leakage of C
cajan
Crop times Irrigation interval 30379 1 30379 0184 ns
Error 1316923 8 16461
Total 50381 12
Chlorophyll a
of C cajan
Crop 0101 1 0101 7957 0022
Irrigation interval 0062 1 0062 4893 ns
Crop times Irrigation interval 0199 1 0199 15600 0004
Error 0102 8 0012
Total 5060 12
Chlorophyll b
of C cajan
Crop 0017 1 0017 7758 0023
Irrigation interval 0027 1 0027 12389 0007
Crop times Irrigation interval 0056 1 0056 25313 0001
Error 0017 8 0002
Total 1727 12
Total
chlorophyll of C cajan
Crop 0178 1 0178 14819 0004
Irrigation interval 0198 1 0198 16520 0003
Crop times Irrigation interval 0509 1 0509 42379 0000
Error 0096 8 0012
Total 13217 12
Chlorophyll a b
ratio of C cajan
Crop 0065 1 0065 0691 ns
Irrigation interval 0033 1 0033 0357 ns
Crop times Irrigation interval 0016 1 0016 0173 ns
Error 0756 8 0094
Total 35143 12
Carotenoids of C cajan
Crop 0021 1 0021 19599 0002
Irrigation interval 0028 1 0028 26616 0000
Crop times Irrigation interval 0041 1 0041 38531 0000
Error 0008 8 0001
Total 1443 12
Phenol of C cajan
Crop 0799 1 0799 3171 ns
Irrigation interval 0040 1 0040 0159 ns
Crop times Irrigation interval 0911 1 0911 3617 ns
Error 2016 8 0252
Total 970313 12
Proline of C cajan
Crop 0008 1 0008 14867 0004
Irrigation interval 0019 1 0019 34536 0000
Crop times Irrigation interval 0008 1 0008 14969 0004
Error 0004 8 0000
Total 0155 12
Protein of C
cajan
Crop 116376 1 116376 3990 ns
Irrigation interval 434523 1 434524 14899 0048
Crop times Irrigation interval 33166 1 33166 1137 ns
Error 233303 8 29163
Total 817371 11
CAT enzyme
of C cajan
Crop 0249 1 0249 0121 ns
Irrigation interval 2803 1 2803 13702 ns
Crop times Irrigation interval 92392 1 9239 4517 ns
Error 16362 8 2045
Total 28654 11
APX enzyme
of C cajan
Crop 855939 1 855939 4073 ns
Irrigation interval 1078226 1 1078226 5130 ns
Crop times Irrigation interval 13522 1 13522 64349 000
Error 1681112 8 210139
Total 17137 11
GPX enzyme
of C cajan
Crop 0965 1 0965 9265 0160
Irrigation interval 1167 1 1167 11195 0101
Crop times Irrigation interval 0887 1 0887 8514 0194
Error 0833 8 0104
Total 3854 11
SOD enzyme
of C cajan
Crop 4125 1 4125 9731 0142
Irrigation interval 4865 1 4865 11477 0095
Crop times Irrigation interval 20421 1 20421 48172 0001
Error 3391 8 0423
Total 32804 11
Nitrate
reductase
enzyme
Crop 0053 1 0053 0034 ns
Irrigation interval 0001 1 0001 0000 ns
Crop times Irrigation interval 10329 1 10329 6650 0327
177
of C cajan Error 12424 8 1553
Total 22808 11
Nitrate of
C cajan
Crop 0039 1 0039 0576 ns
Irrigation interval 0083 1 0083 1222 ns
Crop times Irrigation interval 0003 1 0003 0005 ns
Error 0545 8 0068
Total 0668 11
Appendix-XV Two way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C
cajan intercropped on marginal land under field condition
Variables Source Sum of Squares df Mean Square F-value P
Height of Z mauritiana
Time 79704 3 26568 77303 0000
Treatment 979209 1 979209 4702 0455
Time times Treatment 756019 3 252006 1210 3381 ns
Error 3332 16 208259
Total 90366 39
Canopy volume of Z mauritiana
Time 1049 3 3498 115444 0000
Treatment 3509 1 3509 5966 0266
Time times Treatment 3374 3 1124 1911 1684 ns
Error 9413 16 5883
Total 1284 39
flowers numbers of Z
mauritiana
Time 1794893 3 598297 770043 0000
Treatment 19980 1 19980 10152 0057
Time times Treatment 21017 3 7005 3559 0381
Error 31488 16 1968
Total 1882468 39
Fruits numbers
of Z mauritiana
Time 324096 3 108032 297941 0000
Treatment 10824 1 10824 64081 0000
Time times Treatment 7141 3 2380 14093 0001
Error 2702 16 168913
Total 351833 39
Appendix-XVI One way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C cajan intercropped on marginal land under field condition
Variables Source Sum of Squares df Mean Square F-value P
Weight of ten
fruits (FW) of
Z mauritiana
Treatment 557113 1 557113 6663 0032
Error 668923 8 83615
Total 1226036 9
Weight of ten fruits (DW) of
Z mauritiana
Treatment 4356 1 4356 0321 ns
Error 10862 8 13577
Total 112976 9
diameter of fruit of Zmauritiana
Treatment 0534 1 0534 0946 ns
Error 4514 8 0564
Total 5048 9
Fruit weight per plant of
Z mauritiana
Treatment 0739 1 0739 4022 ns
Error 1471 8 0184
Total 2211 9
Fruit sugar
(soluble) of
Z mauritiana
Treatment 5041 1 5041 0081 ns
Error 497328 8 62166
Total 502369 9
Fruit sugar (extractable) of
Z mauritiana
Treatment 32041 1 32041 0424 ns
Error 604384 8 75548
Total 636425 9
Total fruit
sugars of Z mauritiana
Treatment 16 1 16 0780 ns
Error 164 8 205
Total 18 9
Chlorophyll a of
Z mauritiana
Treatment 0082 1 0082 1384 0020
Error 0024 4 0006
Total 0105 5
Chlorophyll b
of Z mauritiana
Treatment 0011 1 0011 8469 0043
Error 0005 4 0001
Total 0016 5
Total chlorophyll of
Z mauritiana
Treatment 0152 1 0152 11927 0025
Error 0051 4 0013
Total 0203 5
Treatment 0015 1 0015 0867 ns
Error 0067 4 0017
178
Chlorophyll a b
ratio of Z mauritiana
Total 0082 5
Carotinoids of Z mauritiana
Treatment 0011 1 0011 9719 0035
Error 0004 4 0001
Total 0015 5
Leaf protein of
Z mauritiana
Treatment 0106 1 0106 4 ns
Error 0106 4 0027
Total 0213 5
Leaf sugars
(soluble) of
Z mauritiana
Treatment 054 1 054 0025 ns
Error 848 4 212
Total 8534 5
Leaf sugars
(Extractable) of Z mauritiana
Treatment 486 1 486 8055 0046
Error 2413 4 0603
Total 7273 5
Total sugars in
leaf of Z
mauritiana
Treatment 216 1 216 0104 ns
Error 83333 4 20833
Total 85493 5
Leaf phenols of
Z mauritiana
Treatment 8166 1 8166 5665 ns
Error 5766 4 1442
Total 13933 5
Leaf nitrogen of Z mauritiana
Treatment 15 1 15 1939 ns
Error 3093 4 0773333
Total 4593 5
Soil nitrogen of
Z mauritiana
Treatment 0375 1 0375 21634 ns
Error 0693 4 0173
Total 1069 5
Appendix-XVII Two way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C
cajan intercropped on marginal land under field condition
Variables Source Sum of Squares df Mean Square F-value P
Height of Ccajan
Time 700196 2 350098 2716 0000
Treatment 594405 1 594405 16017 0000
Time times Treatment 488829 2 244415 6586 0004
Error 1001996 27 37111
Total 705495 59
Number of branches of
Ccajan
Time 8353 2 4176 1050050 0000
Treatment 24066 1 24066 18672 0000
Time times Treatment 24133 2 12066 9362 0000
Error 348 27 1288
Total 8572 59
Number of flowers of
Ccajan
Time 289297 2 144648 301277 0000
Treatment 365066 1 365066 0701 ns
Time times Treatment 730133 2 365066 0701 ns
Error 14059 27 520733
Total 317415 59
Number of pods
of Ccajan
Time 347682 2 173841 70559 0000
Treatment 159135 1 159135 1558 ns
Time times Treatment 8167 2 40835 0399 ns
Error 27574 27 1021276
Total 447407 59
Appendix-XVIII One way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C
cajan intercropped on marginal land under field condition
Variables Source Sum of Squares df Mean Square F-value P
Shoot weight
(FW) of
Ccajan
Treatment 0 1 0 0 ns
Error 87444 4 21861
Total 87444 5
Shoot weight
(RW) of Ccajan
Treatment 0 1 0 0 ns
Error 13808 4 3452
Total 13808 5
Number of
seeds of
Ccajan
Treatment 245 1 245 0005 ns
Error 940182 18 52232
Total 940427 19
Weight of seeds
of Ccajan
Treatment 02 1 02 0000 ns
Error 7585 18 421406
Total 7585 19
179
Chlorophyll a of
Ccajan
Treatment 0001 1 0001 5442 ns
Error 0001 4 0000
Total 0002 5
Chlorophyll b
of Ccajan
Treatment 0006 1 0006 9079 0039
Error 0002 4 0001
Total 0008 5
Total
chlorophyll of
Ccajan
Treatment 0017 1 0017 51558 0001
Error 0001 4 0000
Total 0019 5
Chlorophyll a b ratio of
Ccajan
Treatment 0183 1 0183 5532 ns
Error 0132 4 0033
Total 0316 5
Leaf protein of Ccajan
Treatment 0001 1 0001 0017 ns
Error 0228 4 0057
Total 0228 5
Leaf sugars of
Ccajan
Treatment 0015 1 0015 0003 ns
Error 1624 4 406
Total 16255 5
Leaf phenols of
Ccajan
Treatment 0201 1 0201 0140 ns
Error 5746 4 1436
Total 5948 5
Leaf nitrogen
of Ccajan
Treatment 1306 1 1306 3062 ns
Error 1706 4 04266
Total 3013 5
Appendix-XIX Two way ANOVA for completely randomized design for investigations on determining range of salt tolerance in Carissa
carandas
Variables Source Sum of Squares df Mean Square F-value P
Height of C carandas
Time 72042 5 14408 55957 0000
Salinity treatment 49345 2 24672 196775 0000
Time times Salinity treatment 16679 10 1667920 13302 000
Error 3009 24 125385
Total 143777 53
Volume of
canopy of
C carandas
Time 3329 4 0832 38126 000
Salinity treatment 1393 2 0696 67129 000
Time times Salinity treatment 0813 8 0102 9792 000
Error 0207 20 0010
Total 5969 44
Appendix-XX One way ANOVA for completely randomized design for investigations on determining range of salt tolerance in Carissa carandas
Variables Source Sum of Squares df Mean Square F-value P
Number of
flowers of C carandas
Salinity treatment 10288 2 5144194 1342937 0000
Error 229833 6 38305
Total 10518 8
Number of fruits of
C carandas
Salinity treatment 18000 2 9000 268215 0000
Error 201333 6 33555
Total 18201 8
Flower shedding
percentage of C carandas
Salinity treatment 1541647 2 770823 53455 0000
Error 86519 6 144199
Total 1628166 8
Weight of ten fruits (FW) of
C carandas
Salinity treatment 82632 2 41316 187678 0000
Error 1321 6 0220
Total 83953 8
Weight of ten
fruits (DW) of
C carandas
Salinity treatment 4355 2 2177 13753 0005
Error 095 6 0158
Total 5305 8
Fruits per plant
(FW) of
C carandas
Salinity treatment 133127 2 66563 278148 0000
Error 1435861 6 239310
Total 134563 8
Fruits per plant
(DW) of C carandas
Salinity treatment 8782 2 439117 117790 0000
Error 223677 6 37279
Total 9006 8
Size of fruits of C carandas
Salinity treatment 1301 2 0651 4125 ns
Error 0946 6 0158
Total 2248 8
Salinity treatment 5607 2 2804 17592 0003
180
Diameter of fruit
of C carandas
Error 0956 6 0159
Total 6563 8
Chlorophyll a of C carandas
Salinity treatment 0112 2 0056 119786 0000
Error 0003 6 0000
Total 0115 8
Chlorophyll b of
C carandas
Salinity treatment 0005 2 0002 434 0000
Error 0000 6 0000
Total 0005 8
Total chlorophyll of C carandas
Salinity treatment 0159 2 0079 104188 0000
Error 0005 6 0001
Total 0164 8
Chlorophyll a b
ratio of C carandas
Salinity treatment 9661 2 4831 324691 0000
Error 0089 6 0015
Total 9751 8
Carotenoids of C carandas
Salinity treatment 0029 2 0014 28822 0000
Error 0003 6 0001
Total 0032 8
Leaf Protein of
C carandas
Salinity treatment 2722 2 1361 98 0012
Error 0833 6 0138
Total 3555 8
Soluble sugar of
C carandas
Salinity treatment 234889 2 117444 12735 0006
Error 55333 6 9222
Total 290222 8
In soluble sugars
of C carandas
Salinity treatment 595395 2 297698 39094 0000
Error 45689 6 7615
Total 641085 8
Total sugar of
C carandas
Salinity treatment 1576898 2 788448 39201 0000
Error 120676 6 20113
Total 1697574 8
Phenols of C carandas
Salinity treatment 14675 2 7338 74202 0000
Error 0593 6 0099
Total 15268 8
Leaf Na+ of
C carandas
Salinity treatment 1346 2 673 673 0000
Error 6 6 1
Total 1352 8
Leaf K+ of C carandas
Salinity treatment 798 2 399 133 0000
Error 18 6 3
Total 816 8
Leaf K+ Na+
ratio of C carandas
Salinity treatment 0305 2 0153 654333 0000
Error 0001 6 0000
Total 0307 8
181
7 Publications
v
DEDICATED TO MY FAMILY
MUHAMMAD HANIF (MY FATHER)
MRS ARIFA (LATE)
(MY BELOVED MOTHER)
SHAHEEN TAYYAB (MY WIFE)
vi
ACKNOWLEDGMENTS
All the praises for almighty Allah and all respects for Prophet Muhammad (Peace be Upon
Him) who has shown me the straight path
I am grateful to my supervisor Prof Dr Rafiq Ahmad for his keen interest
patronage and guidance during this research work which made successful submission of
this thesis
I also obliged to Prof Dr Ehtesham Ul Haque and Prof Dr Javed Zaki (Present
and Former Chairmen Department of Botany respectively) for providing me all the
necessary facilities and administrative support
Being employed as lecturer in Department of Botany Govt Islamia Science
College Karachi I am also thankful to Education and literacy Department Govt of Sindh
(Pakistan) for providing me facilities to perform this study
Thanks are due to Dr D Khan in assessing statistical data analysis and colleague
of Biosaline lab Dr M Azeem Dr Naeem Ahmed and M Wajahat Ali Khan for their
cooperation throughout the course of study
I am also gratefully acknowledged to Mr Noushad Raheem and Mr Noor Uddin
of Fiesta Water Park for providing field plot and facilities to perform this study I am also
thankful to Pakistan Metrological Department for providing environmental data
I am also obliged to Dr M Qasim and Dr M Waseem Abbasi for their suggestions
and support in writing this thesis
Assistance of Abbul Hassan (Lab attendant) Tajwar Khan (Biosaline field
Attendant) and Mr Wahid (Plant Physiology Lab Assistant) is also acknowledged
Thanks are also due to my friends Dr Rafat Saeed Dr Kabir Ahmad Dr Zia Ur
Rehman Farooqi Dr Noor Dr M Yousuf Adnan Asif Bashir Dr A Rauf A Hai Faiz
Ahmed MA Rasheed Jallal Uddin Saadi Ahsan Shaikh Saima Fehmi A Mubeen
Khan Dr Noor Ul Haq Saima Ahmad S Safder Raza SM Akber and my college
colleagues for giving me encouragement during this research work
vii
I can never forget the support and encouragement and good wishes of Mr M
Wilayat Ali Khan Mrs Shahnaz Rukhsana Mr Mansoor Mrs Rabia Mansoor Mrs
Chand Bibi and Mrs Saeeda Anwar
In the last I am highly grateful to my beloved father Muhammad Hanif my loving
mother Arifa (when she alive) my caring wife Shaheen and sweet childrenrsquos Sara and
Sarim my supportive brothers and sisters and all family members for their prayers love
sacrifices and encouragements provided during course of this research work
viii
TABLE OF CONTENTS
No Title Page no
Acknowledgement vi
Summary xix
Urdu translation of summary xxi
General introduction 1
Layout of thesis 11
1 Chapter 1 13
11 Introduction 13
12 Experiment No 1 15
121 Materials and methods 15
1211 Seed collection 15
1212 Experimental Design 15
122 Observations and Results 17
13 Experiment No 2 22
131 Materials and methods 22
1311 Seed germination 22
132 Observations and Results 23
14 Experiment No 3 28
141 Materials and methods 28
1411 Seedling establishment 28
142 Observations and Results 29
1421 Seedling establishment 29
1422 Shoot height 29
15 Experiment No 4 31
151 Materials and methods 31
1511 Drum pot culture 31
1512 Experimental design 31
1513 Vegetative and Reproductive growth 32
1514 Analysis on some biochemical parameters 32
152 Observations and Results 34
1521 Vegetative and Reproductive growth 34
ix
No Title Page no
1522 Study on some biochemical parameters 34
16 Experiment No 5 41
161 Materials and methods 41
1611 Isolation Identification and purification of bacteria 41
1612 Preparation of bacterial cell suspension 41
1613 Study of salt tolerance of Rhizobium isolated from root
nodules of C cajan
41
162 Observations and Results 42
17 Experiment No 6 44
171 Materials and methods 44
1711 Experimental design 44
1712 Vegetative and reproductive growth 45
1713 Analysis on some biochemical parameters 45
172 Observations and Results 46
1721 Vegetative and Reproductive growth 46
1722 Study on some biochemical parameters 46
18 Discussion (Chapter 1) 51
2 Chapter 2 59
21 Introduction 59
22 Experiment No 7 60
221 Materials and Methods 60
2211 Growth and Development 60
2212 Drum pot culture 60
2213 Experimental Design 60
2214 Irrigation Intervals 61
2215 Estimation of Nitrate content 62
2216 Relative Water content (RWC) 62
2217 Electrolyte leakage percentage (EL) 62
2218 Photosynthetic pigments 63
2219 Total soluble sugars 63
22110 Proline content 63
22111 Soluble phenols 64
x
No Title Page no
22112 Total soluble proteins 64
22113 Enzymes Assay 64
222 Observations and Results 67
2221 Vegetative growth 67
2222 Photosynthetic pigments 70
2223 Electrolyte leakage percentage (EL) 70
2224 Phenols 70
2225 Proline 71
2226 Protein and sugars 71
2227 Enzyme essays 71
2228 Vegetative growth 73
2229 Photosynthetic pigments 75
22210 Electrolyte leakage percentage (EL) 76
22211 Phenols 76
22212 Proline 77
22213 Protein and Sugars 77
22214 Enzyme assay 77
23 Experiment No8 90
231 Materials and Methods 90
2311 Selection of plants 90
2312 Experimental field 90
2313 Soil analysis 90
2314 Experimental design 91
2315 Vegetative and reproductive growth 93
2316 Analysis on some biochemical parameters 93
2317 Fruit analysis 94
2318 Nitrogen estimation 94
2319 Land equivalent ratio and Land equivalent coefficient 95
23110 Statistical analysis 95
232 Observations and Results 96
2321 Vegetative parameters 96
2322 Reproductive parameters 96
xi
No Title Page no
2323 Study on some biochemical parameters 97
2324 Nitrogen Contents 98
2325 Land equivalent ratio land equivalent coefficient 98
24 Discussion (Chapter 2) 108
3 Chapter 3 113
31 Introduction 113
32 Experiment No 9 114
321 Materials and methods 114
3211 Drum Pot Culture 114
3212 Plant material 114
3213 Experimental setup 114
3214 Vegetative parameters 115
3215 Analysis on some biochemical parameters 115
3216 Mineral Analysis 116
322 Observations and Result 117
3221 Vegetative parameters 117
3222 Reproductive parameters 117
3223 Study on some biochemical parameters 118
3224 Mineral analysis 118
33 Discussion (Chapter 3) 127
4 Conclusion 129
5 References 130
6 Appendices 168
7 Publications 181
xii
LIST OF FIGURES
Figure Title Page no
11 Effect of irrigation water of different sea salt solutions on seed
germination indices of C cajan
27
12 Effect of irrigating water of different sea salt solutions on
seedling emergence (A) and shoot length (B) of C cajan
30
13 Environmental data of study area during experimental period
(July-November 2009)
36
14 Effect of salinity using irrigation water of different sea salt
concentrations on height of C cajan during 18 weeks treatment
36
15 Effect of salinity using irrigation water of different sea salt
concentrations on initial and final biomass (fresh and dry) of C
cajan
37
16 Percent change in moisture succulence relative growth rate
(RGR) and specific shoot length (SSL) of C cajan under
increasing salinity using irrigating water of different sea salt
concentrations
37
17 Effect of irrigating water of different sea salt solutions on
reproductive growth parameters including number of flowers
pod seeds and seed weight of C cajan
38
18 Effect of irrigating water of different sea salt solutions on leaf
pigments including chlorophyll a chlorophyll b total
chlorophyll and carotenoids of C cajan
39
19 Effect of irrigating water of different sea salt solutions on total
proteins soluble insoluble and total sugars in leaves of C cajan
40
110 Growth of nitrogen fixing bacteria associated with root of C
cajan under different NaCl concentrations
42
111 Photographs showing growth of Rhizobium isolated from the
nodules of C cajan in vitro on YEM agar supplemented with
different concentrations of NaCl
43
xiii
Figure Title Page no
112 Effect of salinity using irrigation water of different sea salt
concentrations on height number of branches fresh weight and
dry weight of shoot of Z mauritiana after 60 and 120 days of
treatment
47
113 Effect of salinity using irrigation water of different sea salt
concentrations on succulence specific shoot length (SSL)
moisture and relative growth rate (RGR) of Z mauritiana
48
114 Effect of salinity using irrigation water of different sea salt
concentrations on number of flowers of Z mauritiana
49
115 Effect of salinity using irrigation water of different sea salt
concentrations on leaf pigments including chlorophyll a
chlorophyll b total chlorophyll and chlorophyll ab ratio of Z
mauritiana
49
116 Effect of salinity using irrigation water of different sea salt
concentrations on total sugars and protein in leaves of Z
mauritiana
50
21 Vegetative parameters of Z mauritiana and C cajan at grand
period of growth under sole and intercropping system at two
irrigation intervals
79
22 Fresh and dry weight of Z mauritiana and C cajan plants under
sole and intercropping system at 4th and 8th day irrigation
intervals
80
23 Leaf weight ratio (LWR) root weight ratio (RWR) shoot weight
ratio (SWR)specific shoot length (SSL) specific root length
(SRL) plant moisture Succulence and relative growth rate
(RGR) of Z mauritiana and C cajan grow plants under sole and
intercropping system at 4th and 8th day irrigation intervals
81
24 Leaf pigments of Z mauritiana and C cajan grow plants under
sole and intercropping system at 4th and 8th day irrigation
intervals
83
xiv
Figure Title Page no
25 Electrolyte leakage phenols and proline of Z mauritiana and C
cajan at grand period of growth plants under sole and
intercropping system at 4th and 8th day irrigation intervals
84
26 Total protein in leaves of Z mauritiana and C cajan plants
under sole and intercropping system at 4th and 8th day irrigation
intervals
86
27 Enzymes activities in leaves of Z mauritiana and C cajan plants
under sole and intercropping system at 4th and 8th day irrigation
intervals
87
28 Nitrate reductase activity and nitrate concentration in leaves of
Z mauritiana and C cajan plants under sole and intercropping
system at 4th and 8th day irrigation intervals
89
29 Soil texture triangle (Source USDA soil classification) 99
210 Vegetative growth of Z mauritiana and C cajan growing under
sole and intercropping system
100
211 Reproductive growth of Z mauritiana and C cajan growing
under sole and intercropping system
101
212 Leaf pigments of Z mauritiana and C cajan growing under sole
and intercropping
102
213 Sugars protein and phenols in leaves of Z mauritiana and C
cajan at grand period of growth under sole and intercropping
system
103
214 Sugars protein and phenols in fruits of Z mauritiana grown
under sole and intercropping system
105
215 Nitrogen in leaves and in soil of Z mauritiana and C cajan
growing under sole and intercrop system
106
31 Chlorophyll a chlorophyll b total chlorophyll chlorophyll a b
ratio carotenoids contents of C carandas growing under
salinities created by irrigation of different dilutions of sea salt
124
xv
Figure Title Page no
32 Total protein sugars and phenolic contents of C carandas
growing under salinities created by irrigation of different
dilutions of sea salt
125
33 Mineral analysis including Na and K ions was done on leaves of
C carandas growing under salinities created by irrigation of
different dilutions of sea salt
126
xvi
LIST OF TABLES
Table Title Page no
11 Electrical conductivities of different sea salt solutions
used in germination of C cajan
18
12 Effect of irrigation water of different sea salt solutions
on germination percentage (GP) per day of C cajan
seeds pre-soaked in non-saline water prior to
germination with duration of time under various salinity
regimes
19
13 Effect of irrigation water of different sea salt solutions
on germination rate (GR) per day of seeds C cajan pre-
soaked in non-saline water prior to germination with
duration of time under various salinity regimes
20
14 Effect of irrigation water of different sea salt solutions
on mean germination rate (GR) coefficient of
germination velocity (GV) mean germination time
(GT) mean germination index (GI) and final
germination (FG) of C cajan seeds pre-soaked in non-
saline water prior to germination under various salinity
regimes
21
15 Electrical conductivities of different sea salt solutions
used in germination of C cajan
24
16 Effect of irrigation water of different sea salt solutions
on germination percentage (GP) per day of C cajan
seeds pre-soaked in respective sea salt concentrations
with duration of time
25
17 Effect of irrigation water of different sea salt solutions
on germination rate (GR) per day of C cajan seeds pre-
soaked in respective sea salt concentrations with
duration of time
26
xvii
Table Title Page no
18 Electrical conductivities of different Sea salt
concentrations and ECe of soil saturated paste at the end
of experiment
30
21 Soil analysis data of Fiesta Water Park experimental
field
99
22 Land equivalent ratio (LER) and Land equivalent
coefficient (LEC) with reference to height chlorophyll
and yield of Z mauritiana and C cajan growing under
sole and intercropping system
107
31 Electrical conductivities of different sea salt
concentration used for determining their effect on
growth of C carandas
119
32 Vegetative growth in terms of height and volume of
canopy of C carandas growing under salinities created
by irrigation of different dilutions of sea salt
120
33 Vegetative growth in terms of height and volume of
canopy of C carandas growing under salinities created
by irrigation of different dilutions of sea salt
121
34 Reproductive growth in terms of flowers and fruits
numbers flower shedding percentage fresh and dry
weight of ten fruit and their totals per plant fruit length
and diameter of C carandas growing under salinities
created by irrigation of different dilutions of sea salt
123
xviii
LIST OF ABBREVIATIONS
APX Ascorbate peroxidase
CAT Catalase
DAP Diammonium Phosphate (fertilizer)
dSm-1 Deci Siemens per meter
ECe Electrical conductivity of the Soil saturated extract
ECiw Electrical conductivity of the irrigation water
GPX Guaiacol Peroxidase
GR Glutathione reductase
GSH Reduced glutathione
LEC Land equivalent coefficient
LER Land equivalent ratio
NPK Nitrogen Phosphate Potash (fertilizer)
NR Nitrate reductase
RGR Relative growth rate
ROS Reactive oxygen species
RWR Root weight ratio
SOD Superoxide dismutase
SRL Specific Root Length
SSL Specific Shoot Length
SWR Shoot weight ratio
xix
Summary
Salinity is a growing threat to crop production which affects sustainability of agriculture
in aridsemiarid areas Growth responses of plant to salinity vary considerably among
species Cajanus cajan Ziziphus mauritiana and Carissa carandas are sub-tropical crops
grown worldwide particularly in Asian subcontinent for edible and fodder purposes but
not much is known about their salinity tolerance and intercropping
Effect of salinity has been initially studied in present work at germination of C cajan
under different sea salt salinities using presoaked seeds with water and respective salt
solutions Seed germination decreased with increasing salinity and it was more sever in
presoaking under water of different salinities The 50 threshold reduction started at
ECiw= 35 dSm-1 sea salt in presoaking treatments However this threshold was decreased
up to ECiw= 168 dSm-1 sea salt at further seedling establishment stage Growth experiment
of C cajan in drum pot culture (Lysimeter) also showed a salt induced growth reduction
in which plant tolerate salinity up to 42 dSm-1 At this salinity leaf pigments (chlorophylls
and carotenoids) proteins and insoluble sugars decreased up to 50 whereas soluble
sugars were increased (~25) Reproductive growth was also affected at this salinity in
which at least 70 reduction in flowers pods and seeds were observed
Salt tolerance of symbiotic nitrogen fixing bacteria associated with root of C cajan
showed salinity tolerance up to ECw= 366 dSm-1 NaCl salinity invitro environment For
intercropping experiments Ziziphus mauritiana (grafted variety) was selected with C
cajan Preliminary investigations showed a growth promotion in Z mauritiana at low
salinity (ECe= 72 dSm-1) and growth was remained unaffected up to ECe= 111 dSm-1
Intercropping of C cajan with Z mauritiana was primarily done in drum pot (Lysimeter)
culture Result showed better growth responses of both species when growing together as
intercrops than sole in which encouraging results were found in 8th day irrigation interval
rather than of 4th day Biochemical parameters eg photosynthetic pigments protein
phenols electrolyte leakage and sugars of these species displayed increase or decrease
according to their growth responses Increased activity of antioxidant enzymes and that of
nitrate reductase and its substrate (NO3) also contributed in enhancement of growth
Field experiment of intercropping of above mentioned plants at marginal land
irrigated with underground water (Eciw= 28 dSm-1) showed better vegetative growth of
xx
both species than sole crop The overall reproductive growth remained unaffected
although the numbers size and weight of fruit were better in intercropping system
Photosynthetic pigments were mostly increased whereas leaf protein and sugars remained
unchanged In addition higher values of LER and LEC (gt 1) indicated the success of
intercropping system
Experiment on salinity tolerance of Carissa carandas (varn karonda) using drum
pots culture showed improvement at low salinity (up to ECiw= 42 dSm-1 sea salt) whereas
higher salinity (ECiw= 129 dSm-1 sea salt) adversely affected vegetative and reproductive
growth Plant managed to tolerate up to ECiw= 99 dSm-1 sea salt Salinity severely affected
biochemical parameters including photosynthetic pigments proteins and sugars whereas
leaf phenolics were increased Leaf accumulated high amount of Na+ whereas affect
absorption of essential minerals like K+ was decreased
In the light of above mentioned investigations it appears that C cajan can be
propagated in saline soils with good presoaking techniques in non-saline water which
would helped to grow at moderately saline conditions It could be a good option used as
intercrop species because of its ability to improve soil fertility even under water deficit
conditions The proposed Cajanus-Ziziphus intercropping system could help poor farmers
to generate income from unproductive soils by obtaining sufficient fodder from C cajan
for their cattle and producing delicious edible fruits from Z mauritiana for commercial
purposes Carissa carandas could also be introduced as new crop for producing fruits from
moderate saline waste lands and used for preparing prickle jam and jelly for industrial
purposes
xxi
لاصہ خ
کا عمل ے ں ب ڑھئ لف پ ودوں می ی ےمخ طرہ ہ
وا خ ا ہ ے ب ڑھی لئ داوار کے ی ں زرعی ب وں می
ر علاق ج
ن ی م ب
ر و ب ج ن کھاری پ ن کھاری پ ن ب
دا کروت ی ر اور ر ب ے ارہ ا ہ وت لف ہ ی ی مخ کاف ں ودگی می اص Subtropical کی موج ا اور خ ی و پ وری دب ں ج ی ں ہ صلی
کی ف طے
خ
وراک و ں ج می
ی ملکوں
ائ ی ش کھاکر ای کی ی ان پ ودوں کم لوگ ہ ہت کن ب ں لی ی ی ہ
وئ عمال ہ
ارے کے طور ب ر است ری پ ن سے خ
ں ی ے ہ ں علم رکھئ ارے می ے عمل کے ت گئ ے گائ
کر ا ھ ملا
ی سات ک ہ رواداری اور ات
وں ج ن ر کےب ے ارہ
ھگوئ ہلے سے ت ں ب کاز والے محلول می لف ارت ی
مک کے مخ
دری ں ں سمی ی مطالعہ می
دائ ی کھاری اب کا
کہ پ ن کے و ی ج وئ ع ہ
کمی واف ں ی ت می ب
کی طن وں ج ن
ھ ب ہ کے سات
اف ں اض کھاری پ ن می ا گی ا کی دہ اہ کا مش رات
iwEC =اب
1-35 dSm می خ ی کہ ت ی ج مک کے ب راب ررہ
دری ں زی سمی کا
ہ ارت ں ی ام می ی ت صدی dSm= iwEC 168-1پ ودوں کے ق
ق
ی ک رہ ں Lysemeterت ے والے پ ودوں می ڑھئ ں ب روان چ می 1-dSm 24 ں جوضلہ مک محلول می
دری ں زی سمی کا
ارت
ں کر می ر خل ب زب ر س ی
ات اور غ روز مادوں لمخی
گ اف الت ف کے رت ی ت
ائ ی ں ض کھاری پ ن می ی اس
گئ کھی
ت ت د زا ب رداش
ت صدی 05اف
ق
ی ش کم وب ں کر می ی کہ خل ب زب ر س ں 50کمی ج وں می ج ن
ھلی اورب ھول ت ں ت ن می ری ج دی ب ڑھوب ولی
ا پ ا رہ مات
ہ ں اف ت صدی اض
05ق
ی گئ کھی
ت ح طور د
کمی واض ت صدی
ق
ی وی شلک سہب ڑ سے می کی چ ر مک (Symbiotic)ارہ
کی ں ا رت ی
کٹ ی ے والے ب
کرئ مد خ
ن من روج ی
اب سے (NaCl)ت
ی ر کے سا dSmwEC 366 =-1رواداری ں ب ری ہ می ج ے عمل کے ت گئ ے
گائ
کر ا ھ ملا
ی سات ک ہ یات
گئ کھی
ت ک د ر ت ھ ارہ
ت
بی ق کے ب
حق ی ت دائ ی ا اب گی ا ی
کھاری پ ن کو ج کم ں ے می ج ں dSme (Ec 72 =-1(ن ی کہ می ری ج ں ب ڑھوب ی ر می e (Ec =ب
)1-111 dSm ہل ہلے ب ے عمل ب گئ ے
گائ
کر ا ھ ملا
ی سات ک ہ کو ات ر ی ر اور ب ی ارہ
ر رہ اب ر می ی
ک غ کی خد ت
Lysemeter ج ب رآم ت ا ی زا ب ی کے جوضلہ اف
اش ی ے سے آب
ف ف ھ دن کے و
سی ت آت
کی ی ار دن ی خ
گئ کی ں ں دمی ن می ے ج
وئ ہ
ے عمل گئ ے گائ
کر ا ھ ملا
ی سات ک ہ سی ت ات
کی ی ے پ ودوں
گائ
ن ہا ا کی پ ودوں ب شام
وں اق
ے دوپ ج گئ
ت ا ی زا ب ادہ جوضلہ اف ں زت می
ی ول ب ات ف روزمادوں لخمی
گ اف الت ف کے رت ی ت
ائ ی ضلاات می درخ ی می
ائ کی می ی
ائ ےجی
وئ Electrolyteب رآمد ہ
Leakage کی کر ں س ی وں می ب ی ان پ ودوںاور ب
ی ش کمی ب ں دار می ی دپ ں مق
ں دکھائ ر می
اظ ی ری کے ب
کے ب ڑھوب
xxii
Antioxidant ی ظرح سے ہ اور اس ہ اف ں اض کی سرگرمی وں می امروں
اور اس کے Nitrate Reeducatesخ
Substrate )3(NO ا ی کا سی ب ب ہ اف ں اض ما می وں
ش ھی ی
ت
ےdSmiw(Ec 28 =-1(معمولی گئ ے ئ کب راب ں سی ی می ائ ہ ت والے ت درج ں می ری ہ می ج
ی ت ئ ن ہا زمب کی ب الا پ ودوں
ے عمل گئ ے گائ
کر ا ھ ملا
ی سات ک ہ سی ت ات
کی ی ے پ ودوں
ادوں ب ر لگائ ی
ب ما ب وں
ش دی ی ولی
ے پ
وئ ج خاضل ہ
ت ا ی ر بہی ادہ ب ں زت می
ےض ر رہ ہی ں ب ام می ط ے ت گئ ے
گائ
کر ا ھ ملا
ی سات ک ہ شامت اور وزن ات عداد ج
کی ت ھلوں ی کہ ت ی ج ر رہ اب ر می ی
الت ف ی غ ی ت
ائ
ی وئ ں ہ ہی
ع ب ی دت لی واف ی ب
کوئ ں دار می کی مق کر
ات اور س ں لمخی ی وں می ب ی کہ ب ہ ج
اف ا اض مات
ں ں روزمادوں می
گ اف د کے رت LER مزت
ے LEC (gt1)اور ی ہ کرئ ارہ کی ظرف اس ی ائ کامی کی ام
ط ے ت گئ ے
گائ
کر ا ھ ملا
ی سات ری ات ک ہ
کی ب ڑھوب
ک دا کروت ں ری ہ می ج کھاری پ ن ) Lysemeterو کھاری پ ن روداری کے ت ا کم گی ا ں اگات iwEC = 142می
1-dSm ( کھاری پ ن ادہ ی کہ زت ی ج وئ ری ہ ہی ں ب مک( می
دری ں زی سمی کا
زی dSm= iwEC 129-1 ارت کا دری ارت سمی
ی وئ ر ہ
اب ری ب ری ظرح می
دی ب ڑھوب ولی
ی اور پ
ائ علی
ں ف مک( می
ی کہ ں ک dSm9= iw(Ec 9-1(ج مک ت
دری ں زی سمی کا
ارت
ت کب رداش ات اور س روز مادوں لخمی گ اف الت ف کے رت ی ت
ائ ی ضلاات می درخ ی می
ائ کی می ی
ائ ےجی اب رہ کامی ں ےمی
ر ب ری ظرح کرئ
ں ی وں می ب وا ب ہ ہ
اف ں اض ی ول می ب
ں ف ی وں می
ب ی کہ ب ں ج ی
وب ر ہ اب می
+Na ہ سے کی وج مع ی ج اف رلز کے K+اض روری می
ی سے ض ج
ی وئ ر ہ
اب کی ضلاجی ت می ے
کرئ زب چ
ا ت ق حق الا ت ہ ت درج ے ظر می
وئ ےہ
ھگوئ ں ت ی می
ائ ہلے سے ت کہ ب ی
ے آئ مئ ں ی ہ ت ات سا ی می
ئ کی روش ر ت ہ سے ارہ کی وج ے
ت ف
ھی مدد دے س ں ت ے می گئ ں ا ن می ن زمی مکی دل ں وکہ معی ے ج ا ہ اسکی ا خ ھی لگات
ں ت ن خالات می مکی کو ں وں ج ن
وزہ کے ب ے مج ا ہ کی
داواری ی ر ب ی ے عمل غ گئ ے
گائ
کر ا ھ ملا
ی سات ک ہ ی ر ات ر اور ب ی ضلاجی ت والی ارہ
اف ے اض لئ وروں کے
اپ کی صور ت خ ر ن ارہ زمی
ھی دا ت کروت ے ا ہ وسکی ت ہ اب کا ذرت عہ ت ے ی ب ڑھائ
کی آمدئ وں
کشاپ ی صورت
ارئ ح کی ت ل
ھ ی ت وردئ دار ج ی ر سے مزت ارہ اور ب ی خ
عئصت
صل کے طور ب ی ف ئے ب لئ ے کے
کرئ دا ی ھل ب ن سے ت کارآمد زمی ر ی
ن اور غ مکی
دل ں ے معی
لئ اضد کے ے رمق ا ہ اسکی ا خ کی ی ش ب
1
General Introduction
Intercropping is a major resource conservation technique for sustainable agriculture under
various climatic conditions (Zhang et al 2010 Li et al 2014) It can reduced operational
cost for the production of multiple crops with maintained or even higher level of
productivity (Vandermeer 2010 Perfecto and Vandermeer 2010) It can enhance the
water use efficiency by saving 20 to 40 irrigation water with improved fertilizer
management (Fahong et al 2004 Jat et al 2005 Jani et al 2008) Intercropping system
is more suitable in marginal areas with lower mechanization and cultivation input by
farmers on small tracts of farmlands (Ngwira et al 2012) It can enhance the cumulative
production per unit area and protect the small farmers against market fluctuations or crop
failure ensure the income improve soil fertility and food demands (Rusinamhodzi et al
2012) In this system dominating more compatible and productive species are selected or
replaced in which complementarity effects and beneficial interactions resulting enhanced
yield as compared to monoculture (Huston 1997 Loreau and Hector 2001) It was
estimated that in species diverse systems biomass production is 17 times higher as
compared to monoculture (Cardinale et al 2007)
It is suggested that intercropping is the best suitable cropping system which can
improve the resource-use efficiency by procurement of limiting resources enhanced
phyto-availability and effective plants interactions (Marschner 2012 White and
Greenwood 2013 Ehrmann and Ritz 2014) It is widespread in many areas of world
particularly in latin America it is estimated about 70-90 by small farmers which mainly
grow maiz potatoes beans and other crops under this system whereas intercropping of
maiz with different crops is estimated about 60 (Francis 1986) Additionally
agroforestry is more than 1 billion ha in this area (Zomer et al 2009) The land used for
intercropping system of various crops is greatly varied from 17 in India to 98 in Africa
(Vandermeer 1989 1992 Dupraz and Liagre 2011)
In intercropping system two or more crops or genotypes coexist and growing
together at a same time on a similar habitat (Li et al 2013) It may be divided into various
types such as in mixed intercropping system two or more crops simultaneously growing
without or with limited distinct arrangements whereas in relay intercropping system
second crop is planted when the first is matured while in strip intercropping both the crops
2
are simultaneously growing in strips which can facilitate the cultivation and crop
interactions (Ram et al 2005 Sayre and Hobbs 2004)
Several less-conventional fruit tress including Manilkara zapota (Chicko)
Ziziphus mauritiana (Jujubar) Carissa carndas (Karanda) Annona squamosa (Sugar
apple) and Grewia asiatica (Falsa) has been reported with high nutritional value with
capability to grow at marginal lands (Mass and hoffman 1997) Qureshi and Barrett-
Lennard (1998) suggested few grafted plants that can widely use to improve the quality
and productivity of fruits Grafting is also used to induce stress tolerance in plants against
various abiotic and biotic stresses including salinity stress (Rivero et al 2003) Both root
stocks and shoot stocks contribute to increase the tolerance level of plants Root stocks
represent the first part of defense to control the uptake and translocation of nutrients and
salts throughout the plant (Munns 2002 Santa-cruz et al 2002 Zrig et al 2011) while
shoot stocks develops physiological and biochemical changes to promote plant growth
under stress conditions (Moya et al 2002 Chen et al 2003)
Ziziphus mauritiana Lamk (varn grafted ber) belongs to the family Rhamnaceae
grows widely in most of the dry tropical and subtropical regions around the world Various
grafting methods are used for their propagation including wedge and whip or tongue
methods (Nerd and Mizrahi 1998) Intercropping of these grafted fruit trees with various
leguminous crops is also being successfully practiced in many countries thought the world
Leguminous crops are considered excellent symbiotic nitrogen fixing crops It can
effectively improve soil fertility and offset the critical problems of sub-tropical areas to
fight against desertification and soil degradation These plants are considered as an
excellent source of proteins for humans and animals They can fix the 90 of atmospheric
nitrogen and contribute 40 nitrogen to the soil thus increase the soil fertility (Peoples et
al 1995) However most of the leguminous plants are not salt tolerant while some
species are better drought tolerant and effectively contribute in marginal lands (Zahran
1999)
Among the leguminous plants Pigeon pea (Cajanus cajan (L) Millspaugh) of the
family Fabaceae is widely grown for food fodder and fuel production particularly in
semiarid areas The salinity tolerance of this specie is not well documented both at
germination and seedling stages This crop is still underexploited due to its edible and
3
economic importance While limited investigations has been made to uncover its
nutritional quality medicinal uses and drought tolerance
The identical physiological traits are important in both the mono and intercropping
systems to maximize the resource acquisition The exploitation of best possible
combination of traits of different plants in intercropping system is very important to
maximize the overall performance in intercropping system It depends on the above ground
beneficial plant interactions for light space and optimal temperatures (Wojtkowski 2006
Zhang et al 2010 Shen et al 2013 Ehrmann and Ritz 2014) as well as the
complementary below ground plant interactions with soil biotic factors (Bennett et al
2013 Li et al 2014)
Water is also a major limiting factor intercropping can enhanced the acquisition
of water by root architecture and distribution in the soil profile for effective utilization of
rainfall (Zegada-Lizarazu et al 2006 De Barros et al 2007) and enhanced the water use
efficiency for effective hydraulic redistribution by deep rooted crops and water stored in
the soil profile (Morris and Garrity 1993 Xu et al 2008) Mycorrhizal networks around
the roots of intercrop plants also enhanced the availability of water and available resources
and reduced the surface runoff (Caldwell et al 1998 Van-Duivenbooden et al 2000
Prieto et al 2012)
Intercropping with leguminous plants can enhanced the agricultural productivity in
less productive soils due to enhanced nitrogen availability and also improved the soil
fertility by effective nitrogen fixation (Seran and Brintha 2010 Altieri et al 2012) Due
to weaker soil nitrogen competition intercropping with legumes enhanced the nitrogen
availability to the non-leguminous intercrop which also absorbs the additional nitrogen
released in the soil or root nodules of the leguminous plant (Li et al 2013 White et al
2013a) The use of legumes in many intercropping systems is pivotal According to the
listing of Hauggaard-Nielsen and Jensen (2005) seven out of ten are the legumes among
the most frequently used intercrops around the world
The ecological range of adaptability of legumes reaches from the inner tropics to
arctic regions with individual species expressing tolerance to drought temperature
nutrient deficiency in soil water logging salinity and other environmental conditions
(Craig et al 1990 Hansen 1996) The woody perennial leguminous plants have a number
4
of purposes they can be used to reclaim degraded wastelands retard erosion and provide
shade fuel wood timber and green manure (Giller and Wilson 1991)
Trees with nitrogen fixing capability play an important role to offset the critical
problems of tropical and sub-tropical regions in their fight against desert encroachment
and soil impoverishment These plants are capable to live in N-poor soils through their
association with Rhizobium that fix atmospheric nitrogen Nitrogen fixing activity in the
field depends both on their N2-fixing potential and on their tolerance to existing
environmental stresses (Galiana et al 2002) Symbiotic N2 fixation in leguminous plants
can mainly be considered an excellent source of protein supply for human and animal
consumption They range from extensive pasture legumes to intensive grain legumes and
are estimated to contribution up to 40 of their nitrogen to the soil (Simpson 1987)
The traits in the monocropping system in the selected crop extensively exploit the
acquisition of limiting resources in the environment and continuously focused on the
availably of similar resources for the successful crop production (White et al 2013 ab)
whereas in intercropping with different crops cycling of resources can be optimized to
the complementarity or facilitation traits (Costanzo and Barberi 2014) to overcome
resource limitations during the growing season (Hill 1996 George et al 2014)
For the long term sustainable agriculture and food production in resource limiting
areas with lower input Intercropping systems have the potential to increase the
productivity With efficient mechanization cultural practices and optimized nutrient
management rapid improvements are also possible through this system In future
perspective intercrops with higher resource use efficiency through plant breeding and
genetics is likely to be the most effective option for sustainable agriculture and
development
Increase of world population and demand of additional food production
The demand and production gap of food fodder fuel wood and livestock products is
increasing day by day due to global population which will increase from about 7 billion
(FAO 2014) to 9 billion by 2050 (Haub 2013) The increasing urbanization further
intensifies the problem which will increase from 54 to 66 expected in 2050 (UN
2014) Majority of this rise in urbanization will occur in developing countries around the
5
globe The major problem is to meet the challenge of increasing food demand for this ever
growing population up to 70 more food crops to feed the additional 23 billion population
worldwide by 2050 (FAO 2010 2011) Hence there is great need to increase the re-
vegetation for fuel wood and fodder production (Thomson 1987) An increase in
production could be envisaged through increasing the yield of already productive land or
through more extensive use of unproductive land The high concentration of salts in soil
or water does not let the conventional crops grow and give feasible economic return
Hence it is necessary to search for unconventional crops for foods fodder and fuel which
could give profitable yield under saline conditions (Ahmad and Ismail 1993) Reclamation
of this land through chemical and engineering treatments is very expensive The most
appropriate use of saline wasteland is the production of high yielding salt tolerance fuel
wood timber and forage species (Qureshi et al 1993) Therefore the most attractive
option is to screen a range of species and identify those which have potential of being
commercially valuable for the degraded environments (Ismail et al 1993)
Pakistan is in semi-arid region and the 6th most populated county of the world
Population drastically increased in Pakistan which was 80 million in 1980 and annual
increase in population is about 4 million (UNDES 2011) This is continuously
overburdened and it is estimated that in 2025 it will reach to 250 million and 335 million
in 2050 which decrease the available water per capita to less than 600 m3 resulting 32
shortfall of water requirements causing an alarming condition particularly for Pakistan
Furthermore this shortfall in 2050 leading to severe food shortage upto 70 million tones
which indicates the further development and serious measures for the new resources
(ADB 2002) Subsequent severe food and fodder crises along with all the resource
limitations with continuous increase in urbanization from the current 35 to 52 in 2025
will further intensity the agriculture production and demand
Shortage of good quality irrigation water
On earth surface the major resources of available fresh water is deposited in the form of
ponds lakes rivers ice sheets and caps streams and glaciers whereas underground water
as underground streams and aquifers With the drastic increase in population the water
consumption rise as the twice of the speed of population growth The scarcity of water is
widespread to many countries of different regions Majority of population in developing
countries suffering from seasonal or year round water shortage which will increase with
6
expected climatic changes Currently almost 50 countries around the globe are facing
moderate to severe shortage of water
Due to the greenhouse effect it is estimated that since the start of 20th century 14
degF temperature is already risen which will likely rise at least another 2degF and over the next
100 years it is estimated about more than 11degF due to the consequences of biogenic gases
(El-Sharkawy 2014) This is mainly due to the product of human activities including
industrial malpractices excess fossil fuel consumption deforestation poor land use and
cultural practices
Rising in atmospheric CO2 concentration which probably reached 700 μmol (CO2)
molminus1 resulting severe climatic changes It will accelerate the melting of ice and glacier
resulting the rising rainfall and storms in tropics and high latitude consequently 06 to 1
meter rise in sea level on the expense of costal lowlands across the continents After this
initial high flows the decrease in inflow was very terrifying Due to these climatic changes
humans suffering from socioeconomic changes including degradation of lands with lower
agricultural output and degradation of natural resources will further enhanced the poverty
and hunger resulting dislocation and human migrations (Randalls 2010)
In the mean while scarcity of good quality water is increasing day by day with the
demands of water for domestic agricultural and industrial utilization which will further
increase up to 10 of the total available resources as estimated by 2025 which needs
serious water managements (Bhutta 1999) It is very challenging for the modern
agriculture to ensure the increasing demand of more arable and overburdened population
with the limiting resources including the unavailability of good quality water and
deterioration of even previously productive land (Du et al 2015)
In Pakistan Indus River basin is the back bone of agriculture and socioeconomic
development which contributes 65 of the total river flows and 90 for the food
production with a share of 25 to the GDP It is estimated that about 30-40 of its surface
storage capacity will reduce by 2025 due to siltation of reservoirs and climatic changes It
will impose serious threat to irrigated agriculture in near future consequently with
decreases in groundwater resources resulting shortage of fresh water and 15-20
reduction in grain yield in Pakistan (World Bank 2006)
7
Spread of saline soil and reduction in agricultural yield
Along with scarcity of water soil salinity is one of the major environmental stresses which
severely threaten the agriculture The damages of salinity is widespread around the world
which is so far effected the more than 800 million hectare (more than 6) of land
worldwide including 397 million ha by salinity associated with 434 million ha by sodicity
(FAO 2010) The out of total 230 million hactares of irrigated land more than 45 million
hactares (20) is so far effected by salinity which is about the 15 of total cultivated land
(Munns and Tester 2008)
In Pakistan out of 2036 million hectares of cultivated land more than 6 million
hectares is affected by salinity and water logging of various degrees (Qureshi et al 2004)
About 16 million hectares of tropical arid plains which have been put under crop
cultivation depend extensively on canal irrigation network This area (about 60) is now
seriously affected by water logging and salinity (Qureshi et al 2004) The rise of subsoil
water levels accompanied by its subsequent decline due to irrigation combined with
insufficient drainage has led to salinization of valuable agricultural land in arid zones all
over the world (Ahmad and Abdullah 1982) The dominated cation in salt-affected soil is
Na+ followed by Ca2+ and Mg2+ while the anions Cl and SO4 are almost equal in
occurrence (Qureshi et al 1993) Salt content varies in different regions of the salt-
affected areas but at certain sites could reach up to an ECe of 90-102 dSm-1 (Ahmad and
Ismail 1993)
Salinity is a chief anxiety to meet the ever growing demands of food crops Salinity
adversely affects the plant growth and productivity Plants differentially respond to salt
stress and categories into four classes Salt sensitive moderately salt sensitive moderately
salt tolerant and highly salt tolerant plants on the basis of their tolerance limits Whereas
mainly plants are divided into halophytes (salt tolerant) and glycophytes (salt sensitive) on
the basis of adaptive evolution (Flowers 2004 Munns and Tester 2008) Unfortunately
majority of cultivated crops are not able to withstand in higher salinity regimes and
eventually die under higher saline conditions which proposed serious attentions to manage
the dissemination of salinity (James et al 2011 Rozema and Flowers 2008)
Excessive accumulation of salts in rhizosphere initially reduced the water
absorption capacity of roots leading to hyperosmotic stress followed by specific ion
8
toxicity (Munns 2008 Rahnama et al 2010) Plants initially manage the overloaded salt
by various excluding and avoidance mechanisms depending on their tolerance levels The
management of salt inside the cytosol is depends on the compartmentalization capacity of
plants followed by osmotic adjustments and efficient antioxidant defense mechanisms
Whereas higher salt beyond the tolerance impose injurious effects on various
physiological mechanisms These are including disruption of membrane integrity
increased membrane injuries nutrient ion imbalances osmotic disturbance
overproduction of reactive oxygen species (ROS) compromised photosynthesis and
respiration due to stomatal closure and damages of enzymatic machinery (Munns and
Tester 2008) In specific ion toxicity Na+ and Cl- are the chief contributors in
physiological disorders Excessive Na+ in rhizosphere antagonize the uptake of K+
resulting lower growth and productivity (James et al 2011) Salt load in the cytosol trigger
the overproduction of ROS including H2O2 OH- super oxides and singlet oxygen They
are involved in sever oxidative damages to various vital cellular components including
DNA RNA lipids and proteins (Apel and Hirt 2004 Ahmad and Umar 2011)
Strategies to cope up the salinity problem
The development and cultivation of highly salt tolerant crop varieties for salt affected areas
is the major necessity to meet the future demands of food production whereas the majority
of available food crops are glycophytes Therefore it is an emergent need of crop
improvement methods which are more efficient cost effective and grow on limiting
resource The use of poor quality water for irrigation is also very important under the
proposed shortage of fresh water in near future For the development of salt tolerant
varieties more understanding of stress mechanisms are required at whole plant molecular
and cellular levels
The variability in stress tolerance of salt sensitive genotypes (glycophytes) and
highly salt tolerant plants (halophytes) showed genetic basis of salt tolerance It indicate
that salt tolerance is a multigenic trait which involves variety of gene expressions and
related mechanisms Salt stress induces both the qualitative and quantitative changes in
gene expression (Manchanda and Garg 2008) These multigenetic expressions play a key
role in upregulation of various proteins and metabolites responsible for the management
of anti-stress mechanisms (Bhatnagar-Mathur et al 2008) Plant breeding and transgenic
strategies are intensively used for decades to improve the crop performance under salinity
9
and aridity conditions Few stress tolerant varieties are so far released for commercial
production whereas in natural condition where plant exposed to variety of climatic
conditions the overall performance of plant have changed as compared to controlled in
invitro conditions (Schubert et al 2009 and Dodd and Perez-Alfocea 2012) The success
stories about transgenic approaches for crop improvement under stressful environments
are still very scanty because of the insufficient understanding about the sophisticated
mechanisms of stress tolerance (Joseph and Jini 2010) It indicates that there is less
correlation between the assessment of stress tolerance in invitro and invivo conditions
Although there have been some achievement in this connection in some model plants
including rice tobacco and Arabidopsis (Grover et al 2003) which proposed the
possibilities of success in other crops in future Variety of technicalities and associated
financial challenges are still associated with this strategy
In conventional cultivation practices continuous irrigation with poor quality water
can enhanced the salinization due to evapotranspiration leading to increased saline andor
sodic soils This problem can be cope up by intercropping system in which high salt
tolerant or salt accumulator plants are intercropped with salt sensitive crops which can
accumulate salt thus can reduce the risk of salt increment in soil Additionally better
cultivation practices including the micro-jet or drip irrigation and partial root zone drying
technique is also very fruitful to optimize the water requirements and avoid the risks
associated with conventional flooding irrigation system
In dry land agriculture plantation of deep rooted perennials during off season or
annuals can reduced the risk of salinization They continuously grown and utilize excess
amount of water create a balance between water utilization and rail fall Thus prevent the
chance of salt accumulation on soil surface due to increased water table and
evapotranspiration (Manchanda and Garg 2008) The efficient irrigation and
intercropping strategy is seemed quite attractive cost effective and very beneficial in less
mechanized poor marginal areas It can ameliorate the injurious effects of salinity and
increased production per unit area thus ensure the sustainable agriculture in semi-arid or
marginal lands (Venkateswarlu and Shanker 2009)
A number of plant species are available that are highly compatible with saline
sodic and marginal lands The cultivation of these species with proposed intercropping
system is economically feasible to grow in marginal soil Some plants including Carissa
10
carandus Ziziphus mauritiana and Cajanus cajan was selected to revealed their potential
for intercropping under saline marginal lands These are important plants which can
established well at tropical and subtropical arid zone under high temperatures Hence their
range of salt tolerance and suitability for cultivation at waste saline land or with saline
water irrigation is being undertaken for commercial exploitation
Objective of present investigation
The plan of present investigation has been worked out to look into possibility of increasing
production of an unconventional salt tolerant fruit tree (Z mauritiana) by intercropping
with a legume ( C cajan) which apart from increasing fertility of soil could be able to
provide fodder for grazing animals from salt effected waste land Possibility of making
use of saline water for irrigation has also been considered for growing leguminous plant
(C cajan) and salt tolerant unconventional fruit tree (Crissa carandas) under saline
condition
11
LAYOUT OF THESIS
Chapter 1 Monoculture of Cajanus cajan (Vern Arhar) and Ziziphus mauritiana
(Varn Ber) under different range of salinities created by irrigation of
various sea salt concentrations
A Experiments on Cajanus cajan
Following experiments were performed under A
Experiment No 1 Effect of Pre-soaked seeds of C cajan in distilled water for
germination in water of different sea salt concentrations
Experiment No 2 Effect of Pre-soaked seeds of C cajan in various dilutions of sea salt
for germination in water of respective sea salt concentrations
Experiment No 3 Seedling establishment experiment of C cajan on soil irrigated with
sea salt of different concentrations
Experiment No 4 Growth and development of C cajan in Lysimeter (Drum pot culture)
being irrigated with water of different sea salt concentrations
Experiment No 5 Range of salt tolerance of nitrogen fixing symbiotic bacteria
associated with root of C cajan
B Experiments on Ziziphus mauritiana
Experiment No 6 Growth and development of Z mauritiana in large size clay pot being
irrigated with water of two different sea salt concentrations
Discussion (Chapter 1)
Chapter 2 Intercropping of Ziziphus mauritiana with Cajanus cajan
Experiment No 7 Physiological investigations on Growth of Ziziphus mauritiana and
Cajanus cajan intercropped in drum pot (Lysimeter) culture being
irrigated with water of sea salt concentration at two irrigation
intervals
Experiment No 8 Investigations of intercropping Ziziphus mauritiana with Cajanus
cajan on marginal land under field conditions
12
Discussion (Chapter 2)
Chapter 3 Investigations on rang of salt tolerance in Carissa carandas (varn
karonda) for determining possibility of growing at waste saline land
Experiment No 9 Investigation on the effect of higher range of salinities on growth of
Carissa carandas (varn karonda) created by irrigation of different
dilutions of sea salt
Discussion (Chapter 3)
13
1 Chapter 1
Monoculture of Cajanus cajan (Vern Arhar) and Ziziphus mauritiana
(Varn Ber) under different range of salinity created by irrigation of
various sea salt concentrations
11 Introduction
Scarcity of good quality water enforced the growers to irrigate the crops with
lowmoderately saline water at marginal lands which ultimately enhance soil salinity due
to high evapo-transpiration (Azeem and Ahmad 2011) To overcome this situation people
are now focusing on less-conventional plants which can grow on resource limited areas
and can produce edible biomass for human and animal consumption
Ziziphus mauritiana (varn grafted ber) is salt and drought tolerant plant which can
grow on marginal and degraded land (Morton 1987) It has wide spread crown and a short
bole fast growing tree with average bearing life of 25 years The ripe fruit (drupe) is juicy
hard or soft sweet-tasting pulp has high sugar content vitamins A amp C carotene
phosphorus and calcium (Nyanga et al 2013 2008 Pareek 2013) The leaves contain 6
digestible crude protein and an excellent source of ascorbic acid and carotenoids The
leaves are used as forage for cattlesheepgoats and also palatable for human consumption
(Sharma et al 1982 Bal and Mann 1978 Agrawal et al 2013) The timber is very hard
can be worked to make boats charcoal and poles for house building Roots bark leaves
wood seeds and fruits are reputed to have medicinal properties The tree also used as a
source of tannins dyes silk (via silkworm fodder) shellac and nectar (Dahiru et al 2006
Chrovatia et al 1993 Gupta 1993)
Some atmospherics nitrogen fixing bacterial associated deep rooted drought
tolerent leguminious plants like Cajanus cajan can fix up to 200 Kg nitrogen ha-1 year-1
due to symbiotic association of Rhizobium with its deep penetrating roots (Bhattacharyya
et al 1995) Total cultivated area of Pigeon pea is about 622 million hectare and global
annual crop production is around 474 million tonnes whereas total seed production of
this crop is about 015 million tonnes (FAOSTAT 2013) Its seeds are an excellent source
of good quality protein (up to 24) and foliage is used as animal fodder with high
nutritional value (Pandey et al 2014) Besides being used as food and fodder this plant
14
also have therapeutic value and it is used against diabetes fever dysentery hepatitis and
measles (Grover et al 2002) It also use traditionally as a laxative and was identified as
an anti-malarial remedy beside other medicinal species (Ajaiyeoba et al 2013 Qasim et
al 2010 2011 2014)
Following experiments were conducted to evaluate the seed germination seedling
establishment and growth of C cajan as well as grafted sapling of Z mauritiana under
various salinity regimes Investigations were also undertaken to find-out of their
intercropping has any beneficial effect on growth at marginal saline land saline
environment
15
12 Experiment No 1
Effect of Pre-soaked seeds of Cajanus cajan in distilled water for
germination in water of different sea salt concentrations
121 Materials and methods
1211 Seed collection
Seeds of C cajan were purchased from local seed market Mirpurkhas Sindh and were
tested to determine the effect of salinity on germination at the biosaline laboratory Botany
department Karachi University Karachi The best lot of healthy seeds having 100
germination was selected for further experiments
1212 Experimental Design
Seeds of C cajan were surface sterilized with 01 sodium hypochlorite solution for 2-3
minutes washed in running tap water then soaked in sterilized distilled water for one hour
(Saeed et al 2014) Sterilized glass petri plates (9cm) lined with filter paper were moist
with 10 ml of distilled water at different saline water of different sea salt concentrations
and their germination percentage was observed Their electrical conductivities on these
sea salt dilutions are mentioned in Table 11 Three replicates were used for each treatment
Ten seed were placed in each petri plate which were kept in temperature controlled
incubator (EYELA LTI-1000 Japan) at 28 plusmn 1ordmC in dark Experiment was continued for 7
days Data were recorded on daily bases Analyses of varience by using repeated measures
and the significant differences between treatment means were examined by least
significant difference (Zar 2010) All statistical analysis was performed using SPSS for
windows version 14 and graphs were plotted using Sigma plot 2000
Germination percentage of C cajan was recorded every 24 hours per seedling
evaluation procedure up to 07 days The final percent germination related with salinity in
accordance with Maas and Hoffman (1977) The percent germination was calculated using
the following formula (Cokkizgin and Cokkizgin 2010)
16
Germination index for C cajan was recorded according to AOSA (1990) by using
following formula
Where Gt is the number of germinated seed on day t and Dt is the total number of
days (1 - 7)
Coefficient of germination velocity of C cajan was calculated described by Maguire
(1962)
Where G represents the number of germinated seeds counted per day till the end of
experiment
Mean germination time of C cajan was calculated by Ellis and Roberts (1981) by
using following formula
Where lsquonrsquo is the number of germinated seeds in day d whereas Σn is the total
germinated seeds during experimental period
Germination rate was of C cajan determined according to following formula
(Shipley and Parent 1991)
Where numbers of germinated seeds were recorded from 1 to 7
17
122 Observations and Results
Cajanus cajan (imbibed in distilled water) grown at different salinity regimes showed 50
reduction at 16 salt concentration corresponding ECiw 168 dSm-1 (Table 1 2 Appendix
I)
Rate of germination was inversely correlated with sea salt concentration It was
significantly (p lt 0001) decreased from first day to final (day 7) of observation Higher
germination rate was recorded in control and at lower concentrations of sea salt in early
days of seed incubation with contrast to higher concentrations of sea salt which was
reduced with increasing day of incubation (Table 13 Appendix I)
A significant decrease (p lt 0001) in coefficient of germination velocity was
observed with increasing salinity (Table 14 Appendix I)
A significantly increase (p lt 0001) in mean germination time of seeds was observed
with increasing sea salt concentrations However the difference was insignificant at lower
salinities (Table 14 Appendix I)
A significant decrease (p lt 0001) in mean germination index was observed with
increasing salt concentrations except lower salinities More reduction was observed
byhond 16 and onward sea salt concentration (Table 14 Appendix I)
18
Table 11 Electrical conductivities of different sea salt solutions used in germination of C cajan
Sea salt () ECiw (dSm-1)
Non saline control 06
01 09
02 16
03 35
04 42
05 58
06 62
07 79
08 88
09 99
10 101
11 112
12 128
13 131
14 145
15 159
16 168
ECiw is the electrical conductivity of irrigation water measured in deci semen per meter
19
Table 12 Effect of irrigation water of different sea salt solutions on germination percentage (GP) per day
of C cajan seeds pre-soaked in non-saline water prior to germination with duration of time under
various salinity regimes
Sea Salt
(ECiw= dSm-1)
GP
1st day
GP
2nd day
GP
3rd day
GP
4th day
GP
5th day
GP
6th day
GP
7th day
Control 8333plusmn667 90plusmn00 9333plusmn333 9333plusmn333 9333plusmn333 9333plusmn333 9333plusmn333
09 8667plusmn333 9333plusmn333 9667plusmn333 9667plusmn333 100plusmn00 100plusmn00 100plusmn00
16 7667plusmn667 80plusmn10 8333plusmn882 8333plusmn882 8333plusmn882 8333plusmn882 8667plusmn667
35 6667plusmn333 9333plusmn333 9333plusmn333 9333plusmn333 9333plusmn333 9333plusmn333 9333plusmn333
42 70plusmn00 8667plusmn333 90 plusmn00 90 plusmn00 90 plusmn00 90 plusmn00 90 plusmn00
58 6333plusmn667 7333plusmn333 8333plusmn333 90 plusmn00 90 plusmn00 90 plusmn00 90 plusmn00
62 5667plusmn667 80plusmn577 90 plusmn00 90plusmn00 90 plusmn00 90 plusmn00 90plusmn00
79 5333plusmn333 70plusmn00 8333plusmn333 8333plusmn333 8333plusmn333 8333plusmn333 8333plusmn333
88 4000plusmn00 6667plusmn667 8333plusmn333 8333plusmn333 8333plusmn333 8333plusmn333 8333plusmn333
99 2667plusmn333 60 plusmn00 90 plusmn00 90plusmn00 90 plusmn00 90 plusmn00 90 plusmn00
101 2333plusmn333 70plusmn577 7333plusmn333 7333plusmn333 7333plusmn333 7333plusmn333 7333plusmn333
112 70plusmn577 7667plusmn333 80plusmn00 8333plusmn333 8333plusmn333 8333plusmn333 8333plusmn333
128 6667plusmn333 6667plusmn333 6667plusmn333 6667plusmn333 6667plusmn333 6667plusmn333 6667plusmn333
131 3333plusmn882 50plusmn00 5333plusmn333 5333plusmn333 5333plusmn333 5333plusmn333 5667plusmn333
145 3333plusmn667 40 plusmn00 50 plusmn577 50plusmn577 50 plusmn577 5333plusmn333 5333plusmn333
156 3667plusmn667 40plusmn577 4667plusmn882 4667plusmn882 50plusmn577 50plusmn577 5333plusmn667
168 1667plusmn882 3333plusmn333 3333plusmn333 3333plusmn333 3667plusmn333 3667plusmn333 4333plusmn333
LSD 005 Salinity 18496
Time (days) 13322
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and LSD among the treatments is recorded at p lt 005
20
Table 13 Effect of irrigation water of different sea salt solutions on germination rate (GR) per day
of seeds C cajan pre-soaked in non-saline water prior to germination with duration of
time under various salinity regimes
Sea Salt
(ECiw= dSm-1)
GR
1st day
GR
2nd day
GR
3rd day
GR
4th day
GR
5th day
GR
6th day
GR
7th day
Control 833plusmn067 450plusmn00 311plusmn011 233plusmn008 187plusmn007 156plusmn006 133plusmn005
09 867plusmn033 467plusmn017 322plusmn011 242plusmn008 200plusmn00 167plusmn00 143plusmn00
16 767plusmn067 400plusmn050 278plusmn029 208plusmn022 167plusmn018 139plusmn015 124plusmn010
35 667plusmn033 467plusmn017 311plusmn011 233plusmn008 187plusmn007 156plusmn006 133plusmn005
42 700plusmn00 433plusmn017 300plusmn00 975plusmn750 180plusmn00 150plusmn00 129plusmn00
58 633plusmn067 367plusmn017 278plusmn011 225plusmn00 180plusmn00 150plusmn00 129plusmn00
62 567plusmn067 400plusmn029 300plusmn00 225plusmn00 180plusmn00 150plusmn00 129plusmn00
79 533plusmn033 350plusmn00 278plusmn011 208plusmn008 167plusmn007 139plusmn006 119plusmn005
88 400plusmn00 333plusmn033 278plusmn011 208plusmn008 167plusmn007 139plusmn006 119plusmn005
99 267plusmn033 300plusmn00 300plusmn00 225plusmn00 180plusmn00 150plusmn00 129plusmn00
101 233plusmn033 350plusmn029 244plusmn011 183plusmn008 147plusmn007 122plusmn006 105plusmn005
112 700plusmn058 383plusmn017 267plusmn00 208plusmn008 167plusmn007 139plusmn006 119plusmn005
128 667plusmn033 333plusmn017 222plusmn011 167plusmn008 133plusmn007 111plusmn006 095plusmn005
131 333plusmn088 250plusmn00 178plusmn011 133plusmn008 107plusmn007 089plusmn006 081plusmn005
145 333plusmn067 200plusmn00 167plusmn019 125plusmn014 100plusmn012 089plusmn006 076plusmn005
156 367plusmn067 200plusmn029 156plusmn029 117plusmn022 100plusmn012 083plusmn010 076plusmn010
168 167plusmn088 167plusmn017 111plusmn011 083plusmn008 073plusmn007 061plusmn006 062plusmn005
LSD 005 Salinity 0481
Time (days) 0378
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and LSD among the treatments is recorded at p lt 005
21
Table 14 Effect of irrigation water of different sea salt solutions on mean germination rate (GR)
coefficient of germination velocity (GV) mean germination time (GT) mean
germination index (GI) and final germination (FG) of C cajan seeds pre-soaked in non-
saline water prior to germination under various salinity regimes
Sea Salt
(ECiw= dSm-1) GR GV GT GI FG
Control 2624plusmn100 369plusmn005 027plusmn00 2624plusmn100 9667plusmn333
09 2743plusmn063 365plusmn009 027plusmn001 2743plusmn063 100plusmn00
16 2398plusmn218 423plusmn036 024plusmn002 2398plusmn218 8333plusmn882
35 2467plusmn086 378plusmn005 026plusmn00 2467plusmn086 9333plusmn333
42 3169plusmn733 311plusmn058 035plusmn008 3169plusmn733 9333plusmn333
58 2264plusmn081 399plusmn015 025plusmn001 2264plusmn081 90plusmn00
62 2253plusmn073 400plusmn013 025plusmn001 2253plusmn073 9333plusmn333
79 2074plusmn081 402plusmn00 025plusmn00 2074plusmn081 8333plusmn333
88 1927plusmn043 449plusmn008 022plusmn00 1927plusmn043 90plusmn577
99 1853plusmn033 486plusmn009 021plusmn00 1853plusmn033 90plusmn00
101 1635plusmn056 470plusmn022 021plusmn001 1635plusmn056 8667plusmn882
112 2263plusmn042 369plusmn020 027plusmn001 2263plusmn042 9667plusmn333
128 1953plusmn098 341plusmn00 029plusmn00 1953plusmn098 9667plusmn333
131 1368plusmn059 440plusmn018 023plusmn001 1368plusmn059 6667plusmn333
145 1276plusmn099 446plusmn019 023plusmn001 1276plusmn099 60plusmn577
156 1289plusmn153 447plusmn030 023plusmn002 1289plusmn153 8000plusmn100
168 876plusmn104 589plusmn078 018plusmn002 876plusmn104 8667plusmn333
LSD005 5344 3312 0064 5344 1313
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and LSD among the treatments is recorded at p lt 005
22
13 Experiment No 2
Effect of Pre-soaked seeds of Cajanus cajan in various dilutions of sea
salt for germination in water of respective sea salt concentrations
131 Materials and methods
1311 Seed germination
Procedure of seed germination has been mentioned in Experiment No 1 earlier The seeds
were pre-soaked in various sea salt concentrations instead of non-saline water and
germinated in respective sea salt concentrations Their electrical conductivities mentioned
in Table 15 Data were calculated and analysed according to formulas given in Experiment
No 1
Since these pre-soaked seeds in different sea salt concentration showed 50
germination at 03 equivalent to ECiw= 42dSm-1 sea salt solution any further work
beyond ECiw= 42dSm-1was not continued
132 Observations and Results
The final percent germination related with salinity in accordance with Maas and
Hoffman (1977) linear relative threshold response model as follows
Relative Final Germination = 100-200 (Ke ndash 005)
Where threshold salt concentration was 005 and Ke is the concentration of salts
at which relative final germination may be predicted This model indicated 50
declined in final germination at 030 salt concentration corresponding to ECiw= 42
dSm-1 (Table 16 Appendix II)
Rate of germination was significantly decreased (p lt 0001) from first day to final
(day 07) of observation and it was inversely correlated with sea salt concentration High
germination rate was recorded in control and low sea salt concentrations in early days of
seed incubation compared to higher sea salt concentrations but the difference in rate was
reduced (Table 17 Appendix II)
23
A progressive decline (p lt 0001) in coefficient of germination velocity was
observed with increasing salinity and fifty percent reduction was observed at 021 sea
salt concentration (ECiw = 319 dSm-1 Figure 11 Appendix II)
Final germination percentage was decreased significantly with increasing sea salt
concentrations However the difference was insignificant at lower (ECiw = 16 dSm-1)
salinity (Figure 11 Appendix II)
Mean germination time of seeds was increased significantly (p lt 0001) with
increasing sea salt concentrations However the difference was insignificant at lowest
(ECiw = 09 dSm-1) salinity (Figure 11 Appendix II)
Mean germination index was also significantly decreased (plt0001) with
increasing salt concentrations except for ECiw = 09 dSm-1 salinity Fifty percent reduction
in mean germination index was observed at 0188 sea salt concentration (ECiw = 289
dSm-1 Figure 11 Appendix II)
24
Table 15 Electrical conductivities of different sea salt solutions used in germination of C cajan
Sea salt () ECiw (dSm-1)
0 04
005 09
01 16
015 24
02 32
025 39
03 42
ECiw is the electrical conductivity of irrigation water measured in deci semen per meter
25
Table 16 Effect of irrigation water of different sea salt solutions on germination percentage (GP) per day of C cajan seeds pre-soaked in respective sea salt concentrations
with duration of time
Sea salt
ECiw (dSm-1)
GP
1st day
GP
2nd day
GP
3rd day
GP
4th day
GP
5th day
GP
6th day
GP
7th day
Control 6667plusmn333 8667plusmn333 10000plusmn000 10000plusmn000 10000plusmn000 10000plusmn000 10000plusmn000
09 7000plusmn000 7667plusmn333 9000plusmn000 10000plusmn000 10000plusmn000 10000plusmn000 10000plusmn000
16 4667plusmn333 6000plusmn000 7333plusmn333 8000plusmn000 8667plusmn333 8667plusmn333 9000plusmn577
24 4333plusmn333 5000plusmn000 6000plusmn577 6667plusmn333 7333plusmn333 7333plusmn333 8000plusmn000
32 3000plusmn000 3333plusmn333 3667plusmn333 4333plusmn333 5000plusmn577 6000plusmn577 7000plusmn577
39 1667plusmn333 2333plusmn333 2333plusmn333 4000plusmn577 4333plusmn333 5000plusmn000 6000plusmn000
42 667plusmn333 1333plusmn333 2333plusmn333 2333plusmn333 3333plusmn333 3667plusmn333 5000plusmn000
LSD 005 Salinity 327 Time 327
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and LSD among the treatments was recorded at p lt 005
25
26
Table 17 Effect of irrigation water of different sea salt solutions on germination rate (GR) per day of Ccajan
seeds pre-soaked in respective sea salt concentrations with duration of time
Sea salt
(ECiw= dSm-1)
GR
1st day
GR
2nd day
GR
3rd day
GR
4th day
GR
5th day
GR
6th day
GR
7th day
Control 667plusmn033 433plusmn017 333plusmn000 250plusmn000 200plusmn000 167plusmn000 143plusmn000
09 700plusmn000 383plusmn017 300plusmn000 250plusmn000 200plusmn000 167plusmn000 143plusmn000
16 467plusmn033 300plusmn000 244plusmn011 200plusmn000 173plusmn007 144plusmn006 129plusmn008
24 433plusmn033 250plusmn000 200plusmn019 167plusmn008 147plusmn007 122plusmn006 114plusmn000
32 300plusmn000 167plusmn017 122plusmn011 108plusmn008 100plusmn012 100plusmn010 100plusmn008
39 167plusmn033 117plusmn017 078plusmn011 100plusmn014 087plusmn007 083plusmn000 086plusmn000
42 067plusmn033 067plusmn017 078plusmn011 058plusmn008 067plusmn007 061plusmn006 071plusmn000
LSD 005 Salinity 014
Time 014 Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and LSD among the treatments is recorded at p lt 005)
27
Sea salt (ECiw = dSm-1
)
Contr
ol
09
16
24
32
39
42
Germ
ination Index(s
eedd
ays
-1)
0
2
4
6
8
Fin
al germ
ination (
)
0
20
40
60
80
100
Coeff
icie
nt of
germ
ination v
elo
city
(seedd
ays
-1)
00
01
02
03
04
05
06
07
Sea salt (ECiw = dSm-1
)
Contr
ol
09
16
24
32
39
42G
erm
ination tim
e (
Days
)
0
1
2
3
4
LSD005 = 0086
a = 0664 b = 1572
R2 = 0905 n =21
LSD005 = 062
a = 1239
b = 9836
R2 = 0894 n=21
LSD005 = 053
a = 8560b = -2272
R2 = 0969 n=21
RGF = 100-200 (Ke -005) Ke = 030
Figure 11 Effect of irrigation water of different sea salt solutions on seed germination indices of C cajan
(Bars represent means plusmn standard error of each treatment and significance among the treatments
was recorded at p lt 005)
28
14 Experiment No 3
Seedling establishment experiment of Cajanus cajan on soil irrigated with
sea salt of different concentrations
141 Materials and methods
1411 Seedling establishment
Seedling establishment experiment was carried out in Biosaline research field Department
of Botany University of Karachi Surface sterilized seeds pre-soaked were sown in small
plastic pots filled with 15 Kg sandy loam soil provided with farm manure at 91 ratio (30
water holding capacity) Sea salt solutions of different concentrations mentioned above
were used for irrigation The electrical conductivity of soil saturated paste (ECe) was also
determined at the end of the experiment (Table 18) Data on seedlings emergence was
recorded and their height were measured after 14 days of salinity treatment EC of the soil
(ECe) was initially 054 dSm-1 Statistical analyses were done according to the procedures
given in Experiment No 1
Since germination percentage of seeds pre-soaked in non-saline water was found
better under different concentrations of sea salt the seeds sown in soil for taking for
seedling establishment were pre-soaked in distilled water
29
142 Observations and Results
1421 Seedling establishment
Seedling emergence from soil was reduced significantly (p lt 0001) with increasing salt
concentration of irrigation water Not a single seedling emerged from soil in ge ECiw= 39
dSm-1 saline water irrigation However lower salinities (ECiw= 09 16 dSm-1) showed
slight decrease in seedling emergence with respect to controls Seedling emergence related
with salinity in accordance with a quadratic model as follows
Equation for seedling emergence () = 977751+ 44344 salt ndash 22215238 (salt)2 plusmn
6578 r = 09810 F = 15358 (p lt 00001)
Fifty percent reduction in seedling emergence was noticed at 016 sea salt
concentration (ECiw = 241 dSm-1 Figure 12 Appendix III)
1422 Shoot height
Shoot height was measured after fourteen days of irrigation Shoot length was
significantly decreased (p lt 0001) with increasing salinity A lower decrease was
observed in low sea salt salinity (ECiw= 09 and 16 dSm-1) compared to controls while
higher decrease in shoot height was noticed from ECiw= 2 dSm-1sea salt concentration
Shoot height related with salinity as follows
Equation for shoot height (cm) = 9116714 ndash 3420286 salt plusmn 09221 r = 0968 F =
128893 (p lt 0001)
Fifty percent reduction in shoot height was estimated at 013 sea salt concentration
(ECiw = 210 dSm-1) (Figure 12 Appendix III)
30
Table 18 Electrical conductivities of different Sea salt concentrations and ECe of soil saturated paste at the
end of experiment (ECe = 0447 + 1204 (salt ) plusmn 02797 R = 0987 F = 72301 (p lt
000001)
Sea salt () ECiw (dSm-1) ECe (dSm-1)
0 04 05
005 09 161
01 16 278
015 24 354
02 32 433
025 39 483
03 42 552
Electrical conductivity of soil saturated paste determined after 14 days of saline water irrigation in pots
Figure 12 Effect of irrigating water of different sea salt solutions on seedling emergence (A) and shoot
length (B) of C cajan (Bars represent means plusmn standard error of each treatment where similar
letters are not significantly different at p lt 005)
e f
Sea salt (ECiw = dSm-1
)
Contr
ol
16
27
8
35
4
43
3
48
3
Shoot le
ngth
(cm
)
0
2
4
6
8
10ab
c
de
Contr
ol
16
27
8
35
4
43
3
48
3Seedlin
g e
merg
ence (
)
0
20
40
60
80
100a
bb
c
d
A B
31
15 Experiment No 4
Growth and development of Cajanus cajan in Lysimeter (Drum pot
culture) being irrigated with water of different sea salt concentrations
151 Materials and methods
1511 Drum pot culture
A modified drum pot culture (lysimeter) installed by Ahmad amp Abdullah (1982) at
Biosaline research field (Department of Botany University of Karachi) was used in
present experiment Each drum pot (60 cm diameter 90 cm depth) was filled with 200 kg
of sandy loam mixed with cow-dung manure (91) having 28 water holding capacity
They are fixed at cemented platform at slanting position with basal hole to ensure rapid
drain Over irrigation was practiced to avoid the accumulation of salt in the root zone
1511 Experimental design
Growth and development of C cajan in drum pots was carried out in six different drum
pot sets (each in triplicate) and irrigated with sea salt of following concentrations
Drum pot Sets Sea salt
()
ECiw ( dSm-1) of
irrigation water
Resultant ECe (dSm-1) after
end of experiment
Set I Non saline (C) 04 05
Set II 005 sea salt 09 16
Set III 001 sea salt 16 28
Set IV 015 sea salt 24 35
Set V 02 sea salt 28 38
Set VI 025 sea salt 34 43
Note ECiw is the electrical conductivity of irrigation water and ECe is the electrical conductivity of the saturated soil extract taken after
eighteen weeks at the end of experiment
Ten surface sterilized seeds with 01 sodium hypochlorite were sowed in each
drum pot and were thinned to three healthy and equal size seedlings after two weeks of
establishment in their respective sea salt concentration Each drum pot was irrigated with
15 liters non-saline or respective sea salt solution at weekly intervals Electrical
conductivity of soil was measured by EC meter (Jenway 4510) using saturated soil paste
32
at the end of experiment Experiment was conducted for a period of 18 weeks (July to
November 2009) during which environmental data which includes average humidity
(midnight 76 and noon 54) temperature (low 23oC and high 33oC) wind velocity (14
kmph) and rainfall (~4 cm) was recorded (Pakistan Metrological Department Karachi) is
given in Figure 13Statistics were analysed according to the procedures given in
Experiment No 1
1512 Vegetative and Reproductive growth
Shoot height was measured at every two week interval after seedling establishment Fresh
and dry weight of shoot was recorded at final harvest (18th week when pods were fully
matured) Leaf succulence (dry weight basis Abideen et al 2014) Specific shoot length
(SSL Panuccio et al 2014) and relative growth rate (RGR Moinuddin et al 2014) were
measured using following equations
Succulence (g H2O gminus1 DW) = (FW minus DW) DW
SSL = shoot length shoot dry weight
RGR (g gminus1 dayminus1) = (lnW2 - lnW1) (t2 - t1)
Whereas FW fresh weight DW dry weight W1 and W2 initial and final dry weights and
t1 and t2 initial and final time of harvest in days
Reproductive data in terms of number of flowers number of pods number of seeds
and seed weight per plants was recorded during reproductive period
1513 Analysis on some biochemical parameters
Biochemical analysis of leaves was carried out at grand period of growth Following
investigations was undertaken at different biochemical parameters
i Photosynthetic pigments
Fresh and fully expended leaves (at 2nd3rd nodal part) samples (01g) were crushed in 80
chilled acetone and were centrifuged at 3000rpm for 10 minutes Supernatant were
separated and adjusted to 5ml final volume The absorbance was recorded at 663nm and
645 nm on spectrophotometer (Janway 6305 UVVis) for chlorophyll content while 480
33
and 510 nm for carotenoids Chlorophyll ab ratio was calculated after the amount
estimated The chlorophyll and carotenoid contents were determined according to Strain
et al (1971) and Duxbury and Yentsch (1956) respectively
Chlorophyll a (microgml) = 1163 (A665) ndash 239 (A649)
Chlorophyll b (microgml) = 2011 (A649) ndash 518 (A665)
Total Chlorophylls (microgml) = 645 (A665) + 1772 (A649)
Carotenoids (microgml) = 76 (A480) ndash 263 (A510)
ii Total soluble sugars
Dry leaf samples (01g) were homogenized in 5mL of 80 ethanol and were centrifuged
at 4000 g for 10 minutes 10 mL diluted supernatant in 5mL Anthronrsquos reagent was kept
to boil in 100oC water bath for 30 minutes and were cooled in running tap water Optical
density was taken at 620nm for the determination of soluble carbohydrates according to
Fales (1951)Total soluble carbohydrates was estimated against glucose as standard and
was calculated from the equation mentioned and expressed in mgg-1 dry weight
Total carbohydrates (microgmL-1) = 228462 OD 097275 plusmn004455
iii Protein content
Fresh and fully expended leaves at 2nd3rd nodal part were taken for protein estimation
The protein contents were measured according to Bradford Assay reagent method against
Bovine Serum Albumin as standards (Bradford 1976) Dye stock was made to dissolved
50mg comassie blue in 25 ml methanol The solution is added to 50ml of 85 phosphoric
acid and diluted to 100 ml with distilled water 02g fresh leaf samples were mills in 5 ml
phosphate buffer pH7 5ml of assay reagent (diluting 1 volume of dye stock with 4 volume
distilled water) were added in 01 ml leaf extract used for enzyme assay Absorbance was
recorded at 590nm and was expressed in mgg-1 fresh weight Proteins were calculated
from the following best fit standard curve equation
Protein (microgml-1) = -329196 + 1142755 plusmn 53436
34
152 Observations and Results
1521 Vegetative and Reproductive growth
Effect of sea salt on vegetative growth including height fresh and dry weight of Cajanus
cajan is presented in (Figure 14 and 15 Appendix-VI) Comparative analysis showed
that plant growth (all three parameters) was significantly increased with time (plt 0001)
however it was linearly decreased (plt 0001) with increasing salinity (Figure 16
Appendix-VI) shows the water content succulence relative growth rate (RGR) and
specific shoot length (SSL) of Cajanus cajan Under saline conditions all parameters were
significantly reduced in comparison to control however SSL showed decline after ECe38
dSm-1 Salt induced growth reduction was more pronounced at ECe 38 and 43 dSm-1 in
which plants died before reaching the reproductive maturity after 12 and 14 weeks at sea
salt treatments respectively Therefore further analysis was carried out in plant grown up
to ECe= 35 dSm-1 sea salt concentrations
Salinity significantly reduced (plt 0001) reproductive parameters including
number of flowers pods seeds and seed weight (Figure 17 Appendix-VII) Among all
treatments highest reduction was observed in 315 dSm-1 in which number of flowers and
pods reduced up to 7187 and 70 respectively Similar trend was observed in total
number and weight of seeds which showed 80 and 8793 reduction respectively
1522 Study on some biochemical parameters
i Photosynthetic pigments
Figure 18 Appendix-VII shows the effect of salinity on pigments (chlorophyll a b ab
ratio and carotenoids) of C cajan leaves A slight increase in total chlorophyll contents
(1828) and chlorophyll ab ratio (1215) was observed at low salinity (ECe= 16 dSm-
1) however they were significantly reduced (4125 and 3630 respectively) in high salt
treatment (plt 0001) Chlorophyll a was higher than chlorophyll b in all treatments
however chlorophyll b was un-affected by salinity whereas total chlorophyll content and
ab ratio was disturbed due to change in chlorophyll a This reduction was more
pronounced at high salinity (ECe= 35 dSm-1) in which chlorophyll a total chlorophylls
and ab ratio was decreased by 505 412 and 3630 respectively Carotenoid content
was maintained at ECe= 16 dSm-1 and decreased with further increase in salinity
35
ii Total soluble sugars
Total soluble sugars in leaves of C cajan is presented in Figure 19 Appendix-VII Total
leaf sugars in C cajan were remained un-affected at 16 dSm-1 and subsequently decreased
with further increase in medium salinity Although total sugars were decreased at ECe 28
and 35 dSm-1 a significant increase (~25) of soluble sugars was observed at higher
salinities However this increment was accounted for decrease (504 ) in insoluble sugar
content at that salinity levels
iii Protein
Total protein in leaves of C cajan is presented in Figure 19 Appendix-VII An increase
in leaf protein content in C cajan was found at lower salinity regime (ECe= 16 dSm-1)
which was followed by significant reduction with further increase in salinity This decline
was 2040 at 28 which was more pronounced (5646 ) at high salinity level (ECe=
35dSm-1)
36
Months (2009)
Jun Jul Aug Sep Oct Nov Dec
Valu
es
0
10
20
30
40
50
60
70
80
90
Rainfall (cm)Low Temp (
oC)
High Temp (oC)
Humidity at noon () Wind (kmph)
Humidity at midnight ()
Figure 13 Environmental data of study area during experimental period (July-November 2009)
Time (Weeks)
2 4 6 8 10 12 14 16 18
Pla
nt heig
ht (c
m)
0
30
60
90
120
150
180
210
43 38 35 28 16 Control
Figure 14 Effect of salinity using irrigation water of different sea salt concentrations on height of C cajan
during 18 weeks treatment (Lines represent means plusmn standard error of each treatment represents
significant differences at p lt 005)
37
Sea salt (ECe= dSm
-1)
Cont 16 28 35 38 43
Sea salt (ECe= dSm
-1)
Cont 16 28 35 38 43
Fre
sh w
eig
ht (g
)
0
5
10
15
20
25
30
35Initial Final
a
b b
c c cab b
c c cC 16 28 35 38 43
Fre
sh w
eig
ht
(g)
012345 a
bb
bc ca a ab b c c
Dry weightMoisture
Figure 15 Effect of salinity using irrigation water of different sea salt concentrations on initial and final
biomass (fresh and dry) of C cajan (Bars represent means plusmn standard error of each treatment Different
letters represent significant differences at p lt 005)
Mo
istu
re (
)
0
20
40
60
80
100
Succu
lance
(
)
0
20
40
60
80
100
Sea salt (ECe= dSm
-1)
Co
nt
16
28
35
38
43
RG
R (
)
0
20
40
60
80
100
Co
nt
16
28
35
38
43
SS
L (
)
0
20
40
60
80
100
Sea salt (ECe= dSm
-1)
ab
b b
c c
a
b bc c c
a
b b
c c c
a a a ab
c
Figure 16 Percent change (to control) in moisture succulence relative growth rate (RGR) and specific
shoot length (SSL) of C cajan under increasing salinity using irrigating water of different sea
salt concentrations (Bars represent means plusmn standard error of each treatment Different letters
represent significant differences at p lt 005)
38
Sea salt (ECe= dSm-1)
Control 16 28 35
Tota
l seeds (
Pla
nt-1
)
0
20
40
60
80
100
120
140 Seed w
eig
ht (g
pla
nt -1
)
0
5
10
15
20
25
Num
ber
10
20
30
40
50
60
70 a
b
cc
a
a
b
b
b c
c
a
b
a
c c
Flowers
Pods
Seed weightTotal seeds
Figure 17 Effect of irrigating water of different sea salt solutions on reproductive growth parameters
including number of flowers pod seeds and seed weight of C cajan (Values represent means
plusmn standard error of each treatment Different letters represent significant differences at p lt
005)
39
Sea salt (ECe=dSm-1
)
Control 16 28 35
Caro
tinoid
s (
mg g
-1 F
W)
000
005
010
015
020
025
030
Chlo
rophyll
(mg g
-1 F
W)
00
02
04
06
08
ab
ratio
00
05
10
15
20
25
ab
ab
b
a
cd
b
a
c
d
a
b
c
d
a
a
ab
b
Figure 18 Effect of irrigating water of different sea salt solutions on leaf pigments including chlorophyll a
chlorophyll b total chlorophyll and carotenoids of C cajan (Bars represent means plusmn standard
error of each treatment Different letters represent significant differences at p lt 005)
40
Figure 19 Effect of irrigating water of different sea salt solutions on total proteins soluble insoluble and
total sugars in leaves of C cajan (Bars represent means plusmn standard error of each treatment
Different letters represent significant differences at p lt 005)
Sea salt (ECe= dSm
-1)
C 16 28 35
Pro
tein
(m
g g
-1 F
W)
00
01
02
03
04
05
06
Su
gar
s (m
g g
-1 F
W)
00
02
04
06
08
a ab b
a a
b b
a ab b
a
b
ab
c
SoluableInsoluable
41
16 Experiment No 5
Range of salt tolerance of nitrogen fixing symbiotic bacteria associated
with root of Cajanus cajan
161 Materials and methods
1611 Isolation Identification and purification of bacteria
Nodules of C cajan grow in large clay pots and irrigated with running tap water at
biosaline agriculture research field were collected from the lateral roots (about 15 cm soil
depth) Nodules were surface sterilized with sodium hypochloride (2) for 5 min and
vigorously washed with sterilized distilled water Each nodule was crushed with sterilized
rod in 5 ml distilled water The bacterial suspension was streaked on yeast extract mannitol
agar (YEM) (K2HPO4 05 g MgSO 4 025g Na Cl 01 g Manitol 10g Yeast Extract 1g
Agar 20 g in 1000 ml of Distilled water) with the help of sterilized wire lope Colonies
were identified by studying different phenotypic characters as Rhizobium fredii
(Cappuccino and Sherman 1992 Sawada et al 2003) Pure culture of Rhizobium species
was stored at -20oC temperature
1612 Preparation of bacterial cell suspension
Bacteria were multiplied by growing in YEM broth for 48 hrs on shaking incubator (140
rpm) at 37oC in dark The culture in broth was centrifuged at 4000 rpm for 10 min to
obtained bacterial cell pellet Pellet was washed and centrifuged twice with sterilized
distilled water Pellet then re-suspended in sterilized distilled water before use
1613 Study of salt tolerance of Rhizobium isolated from root nodules of
C cajan
Assessment for salinity tolerance of Rhizobium species was assessed on YEM agar
Salinity levels of 0 05 10 15 20 25 and 30 having electrical conductivity 06 90
188 242 306 366 and 423 dSm-1 respectively were maintained with NaCl Bacterial
cell suspension of 01 ml (5times 103 colony forming unitsml) was poured in each sterilized
Petri dish 10 ml of molten YEM agar was poured immediately and shake well before
solidification of agar Petri plates were incubated at 37deg C in dark Colonies were observed
and counted in colony counter after 48 h and photographed (Dubey et al 2012 Singh and
42
Lal 2015) There were three replicates of each treatment and data were transformed to
log10 before analysis
162 Observations and Results
Colonies of Rhizobium on YEM agar at different salinity levels is presented in Figure 110
and 111 Appendix-VIII A significant decrease (plt0001) in rhizobial colonies was
observed with increasing salinity However the difference between non saline control and
90 dSm-1 and as that of 242 dSm-1 and 302 dSm-1 salt (NaCl) concentration showed
nonsignificant difference in rizobial colonies Whereas drastic decreased was observed on
further salinity levels Rhizobial colonies were not found at 423 dSm-1salt concentration
NaCl (ECw= dSm
-1)
06 9 188 242 306 366 423
Rh
izo
bia
l co
lonie
s (l
og
10)
0
1
2
3
4 a a
b
c c
d
e
Figure 110 Growth of nitrogen fixing bacteria associated with root of C cajan under different NaCl
concentrations (Bars represent means plusmn standard error of each treatment among the treatments
is recorded at p lt 005)
43
Figure 111 Photographs showing growth of Rhizobium isolated from the nodules of C cajan invitro on
YEM agar supplemented with different concentrations of NaCl (ECw)
188
423 90
Control
366
306 242
44
17 Experiment No 6
Growth and development of Ziziphus mauritiana in large size clay pot
being irrigated with water of two different sea salt concentrations
171 Materials and methods
1711 Experimental design
The grafted plants obtained from the local nursery of Mirpurkhas Sindh were transported
to the Biosaline Agriculture Research field Department of Botany University of Karachi
and were transplanted carefully in large earthen pots containing 20 Kg sandy loam soil
mixed with cow dung manure at 91 ratio having about 5 liters of water holding capacity
with a basal hole for drainage of excess salts to avoid accumulation in the rhizosphere
Over irrigation with about 15 liters of non-saline saline water was kept weekly in summer
and biweekly in winter to avoid accumulation of salts in rhizosphere Plants were irrigated
to start with non-saline tap water for about two weeks for establishment All the older
leaves were fallen and new leaves were developed during establishment period Following
irrigation schedule of non-saline (control) and saline water was selected in view of Z
mauritiana being moderately salt tolerant plant which includes both low and as well as
higher concentrations of the salt in irrigation
Sea salt () ECiw (dSm-1)
of irrigation water
Average resultant ECe (dSm-1) of soil
with some fluctuation often over
irrigation
Non saline (Control) 06 12
04 63 72
06 101 111
ECiw = Electrical conductivity of irrigation water ECe = Electrical conductivity of saturated soil
Healthy and well established plants were selected of nearly equal height and
divided into three sets each contain three replicates (total nine pots) Salinity was provided
through irrigation water of different sea salt concentrations All pots except non-saline
control were initially irrigated with 01 sea salt solution and then sea salt concentration
45
in irrigation medium was increased gradually upto the required salinity level The salinity
level of soil was monitored by taken the electrical conductivity of saturated soil paste the
end of experiment The electrical conductivity of soil (ECe) maintained at the level of 12
72 and 111 dSm-1 respectively as described by Mass and Hoffman (1977)
1712 Vegetative and reproductive growth
Vegetative growth in terms of shoot height fresh and dry weight of shoot and number of
branches were noted at destructive harvesting at initial (establishment) 60 and 120 days
of growth For dry weight shoots were dried in oven at 70˚C for three days Shoot
succulence specific shoot length (SSL) moisture percentage and relative growth rate
(RGR) was calculated at final harvest by using formulas given in Experiment No 4
Whereas number of flowers in reproductive data were recorded at onset of reproductive
period
As regard of fruit formation the duration of experiment was not sufficient for fruit
setting and furthermore the amount of sol in pots was not sufficient for healthy growth of
this plant Secondly flowering and fruiting is reported to be poor at the time of 1st initiation
of reproductive period (Azam-Ali 2006) Furthermore statistical significance of flower
and fruit count also become far less due to their excess dropping at early stage Hence it
was decided to proceed with study of fruit formation in forthcoming field trials of their
intercropping culture
1713 Analysis on some biochemical parameters
Biochemical analyses were performed at the grand period (at the time of flower initiation)
in fully expended fresh leaves Chlorophyll contents soluble sugar contents and soluble
proteins were analyzed Leaves samples taken from 3rd 4th node below the apex according
to the procedures given in Experiment No 4
46
172 Observations and Results
1721 Vegetative and Reproductive growth
Effect of sea salt on vegetative growth of Z mauritiana including height fresh and dry
weight is presented in (Figure 112 Appendix-IX) Comparative analysis showed that
plant growth (all three parameters) was significantly increased with time (plt 0001)
however number of branches was decreased (plt 0001) with increasing salinity
Figure 113 shows the moisture content succulence relative growth rate (RGR)
and specific shoot length (SSL) of Z mauritiana A non-significant difference in shoot
succulence SSL and moisture content was observed with time salinity and interaction of
both factors However RGR showed decline Salt induced growth reduction was more
pronounced at higher salinities
In Z mauritiana plants number of flowers showed significant decrease (plt0001)
with increasing salinity treatment Flower initiation seems non-significant at early growth
(60 days) period in controls and salinity treatments However drastic decrease was
observed with increasing salinity in 120 days of observation (Figure 114 Appendix-IX)
1722 Study on some biochemical parameters
i Photosynthetic pigments
The effect of Z mauritiana leaves pigments (chlorophyll a b ab ratio) on salinity shower
a slight difference in chlorophyll lsquoarsquo over control However chlorophyll lsquobrsquo contents
showed increase over control in both salinity treatments due to which the total chlorophylls
were also enhanced compared to controls Chlorophyll ab ratio was significantly
(plt0001) decreased in both salinities as compared to control (Figure 115 Appendix-IX)
ii Sugars and protein
In Z mauritiana plant soluble sugars were significantly decreased (plt0001) over controls
whereas proteins showed little decrease under salinity treatments compared to controls
(Figure 116 Appendix-IX)
47
Control 72 111
Fre
sh w
eig
ht (g
)
0
150
300
450
600
750
900
Sea salt (ECe= dSm
-1)
Control 72 111
Dry
weig
ht (g
)
0
150
300
450
600
750
900
Num
ber
of bra
nches
3
6
9
12
15
18
Heig
ht (c
m)
20
40
60
80
100
120
140
160
Initial 60 days 120 days
AcBb
Ba
AcBb Ba
AcBb Ba
Ac
BbBa
Figure 112 Effect of salinity using irrigation water of different sea salt concentrations on height number of
branches fresh weight and dry weight of shoot of Zmauritiana after 60 and 120 days of
treatment (Bars represent means plusmn standard error of each treatment Different letters represent
significant differences at p lt 005)
48
120 days 60 days InitialS
uccula
nce (
g g
-1 D
W)
00
03
06
09
12
Sea salt (ECe= dSm
-1)
SS
L (
cm
g-1
)
00
01
02
03
04
05
Control 72 111
Mois
ture
(
)
0
10
20
30
40
50
60
Control 72 111
RG
R (
mg g
-1 d
ay
-1)
0
5
10
15
20
a a aa a a a a a a
a aa a a a a a
a a aa a a a a a a a
b
b b
c
Figure 113 Effect of salinity using irrigation water of different sea salt concentrations on succulence
specific shoot length (SSL) moisture and relative growth rate (RGR) of Z maritiana (Bars
represent means plusmn standard error of each treatment Different letters represent significant
differences at p lt 005)
49
Sea salt (ECe= dSm
-1)
Control 72 111
Num
ber
of flow
ers
0
20
40
60
80
100
120
140 60 days120 days
Ac
BbBa
Figure 114 Effect of salinity using irrigation water of different sea salt concentrations on number of flowers
of Z mauritiana (Bars represent means plusmn standard error of each treatment Different letters
represent significant differences at p lt 005)
Sea salt (ECe= dSm
-1)
Control 72 111
Ch
loro
ph
yll
(mg g
-1)
00
03
06
09
12
15
18
bba
bba
bb
a
chl b chl a ab
ab
ra
tio
00
05
10
15
20
Figure 115 Effect of salinity using irrigation water of different sea salt concentrations on leaf pigments
including chlorophyll a chlorophyll b total chlorophyll and chlorophyll ab ratio of Z mauritiana (Values
represent means plusmn standard error of each treatment Different letters represent significant differences at p lt
005)
50
Figure 116 Effect of salinity using irrigation water of different sea salt concentrations on total sugars and
protein in leaves of Z mauritiana (Bars represent means plusmn standard error of each treatment
Different letters represent significant differences at p lt 005)
Sea salt (ECe= dSm
-1)
C 04 06
Pro
tein
s (m
g g
-1)
0
10
20
30
40
50
60
70
80
Solu
ble
sugar
s (m
g g
-1)
0
3
6
9
12
15
18a
a
bb
b b
Control 72 111
51
18 Discussion
Seed germination is the protrusion of radicle from the seed which is adversely affected by
salinity stress (Kaymakanova 2009) Salinity imposes the osmotic stress by accumulation
of Na+ and Cl- which decrease soil water potential that ultimately inhibits the imbibition
process (Othman 2005) Effect of seed germination against salinity is reported in linear
threshold response model of Maas and Hoffman (1977) The germination of a salt tolerant
desert legume Indigofera oblongifolia and a desert graminoid Pennisetum divisum are
also reported to behave to salinity in similar manner (Khan and Ahmad 1998 2007) Many
workers used chemical (organic inorganic) salt temperature biological and soil matrix
priming techniques to enhance seed germination percentage and especially germination
rate in saline medium (Ashraf et al 2008 Ashraf and Foolad 2005)Encouraging results
in most of the species of glycophytes and hydrophytes were found by presoaking in pure
water prior to germinating under saline condition Our study supports this finding and
seeds soaked in distilled water prior to germination performed better than those which
were presoaked in sea salt solutions Salinity adversely affects at all germination
parameters (germination percentage germination rate coefficient of germination velocity
and germination index) directly proportional with increasing salinity (Tayyab et al 2015)
With increase in time a delayed germination at higher salinity was found Higher sea salt
(168 dSm-1 for pure water presoaking and 35 dSm-1 for presoaking in respective
salinities) showed 50 or more reduction in all germination indices as compared to control
(Table 13-16 Figure 11)Our results are parallel with the finding of other workers such
as Kafi and Goldani (2001) who found the same trend in chickpea at higher salinities Pujol
et al (2000) reported that increased salinity inhibit the seed germination as well as delays
germination initiation in various halophyte species as well Similar response was also
found in some other crops such as pepper (Khan et al 2009) sunflower (Vashisth and
Nagarjan 2010) and eggplant (Saeed et al 2014) Salt tolerance within species may vary
at germination and other growth phases (Khan and Ahmad 1998)
According to our results C cajan appeared to be a salt sensitive in initial growth
phase specially when presoaked in saline medium (Figure 12) however at later growth
stages it proved relatively salt tolerant Salt stress delays or either seize the metabolic
activities during seed germination in salt sensitive and even in salt tolerant plants (Khan
and Ahmad 1998 Ali et al 2013b) Salinity also imposes the oxidative stress due to
52
overproduction of reactive oxygen species which may alter metabolic activities during
germination growth and developmental stages (Zhu 2001 Munns 2005
Lauchli and Grattan 2007)
In our study seeds of pigeon pea were unable to emerge beyond ECe39 dSm-1 sea
salt concentration Height of seedling was significantly affected by increasing salinity
(Figure 12) Similar results are also reported in Indian mustered (B juncea Almansouri
et al 2001) some Brassica species (Sharma et al 2013) and tomato cultivars (Jamil et
al 2005) Growth retardation with increasing salinity may be due to reduced
photosynthetic efficiency and inhibition of enzymatic and non-enzymatic proteins
(Tavakkoli et al 2011) Furthermore salt stress also limit the DNA and RNA synthesis
leads to reduced cell division and elongation during germination growth and
developmental stage
Khan and Sahito (2014) found variation in salt tolerance within species subspecies
and provenance level Furthermore the salt tolerance of a species may also vary at
germination and growth phases (Khan and Ahmad 1998 Ali et al 2013a) Srivastava et
al (2006) suggested that the genetic variability influences salinity tolerance eg wild
species like Cajanus platycarpus C scaraboides and C sericea showed better salt
tolerance than C cajan In this connection Wardill et al (2006) has also reported genetic
diversity in Acacia nilotica C cajan in this study appeared to be a salt sensitive at
germination in compression with later stages of growth Seedling establishment at saline
solution faces adverse effects when emerging radicle and plumule come in contact with
salt effected soil particle or saline water hence percent seedling establishment remains
less than germination percentage observed at petri plate Ashraf (1994) found that salinity
tolerance of different varieties of C cajan do not much differ at germination and early
growth stages whereas at adult growth stage show improvement in salt tolerance
Soil salinity is a major limiting factor for plant growth and yield production
particularly in leguminous plants (Guasch-Vidal et al 2013 Tayyab et al 2016) In
present study Plant height RGR fresh and dry biomass were severely reduced with
increasing salinity and plant was unable to grow after ECe= 43 dSm-1(Figure 14-16)
This growth inhibition of C cajan may be accounted for individual and synergistic effect
of water stress nutrient imbalances and specific ions toxicities (Hasegawa et al 2000
Silvera et al 2001) Salt induced ion imbalance results in lower osmotic potential which
53
alter physiological biochemical and other metabolic processes leading to overall growth
reduction (Del-Amor et al 2001) Excessive amount of salt in cytoplasm challenge the
compartmentalization capacity of vacuole and disrupts cell division cell elongation and
other cellular processes (Munns 2005 Munns et al 2006) Our results are parallel with
some other studies in which significant growth inhibition of peas chickpea and faba beans
have been reported against salt stress (El-Sheikh and Wood 1990 Delgado et al 1994)
Singla and Garg (2005) also observed a similar salt sensitive growth response in Cicer
arietinum In our study the fresh and dry biomass of C cajan also showed inhibitory
behavior to salt stress (Figure 15) Hernandez et al (1999) also found significant reduction
in dry biomass of pea plant and common bean (40 and 84 respectively) when grown
in saline medium Mehmood et al (2008) also found similar results in Susbania sasban
Salinity also has imposed deleterious effects on reproductive growth of C cajan
Production of flowers and pods are significantly decreased in response to salinity (Figure
19) Increase in flower shedding leads to decreased number of pods indicating salt
sensitivity of plant at reproductive phase which was more pronounced at high salinity
(Vadez et al 2007) Furthermore seed production and weight of seed per plant was also
linearly decreased Salt induced reduction of reproductive growth has also been found in
mung bean in which 60 and 12 less pods and seeds were produced respectively at 06
saline solution (Qados 2010) Similar results are reported in faba bean (De-Pascale and
Barbieri 1997) tomato (Scholberg and Locascio 1999) maiz sunflower (Katerji et al
1996) and watermelon (Colla et al 2006) Salinity reduces reproductive growth by
inhibiting growth of flowers pollen grains and embryo which leads to inappropriate ovule
fertilization and less number of seeds and fruits (Torabi et al 2013)
On biochemical parameters total chlorophyll and chlorophyll ab ratio has
increased in low salinity in contrast the adverse effect at higher salinity could be due to
high Na+ dependent breakdown of these pigments (Li et al 2010 Yang et al 2011)
Chlorophyll a is usually more prone to Na+ concentration and decrease in total chlorophyll
is mainly attributed to the destruction of chlorophyll a (Fang et al 1998 Eckardt 2009)
This diminution could be due to the destruction of enzymes responsible for green pigments
synthesis (Strogonov et al 1973) and increased chlorophyllase activity (Sudhakar et al
1997) Thus insipid of leaf was a visible indicator of salt induced chlorophyll damage
which was well correlated with quantified values as reported in other legume species
54
(Soussi et al 1998 Al-Khanjari et al 2002) In this study chlorophyll a was found to be
more sensitive than chlorophyll b (Figure 18) Garg (2004) also found similar reduction
in chlorophyll pigments (a b and total chlorophyll) in chickpea cultivars under salinity
stress
At low salinity (16 dSm-1) total carotenoids remained unaffected along with
increased total chlorophyll (Figure 18) which may suggest a role of carotenoids in
protection of photosynthetic machinery (Sharma et al 2012) Similar response was found
in Cajanus indicus and Sesamum indicum (Rao and Rao 1981) however
Sivasankaramoorthy (2013) and Ramanjulu et al (1993) reported slight increase of leaf
carotenoids in Zea maiz and mulberry when exposed to NaCl High salinity was destructive
for both leaf pigments (chlorophyll and carotenoids) of C cajan which was in accordance
with Reddy and Vora (1985) who found similar decrease in some other salt sensitive crops
Salinity led to the conversion of beta-carotene to Zeaxanthin which protect plants against
photo-inhibition (Sharma and Hall 1991)
In present study with increasing salinity water content and succulence of C cajan
were significantly reduced which indicated loss of turgor (Figure 16) Our data suggest
that decreased succulence by lowering water content may help in lowering leaf osmotic
potential when exposed to increasing salinity which is in agreement with findings of Parida
and Das (2005) and Abideen et al (2014) In addition increased production and
accumulation of organic substances is also necessary to sustain osmotic pressure which
provide osmotic gradient to absorb water from saline medium (Hasegawa et al 2000
Cha-um et al 2004) Compatible solutes including carbohydrates amino acids proteins
and ammonium compounds play important roles in water relations and cell stabilization
(Ashraf and Harris 2004) In this study C cajan produce more soluble sugars (Figure 18)
which is considered as a typical plant response under saline conditions (Murakeozy et al
2003) Sugars serve as organic osmotica and their available concentration is related to the
degree of salt stress and plantrsquos tolerance (Ashraf 1994 Murakeozy et al 2003) Sugars
are involved in osmoprotection osmoregulations carbon storage and radical scavenging
activities (Pervaiz and Satyawati 2008) On the other hand insoluble and total sugars were
reduced in higher salinity which is also supported by Parida et al (2002) and Gadallah
(1999) who found similar results in Bruguiera parviflora and Vicia faba
55
Total soluble proteins of C cajan were reduced due to deleterious effects of salinity
(Figure 18) The accumulation of Na+ in cytosol disrupts the protein and nucleic acid
synthesis (Bewley and Black 1985) Gill and Sharma (1993) and Muthukumarasamy and
Panneerselvam (1997) also reported decreased protein content with increasing salinity in
Cajanus cajan seedlings Similar results were found when tomato (Azeem and Ahmad
2011) Zingiber officinale (Ahmad et al 2009) and Sorghum bicolor (Ali et al 2013a)
were grown under variable salt concentrations (Figure 19)
Nodule formation of Rhizobium in Legume depends upon interaction between soil
chemistry of salt composition and osmotic regimes of salt and water (Velagaleti et al
1990 Zahran 1991 Zahran and Sprent 1986) Salinity reduces plant growth directly
through ion and osmotic effects and indirectly by inhibiting Legume-Rhizobium
association (El-Shinnawi et al 1989) Studies demonstrated a more sensitive response of
rhizobial N-fixing mechanism than growth of plant to abiotic stresses including salinity
(Mhadhbi et al 2004) In nodules metabolic disturbance initiated with the production of
ROS leading to tissues injury and loss of nodule function (Becana et al 2000) In general
it slow down the nitrogenase activity and decrease nodule protein and leghemoglobin
content which decreased becteroid development (Mhadhbi et al 2008) In consequence
plant suffer directly by salt induced ion toxicity low water uptake and photosynthetic
damage and indirectly through weak association of symbionts due to high energy demand
for nodule function (Pimratch et al 2008) In our study the isolated rhizobial strain from
nodules of C cajan was found to be tolerant to salinity even up to 2 (ECw= 306 dSm-1)
NaCl (Figure 110 and 111) Some of the other species of Rhizobium such as Brady
Rhizobium have been shown salt tolerant even at higher concentration than their
leguminous hosts (Zahran 1999) For instance a number of rhizobial species can tolerate
up to 06 NaCl (Yelton et al 1983) while Rhizobium meliloti can tolerate 175 to
40 NaCl and R leguminosarum can tolerate can tolerate upto 2 NaCl (Abdel-Wahab
and Zahran 1979 Sauvage et al 1983 Breedveld et al 1991 Helemish 1991
Mohammad et al 1991 Embalomatis et al 1994 Mhadhbi et al 2011) Rhizobia
isolated from soybean and chickpea can tolerate up to 2 NaCl with a difference of fast-
growing and slow growing strains (El-Sheikh and Wood 1990 Ghittoni and Bueno 1996)
Similarly Rhizobium from Vigna unguiculata can survive up to up to 55 NaCl
(Mpepereki et al 1997)
56
Present study shows an increase in vegetative growth in terms of plant height and
fresh and dry weight of shoot with increasing time under non-saline and saline conditions
but the increase was rapid at early period of growth (Figure 112) All the vegetative
growth parameters determined were reduced under salinity stress compared to non-saline
control Measurements of shoot moisture succulence specific shoot length and RGR
(Figure 113) indicate that Z mauritiana adjusted in its water relation over coming
negative water and osmotic potential with increase in salinity levels increased There is
evidence that water and osmotic potentials of salt tolerant plants become more negative in
higher salinities (Khan et al 2000) These altered water relations and other physiological
mechanisms help plants to get by adverse abiotic stress like that of drought and salinity
(Harb et al 2010) However the results clearly showed that salinity had an inhibitory
effect on growth but the decline was less at early sixty days and more during later 60-120
days in compression to controls Growth inhibition in shoot has been observed in number
of plants including different species of halophytes (Keiffer and Ungar 1997) chickpea
(Cicer arietinum Kaya et al 2008) and different wheat cultivars (Triticum aestivum
Moud and Maghsoudo 2008)
Salinity also caused reduction in the number of branches and the number of flowers
in Z mauritiana however reduction in the number of flowers is non-significant in ECe=
72 dSm-1 salinity treatment in comparison with non-saline control (Figure 114) The main
reason for this reduction could be attributed to suppression of growth under salinity stress
during the early developmental stages (shooting stage) of the plants These results are
similar to those reported by Ahmad et al (1991) and Khan et al (1998) As affirmed by
Munns and Tester (2008) suppression of plant growth under saline conditions may either
be due to osmotic effect of saline solution which decreases the availability of water for
plants or the ionic effect due to the toxicity of sodium chloride High salt concentration in
rooting medium also reduced the uptake of soil nutrients a phenomenon which affected
the plant growth thus resulting in less number of branches per plant Various abiotic
stresses such as temperature drought salinity light and heavy metals altered plant
metabolism which ultimately affects plant growth and productivity Amongst these
salinity stress is a major problem in arid and semiarid regions of the world (Kumar et al
2010) Salinity has an adverse effect on several plant processes including seed
germination seedling establishment flowering and fruit formation and ripening (Sairam
and Tyagi 2004) Salinity stress also imposes additional energy requirements on plant
57
cells and less carbon is available for growth and flower primordial initiation (Cheesman
1988) The lesser decrease in number of flowers at lower salinity (ECe= 72 dSm-1) has
been attributed to the fact that the cells of apex are un-vacuolated and the incoming salts
accumulated in the cytoplasm Munns (2002) further suggested a well-controlled phloem
transport of toxic ions from these cells prevented any change in reproductive development
Our findings showed an increase in total chlorophyll contents particularly
chlorophyll b contents were enhanced more than chlorophyll a contents under salinity
stress (Figure 115) In general the total chlorophyll contents decreased under high salinity
stress and this may be due to accumulation of toxic ions in photosynthetic tissues and
functional disorder of stomatal opening and closing (Khan et al 2009) The increase in
total chlorophylls appearing at salinity levels is considered as an important indicator of
salinity tolerance in plants (Katsuhara et al 1990 Demiroglu et al 2001) In another
study on Z mauritiana (cv Banara sikarka) the chlorophyll contents has shown decrease
with increasing salinity and sodicity but the seedlings treated with low salinity (ECe of 5
mmhoscm-1) shows slightly higher values than controls (Pandey et al 1991) Our study
also suggests that increase in total chlorophylls adapted this plant increased its tolerance
to salt stress
Slight decrease in protein has been shown under salinity treatments compared to
controls (Figure 16) Proteins play diverse roles in plants including involvement in
metabolic pathways as enzyme catalyst source of reserve energy and regulation of osmotic
potential under salt stress (Pessarakli and Huber 1991 Mansour 2000) Salts may
accumulate in cell cytoplasm and alter their viscosity depending on the response of plant
to salinity stress (Hasegawa et al 2000 Paravaiz and Satyawati 2008) The decrease in
protein contents under increasing salinity has also been documented in several plants
including Lentil lines (Ashraf and Waheed 1993) sorghum (Ali et al 2013a) and sugar
beet (Jamil et al 2014)
Soluble sugars were also decreased with increasing salinity treatments in our study
(Figure 16) Decrease in soluble sugars due to salinity has also been reported in Viciafaba
(Gadallah 1999) some rice genotypes (Alamgir and Ali 1999) Bruguiera parviflora
(Parida et al 2002) and Lentil (Sidari et al 2008) However the accumulation of soluble
sugars under salinity stress is considered as strategy to tolerate stress condition due to their
58
involvement in osmoprotection osmotic adjustment and carbon storage (Parida et al
2002 Parvaiz and Satyawati 2008)
From these experiments it is evident that C cajan is a salt sensitive plant at every
level of its life cycle starting from germination to growth phases Germination capacity
and salt tolerance ability of this species can be enhanced by water presoaking treatment
Growth reduction with increasing salinity could be attributed to physiological and
biochemical disturbances which ultimately affect vegetative and plant reproductive
growth Its roots are well associated with nitrogen fixing rhizobia and these
microorganisms were salt tolerant in in-vitro cultures Another fruit baring species of
marginal lands Z mauritiana showed growth improvement in lower salinity and its growth
was not much affected in high saline mediums owing to its controlled biochemical
responses
59
2 Chapter 2
Intercropping of Z mauritiana with C cajan
21 Introduction
Increasing soil salinity fresh water scarcity and agricultural malpractice creating shortage
of food crops for human and animal consumption (Bhandari et al 2014) and making
prices high Traditional agriculture which has been practiced since centuries using multi
species at a time in a given space could be a potential solution to narrow down the growing
edges of this supply demand scenario Plant species with innate resilience to abiotic
stresses like salinity and drought could be considered suitable to serve this purpose
especially for arid regions where marginal lands can be utilized to generate economy
Presence of such type of local systems in the region highlight their potential advantage in
crop production income generation as well as sustainability (Somashekar et al 2015)
For instance reports are available on successful intercropping of multipurpose trees
shrubs and grasses like millets pulses and some oil seed and fodder crops Green part of
these species usually mixed and used for cattle feed especially during the lean period The
utilization of the inter-row spaces of fruit trees like Ziziphus mauritiana for growing edible
legumes can generate further income by similar input (Dayal et al 2015) As an option
to this Cajanus cajan could serve as better intercropped as it provides protein rich food
nutritious fodder and wood for fuel which helped to uplift the socio-economic condition
of poor farmers Integrated agricultural practices improve the productivity of each crop by
keeping cost of production under sustainable limits (Arabhanvi and Pujar 2015)
Keeping in mind the above mentioned scenario in present study the possibility to
increase production of a non-conventional salt tolerant fruit tree (Z mauritiana) by
intercropping with a leguminous plant (C cajan) was investigated to produce edible fruits
and fodder simultaneously from salt effected waste lands
60
22 Experiment No 7
Physiological investigations on Growth of Ziziphus mauritiana and
Cajanus cajan intercropped in drum pot (Lysimeter) culture being
irrigated with water of sea salt concentration at two irrigation intervals
221 Materials and Methods
2211 Growth and Development
Experiment was designed to investigate the effect of intercropping on growth and
development of Z mauritiana (a fruit tree) and C cajan (a leguminous fodder) in drum
pot culture irrigated with water of 03 sea salt concentrations at two irrigation intervals
2212 Drum pot culture
Drum pot culture as recommended by Boyko (1966) and modified by Ahmed and
Abdullah (1982) was used for the present investigation as described in chapter 1
2213 Experimental Design
Three sets of 18 plastic drums (lysimeter) were used in this experiment One plant of Z
mauritiana were grown in each lysimeter Three replicates were kept for each treatment
comprising of 06 drums in each set which was further divided in two sub-sets First sub-
set was irrigated at every 4th and second subset at every 8th day
Set ldquoArdquo =Ziziphus mauritiana (Sole crop)
Set ldquoBrdquo = Cajanus cajan (Sole crop)
Set ldquoCrdquo = Ziziphus mauritiana + Cajanus cajan (intercropped)
The effect of salinity on sole crops of C cajan and Z mauritiana on salinity created
by various dilutions of sea salt has been investigated in chapter 1 Concentration of 03
sea salt considered equal level to its 50 reduction has been selected in present
experiment In addition irrigation was given in sub-sets in two intervals to investigate to
have some idea of its water conservation
61
2214 Irrigation Intervals
Sub-set 1 Irrigation was given every 4th day
Sub-set 2 Irrigation was given every 8th day
In set lsquoArsquo and lsquoCrsquo six month old saplings of Ziziphus mauritiana (vern grafted
ber) plants of nearly equal height and good health were transplanted in drum pots Plants
were irrigated to start with non-saline tape water for about two weeks for purpose of
establishment All the older leaves fell down and new leaves immerged during
establishment period
In set lsquoBrsquo and lsquoCrsquo Ten healthy sterilized seeds of Cajanus cajan imbibed in distill
water were sown in each drum pot and irrigated to start with tap water and after
establishment of seedlings only six seedlings of equal size with eqal distance (about one
feet) between C cajan and that of Z mauritiana were kept for further study The sowing
time of cajanus cajan seeds in both sets (B and C) was the same In drum pot lsquoCrsquo it was
sown when sapling of Z mauritiana have undergone two weeks of their establishment
period in tap water
When seedlings of C cajan reached at two leaves stage irrigation in all the sets
(ABC ) was started with gradual increase sea salt concentration till it reached to the
salinity level of treatment (03) in which they were kept up to end of experiment Each
drum was irrigated with enough water sea salt solution which retains 15 liters in soil at
field capacity Rest of water drain down with leaching of accumulated salt in root
rhizosphere
Vegetative growth of Z mauritiana plant was noted monthly in terms of height
volume of canopy while in C cajan height and number of branches was noted Shoot
length root length number of leaves fresh and dry weight of leaf stem and root leaf
weight ratio root weight ratio stem weight ratio specific shoot and root length plant
moisture leaves succulence and relative growth rate was observed and calculated at final
harvest in both the plant species growing individually (sole) or as intercropping at two
irrigation intervals
Investigations were undertaken on nitrate content relative water content and
electrolyte leakage at grand period of growth Amount of photosynthetic pigments soluble
62
carbohydrates proline content soluble phenols and Protein contents were also investigated
in fully expended leaves
Activity of catalase (CAT) ascorbate peroxidase (APX) guaiacol peroxidase
(GPX) superoxide dismutase (SOD) (Anti-oxidant enzymes) and nitrate reductase (NR)
activity was also observed in on both the Z mauritiana and C cajan leaves growing as
sole and as intercropped at two different irrigation intervals
The procedures of above mentioned analysis as follows
Leaf succulence (dry weight basis) Specific shoot length (SSL) and relative
growth rate (RGR) were measured according to the equations given in chapter 1
2215 Estimation of Nitrate content
NO3 was estimated through Cataldo et al (1975) 01g fresh leaf samples were boiled in
50 mL distilled water for 10 min 01mL of sample were added to mixed in 04 mL 50
salicylic acid (wv dissolved in 96 H2SO4 ) and allowed to stand for 20 min at room
temperature 95 mL of 2N NaOH was slowly mixed at last The samples were permissible
to cool NO3 concentration was observed at 410 nm and was calculated according to the
standard curve expressed in mg g-1 fresh weight
2216 Relative Water content (RWC)
Young and fully expended leaf was excise from each plant removing dust particles
preceding to Relative water content (RWC) Fresh weights (FW) were taken to all leaf
samples and were immersed in distilled water at 4 degC for 10 hours The soaked leaf samples
were taken out and surfeit water was removed by tissue paper Weighted again these leaf
samples for turgid weight (TW) and were oven dried at 70 degC Dry weight (DW) was
recorded after 24 hrs The RWC of leaf was calculated by the following formula
RWC () = [FW ndash DW] [TW ndash DW] x 100
2217 Electrolyte leakage percentage (EL)
EL was measured according to Sullivon and Ross (1979) Young and fully expended
leaves removing dust particles were taken 20 disc of 6mm diameter were made through
63
porer and were placed in the test tube containing 10ml de-ionized water First electrical
conductivity (EC lsquoarsquo) was record after shaken the tubes These test tubes now were placed
at 45-50oC warmed water bath for 30 min and observed second Electrical conductivity (EC
lsquobrsquo) Finally tubes were placed at 100oC water bath for ten min and obtained third and final
Electrical conductivity (EC lsquocrsquo) The electrolyte leakage was calculated in percentage by
using following formula
EL () = (EC b ndash EC a) EC b x 100
2218 Photosynthetic pigments
Photosynthetic pigments including chlorophyll a chlorophyll b total chlorophyll
chlorophyll ab ratio and carotinoids were estimated according to the procedure given in
chapter 1
2219 Total soluble sugars
Dry leaf samples (01g) were milled in 5mL of 80 ethanol and were centrifuged at 4000
g for 10 minutes and were estimated according to the procedure described in chapter 1
22110 Proline content
The proline contents were determined through Bates et al (1973) Each dried leaf powder
sample (01 g) was grinded and homogenized in 5 ml of 3 (wv) sulphosalicylic acid and
were centrifuged at 5000 g for 20 minutes 2ml supernatant was boiled by adding 2 ml
glacial acetic acid and 2 ml ninhydrin reagent (prepared by dissolving 125 g ninhydrin in
30 ml of glacial acetic acid and 20 ml 6 M phosphoric acid) in caped test tube The tubs
were kept in boiling water bath (100oC) for 1 hour After cooling 4 ml of toluene was
added to each tube and vortex Two layers were appeared the chromophore layer of
toluene was removed and their absorbance was recorded at 590nm against reference blank
of pure toluene The proline concentrations in leaves were determined from a standard
curve prepared from extra pure proline of (Sigma Aldrich) and were calculated from the
equation and were expressed in mgg-1 of leaf dry weight
Proline (microgmL-1) = -074092 + 1660767 (OD) plusmn054031
64
22111 Soluble phenols
The dried leaf powder (01g) was milled in 3ml of 80 methanol and was centrifuged at
10000g for 15 min (Abideen et al 2015) Final volume (5ml) were adjusted by adding
80 methanol Soluble phenols were determined by using Singleton and Rossi (1965) ie
5 ml of Folin-Ciocalteu reagent (19 ratio in distilled water) and 4 ml of 75 Na2CO3
were added to 01 ml supernatant The absorbance was recorded at 765 nm after incubation
of 30 minutes at room temperature The soluble phenols concentration in leaf tissues was
determined from a standard curved prepared from Gallic acid
22112 Total soluble proteins
The protein contents were measured according to Bradford Assay reagent method against
Bovine Serum Albumin as standards (Bradford 1976) Procedure was followed as given
in chapter 1
22113 Enzymes Assay
Enzyme extract prepared as given below was used for study of enzymes mentioned in text
The juvenile and expended leaf excised was frozen in liquid nitrogen and were stored at -
20 degC These leaf samples (100mg) was firmed in liquid nitrogen and were mills in 3 ml
of ice chilled potassium phosphate buffer (pH = 7 01 M) with 1mM EDTA and 1 PVP
(wv) The homogenate was filtered through a four layers of cheesecloth and were
centrifuged at 21000 g using refrigeration centrifuge (Micro 17 TR Hanil Science
Industrial Co Ltd South Korea) at 4 degC for 20 min The supernatant was separated and
stored at -20 degC and used for investigation on following enzymes
i Superoxide dismutase (SOD)
SOD (EC 11511) antioxidant enzymeactivity was measured through Beauchamp and
Fridovich (1971) derived on the inhibition of nitroblue tetrazolium (NBT) reduction by
produced O2minus using riboflavin photo-reduction 50 mM of pH 78 phosphate buffer (with
01mM EDTA 13 mM methionine) 75 microM nitroblue tetrazolium (NBT) 2 microM riboflavin
and 100 microl of enzyme extract was added to 3ml reaction mixture Riboflavin was added at
the last before the reaction was initiated under fluorescent lamps for 10 min Exposed and
un-exposed to florescence lamp without enzyme extract were used to serve as calibration
65
standards Activity was measured at 560nm Unit of SOD activity was defined as the
amount of enzyme required for 50 inhibition of NBT conversion
ii Catalase (CAT)
CAT (EC 11116) antioxidant enzyme activity was precise according to Aebi (1984)
derived on H2O2 reduction at 240nm for 30 s (ε = 36 M-1 cm-1)100mM potassium
phosphate buffer (pH=7) with 30mM H2O2 and 50 microl of diluted enzyme extract (adding in
last) was added to 3ml reaction mixture The decrease in absorbance due to H2O2 reduction
was measured at 240 nm and expressed in micromol of H2O2 reduced m-1g-1 fresh weight at 25
degC
iii Ascorbate peroxidase (APX)
Nakano and Asada (1981) method was used for APX (EC 111111) antioxidant
enzymeactivity by measuring the decrease in ascorbate oxidation by H2O2 The reaction
mixture (3ml) contained potassium phosphate buffer (50mM pH=7) 01mM H2O2 050
mM Ascorbate and 100 microl of enzyme extract and were observed 290 nm for 1 min 25 degC
(extinction coefficient 28 mM-1cm-1)
iv Guaiacol peroxidase (GPX)
GPX (EC 11117) antioxidant enzymeactivity was estimated through Anderson et al
(1995) 3ml of 50 mM potassium phosphate buffer (pH 7) guaiacol 75 mM H2O2 10 mM
reaction mixture with 20 microl of enzyme extract adding at last Increase in absorbance was
observed due to the formation of tetra-guaiacol at 470 nm for 2 min (extinction coefficient
266 mM-1cm-1)
v Nitrate reductase (NR)
The NR activity in leaves was observed through Long and Oaks 1990 Fresh leaf samples
(01g) were placed in 5ml of 100mM potassium phosphate pH 75 (added to 10
Isopropanol and 25mM KNO3) Tubes were vacuumed for 10 min to remove air from the
mixture and were placed in water bath shaker at 33oC for 60 min in dark The tubes were
placed in hot water (100oC) for 5 min 15 mL from the reaction mixture were added in 05
mL 20 sulphanilamide (wv dissolve in 5N HCl) and 025 mL 008 N-1-Napthylene-
66
diamine dihydrochloride Final volume up to 60 ml was made by adding distilled water
Color developed over the next 20 min Absorbance was measured at 540 nm using
spectrophotometer
67
222 Observations and Results
Sole and intercropped Ziziphus mauritiana
2221 Vegetative growth
Growth of Z mauritiana in terms of shoot root and plant length and number of leaves in
two different cropping system (sole and intercrop with C cajan) in two different irrigation
intervals has been presented in Figure 21 Appendix-XII A significant increase (plt0001)
in plant length was observed in 8th day irrigation in both the cropping systems in Z
mauritiana At 4th day of irrigation interval a non-significant increase in length was
observed in intercropped plants compared to sole crop Similarly at 8th day of irrigation
plants attain almost same heights in both the cropping systems
A significant increase (plt001) in root length was observed in sole Z mauritiana
at 8th day of irrigation compared to other treatments Smallest root length revealed in plants
that were irrigated at 4th day under sole crop system
The shoot length was significantly increase (plt0001) in plants which were
irrigated at 8th day under intercropped system However shoot length remains unaffected
when comparing the different cropping system at both the irrigation intervals
A significant increase (plt0001) in number of leaves was observed in intercropped
Z mauritiana plants compared to plants cultivated according to sole system However
more increase was observed in 4th day irrigated intercropped plant as compared to 8th day
The difference in number of leaves in sole crop at both irrigating intervals remains same
i Fresh weight
Figure 22 Appendix-XII showed fresh and dry weight of stem root and leaf of Z
mauritiana plant in two different cropping system (sole and intercrop with C cajan) in
two different irrigation intervals A significant increase (plt0001) in fresh weights of leaf
stem and root was observed in intercropping (with C cajan) 4th and 8th day of irrigation
interval compared to individual cropping of Z mauritiana In 4th day of irrigation the
increment was more pronounced in fresh weights of root (7848) leaves (4130) and
stem (4047) respectively with comparison to the crop growing alone Similarly
intercropping in 8th day of irrigation showed better growth of leaves (28) stem (12)
68
and root (31) against sole crop Whereas decrease in leaves 33 (plt005) stem 70
(plt0001) and root 60 (plt0001) fresh weights were observed in 8th day of irrigation
compared to 4th day intercropping However the difference was non-significant between
two sole crops irrigated at 4th and 8th day interval
ii Dry weight
Intercropping with comparison to the sole crop showed significant (plt0001) increase in
dry weights of leaves root and stem of Z mauritiana at 4th and 8th day of irrigation (Figure
22 Appendix-XII) At 4th day of irrigation intercropping showed an increment in dry
weights of Leaves (4366) stem (4109) and root (754) compared to the sole crop
Similar increase was observed in leaves (plt0001) stem (plt0001) and root (plt0001)
weights after 8th day of irrigation However intercropping at 8th day irrigation showed an
increment in root (19) stem (11) whereas a slight decrease (1) in leaves dry weight
When comparing irrigation time an increase in stem dry weight at 4th day whereas decline
in leaves dry weight was observed Root dry weights were more or less similar at both
irrigation intervals
iii Leaf weight ratio (LWR) root weight ratio (RWR) stem weight
ratio (SWR)
Leaf weight ratio (LWR) root weight ratio (RWR) stem weight ratio (SWR) of Z
mauritiana plant grown in two different cropping system (sole and intercrop with C cajan)
in two different irrigation intervals has been presented in Figure 23 Appendix-XII An
increased in LWR and SWR was recorded at 8th day of irrigation compared to 4th day of
irrigation in both cropping systems whereas decrease in RWR was observed LWR and
SWR remained un-change in sole and inter crop system However RWR increased in
intercrop system compared to sole crop system
iv Specific shoot length (SSL) specific root length (SRL)
Specific shoot length (SSL) specific root length (SRL) of Z mauritiana plant grown in
two different cropping system (sole and intercrop with C cajan) in two different irrigation
intervals has been presented in Figure 23 Appendix-XII SSL was observed higher in 8th
day of irrigation compared to 4th day in both the cropping systems However the increase
69
in SSL was lesser in sole crop compared to intercropping Similarly SRL was recorded
lesser in 4th day of irrigation compared to 8th day of irrigation in both cropping systems
Intercropped plants showed decline in SRL compared to sole crop plants Greatest SRL
revealed in plants that were irrigated after 8th day and planted according to sole crop
system
v Plant moisture
The moisture content of Z mauritiana plant grown in two different cropping system (sole
and intercrop with C cajan) in two different irrigation intervals has been presented in
Figure 23 Appendix-XII The moisture content of plants was significantly decreased
(plt005) in sole crop while increased (plt005) in intercropping at 8th day of irrigation
compared to 4th day At 4th day moisture remained same in both cropping system
However significant increase in moisture contents was observed in inter-crop system
compared to sole crop system after 8th day of irrigation
vi Plant Succulence
Succulence of Z mauritiana plant grown in two different cropping system (sole and
intercrop with C cajan) in two different irrigation intervals has been presented in Figure
23 Appendix-XII Plant succulence in 8th day was significantly reduced in sole crop
whereas increased in intercropping system In 4th day irrigated plants decrease in
succulence was noticed compared to plants that were irrigated at 8th day under sole crop
system However significant increase (plt0001) was observed in intercropped plants
irrigated at 4th day compared to 8th day
vii Relative growth rate (RGR)
Relative growth rate (RGR) of Z mauritiana plant grown in two different cropping system
(sole and intercrop with C cajan) in two different irrigation intervals has been presented
in Figure 23 Appendix-XII Relative growth rate remains unchanged at both irrigation
times under sole crop system However decline in 8th day was observed compared to 4th
day of irrigation under intercrop system Greatest RGR was recorded in plants that were
irrigated at 4th day under intercrop system
70
2222 Photosynthetic pigments
Photosynthetic pigments including Chlorophyll a chlorophyll b total chlorophyll
Chlorophyll ab ratio and carotinoids of Z mauritiana plant grown in two different
cropping system (sole and intercrop with C cajan) in two different irrigation intervals has
been presented in Figure 24 Appendix-XII
i Chlorophyll contents
A significant increase (plt0001) in chlorophyll a b and total chlorophyll was observed in
plants growing as sole crop compared to intercropped system at both the irrigation
intervals Higher chlorophyll contents were also recorded in plants that were irrigated at
8th day compared to 4th day of irrigation The chlorophyll ab ratio increased in 4th day
while decline in 8th day in intercropped system compared to sole crop However overall
results showed non-significant changes
ii Carotinoids
A significant increase (p lt 0001) in leaf carotinoids was observed in sole crop compare
to intercropped system at both irrigation times in Z mauritiana Least carotene content
was estimated in plants that were irrigated at 4th day under intercrop system
2223 Electrolyte leakage percentage (EL)
Electrolyte leakage percentage (EL) of Z mauritiana plant grown in two different
cropping system (sole and intercrop with C cajan) in two different irrigation intervals has
been presented in Figure 25 Appendix-XII A non-significant result was observed in
electrolyte leakage in plant growing at varying cropping system and irrigating intervals
2224 Phenols
Total phenolic contents in leaves of Z mauritiana plant grown in two different cropping
system (sole and intercrop with C cajan) in two different irrigation intervals has been
presented in Figure II25 Appendix-XII A significant increase (plt001) in total phenolic
contents was observed in intercropped growing at both irrigation interval compared to sole
crop However the increase was more pronounced at 8th day of irrigation Maximum
phenolic contents were measured in plants irrigated at 8th day under intercropped plants
71
2225 Proline
Total proline contents in leaves of Z mauritiana plant grown in two different cropping
system (sole and intercrop with C cajan) in two different irrigation intervals has been
presented in Figure 25 Appendix-XII A significant decreased (plt0001) was observed
in Z mauritiana cultivated according to intercropped system in both irrigation intervals
Maximum decrease was observed in intercropped plants irrigated at 8th day whereas
highest phenolic contents were observed in plants irrigated at 4th day under sole crop
system
2226 Protein and sugars
Protein and sugar contents in leaves of Z mauritiana plant grown in two different cropping
system (sole and intercrop with C cajan) in two different irrigation intervals has been
presented in Figure 26 Appendix-XII A nonsignificant difference in total protein and
sugar contents in Z mauritiana plants was observed in two different (4th and 8th day)
irrigation intervals However the interaction with time and irrigation interval also showed
nonsignificant result
2227 Enzyme essays
Antioxidant enzymes like Catalase (CAT) Ascorbate peroxidase (APX) Guaiacol
peroxidase (GPX) Superoxide dismutase (SOD) and Nitrate reductase activity in leaf of
Z mauritiana plant grown in two different cropping system (sole and intercrop with C
cajan) in two different irrigation intervals has been presented in Figure 27 and 28
Appendix-XII
i Catalase (CAT)
A significant decreased (plt0001) in catalase activities was observed in Z mauritiana
leaves in intercropped system in both time interval with compare to sole crop at 4th day
irrigated plant However maximum decline was in sole plants irrigated at 8th day interval
However their interaction with time was nonsignificant
72
ii Ascorbate peroxidase (APX)
A significant increase (plt0001) in APX activity was observed in 8th day irrigation in both
sole and intercropped plants with compare to sole and intercropped at 4th day irrigation
interval More increase (plt0001) was observed in intercropped Z mauritiana at 8th day
Whereas nonsignificant decrease was observed in two different cropping system in 4th day
irrigation interval However interaction between time and the treatments shows significant
values
iii Guaiacol peroxidase (GPX)
A significant (plt0001) increase in GPX was observed in 8th day intercropped Z
mauritiana plant with compare to irrigation intervals as well as cropping system However
at 4th day both cropping system showed nonsignificant difference Whereas more decline
was observed in 8th day sole crop The ANOVA reflects significant (plt005) interaction
between time and the cropped system
iv Superoxide dismutase (SOD)
A nonsignificant increase in SOD was observed in intercropped at 8th day irrigation
interval Whereas there was nonsignificant differences in 4th day intercropped and at both
time intervals of sole crop However interaction between time interval and the two
cropping system shows nonsignificant result
v Nitrate and Nitrate reductase
A significant increase (plt0001) in nitrate content and activity of nitrate reductase was
observed in intercropped plants of both irrigation intervals Increase in activity was
observed (plt0001) in intercropped Z mauritiana at 4th day
73
Sole and intercropped Cajanus cajan
2228 Vegetative growth
Growth of C cajan in terms of shoot root and plant length and number of leaves was
observed in two different cropping system (sole and intercrop with Z mauritiana) in two
different irrigation intervals has been presented in Figure 21 Appendix-XIII XIV A
significant increase (plt001) in plant length was observed in intercropped C cajan
compared to sole crop at both irrigation interval Whereas sole crop at 8th day interval
showed better results as compare to sole of 4th day Similarly root length remains
unaffected and showed non-significant change in both cropping systems and even at two
different irrigation intervals While shoot length was significantly (Plt001) decreased in
sole crop compared to intercropped at 4th day irrigation Whereas non-significant
difference be observed in rest of cropping systems growing at different irrigation interval
A significant increase (plt001) in leaves number was observed in intercropped
plants compared to sole crop at 4th and 8th day irrigation interval However most
significant decrease (plt0001) was observed in sole crop at 4th day
i Fresh weight
Figure 22 Appendix-XIV showed fresh and dry weight of stem root and leaf of C cajan
plant in two different cropping system (sole and intercrop with C cajan) in two different
irrigation intervals A significant increase (plt001) in fresh weight of leaf was observed in
intercropping (with Z mauritiana) at 4th and 8th day of irrigation interval compared to
individual cropping of C cajan The increase in intercropped system compared to sole
crop was more pronounced at 4th day (42) of irrigation than the 8th day (1701) Plants
showed higher leaves fresh weights in 8th day of irrigation compared to 4th day Similarly
the interaction between cropping system and the irrigation interval was significant
(Plt005)
An insignificant difference was observed in stem at 4th (15) and 8th (12) days
fresh weights in both intercropping system at two different irrigation intervals The
interaction between cropping system and the irrigation interval also showed non-
significant result
74
A non-significant difference in root fresh weight was observed in two different
cropping systems (sole and intercropped) in 4th and 8th day of irrigation intervals However
fresh weight of crop at 8th day irrigation interval was significantly increase (plt0001) over
4th day irrigation interval Similar pattern was observed in 4th day irrigated sole and
intercropped C cajan
ii Dry weight
A significant increase in leaves (42) stem (24) and root (18) dry weights were
observed in 4th day irrigation under intercropped system compared to sole However in 8th
day of irrigation this increase of dry weights was not much prominent Under sole crop
system dry weights of leaves stem and root was increased markedly in 8th day compared
to 4th day However in intercrop system the difference in dry weights was insignificant
between 8th and 4th day of irrigation
iii Leaf weight ratio (LWR) root weight ratio (RWR) stem weight
ratio (SWR)
Leaf weight ratio (LWR) root weight ratio (RWR) stem weight ratio (SWR) of C cajan
grown in two different cropping system (sole and intercrop with Z mauritiana) in two
different irrigation intervals has been presented in Figure 23 Appendix-XIV A
significant increase (plt0001) in LWR was observed at 8th day of irrigation compared to
4th day intercropped Similar pattern was noticed in RWR however SWR showed
insignificant difference between 4th and 8th day of irrigation A slight increase in LWR was
noticed in intercropped plants compared to sole Whereas RWR declined in intercrop
compared to sole and SWR remains un-changed
iv Specific shoot (SSL) root length (SRL)
Specific shoot length (SSL) specific root length (SRL) of C cajan grown in two different
cropping system (sole and intercrop with Z mauritiana) in two different irrigation
intervals has been presented in Figure 23 Appendix-XIV SSL and SRL were observed
to increase in sole crop compared to intercrop at 4th day of irrigation However increase
SSL and SRL was recorded in intercropped compared to sole at 8th day of irrigation A
general decline in SSL and SRL was noticed in 8th day of irrigation compared to 4th day
75
v Plant moisture
The moisture content of C cajan plant grown in two different cropping system (sole and
intercrop with Z mauritiana) in two different irrigation intervals has been presented in
Figure 23 Appendix-XIV The moisture content of plants was decreased significantly
(plt005) at 8th day irrigation interval compared to 4th day in sole crop Whereas non-
significant increase was observe in intercrop plants at 8th day of water irrigation
vi Plant succulence
Succulence of C cajan plant grown in two different cropping system (sole and intercrop
with Z mauritiana) in two different irrigation intervals has been presented in Figure 23
Appendix-XIV A significant increase (plt001) was observed in intercropped plants of C
cajan compared to sole crop at both irrigation interval However succulence increased in
sole crop and decreased in intercrop plants at 8th day of irrigation compared to 4th day
vii Relative growth rate (RGR)
Relative growth rate (RGR) of C cajan plant grown in two different cropping system (sole
and intercrop with Z mauritiana) in two different irrigation intervals has been presented
in Figure 23 Appendix-XIV A significant increase in RGR was observed in 8th day
compared to 4th day in both the cropping systems Highest increase was observed in
intercropped at 8th day irrigation At 4th day irrigation intervals intercropped plants
showed better RGR compared to Sole crop
2229 Photosynthetic pigments
Photosynthetic pigments including Chlorophyll a chlorophyll b total chlorophyll
Chlorophyll ab ratio and carotinoids of C cajan plant grown in two different cropping
system (sole and intercrop with Z mauritiana) in two different irrigation intervals has been
presented in Figure 24 Appendix-XIV
i Chlorophyll contents
A significant increase (plt005) in Chlorophyll a b and total chlorophyll was observed in
intercrop plants at 8th day irrigation interval Whereas at 4th day irrigation interval Sole
76
plants showed better results as compare to intercrop plants Plants at 8th day significantly
increase chlorophyll a b and total chlorophyll compared to 4th day of irrigation
Interactions between cropping systems and irrigation intervals were found significant
(chlorophyll a (plt001) chlorophyll b (plt001) and total chlorophyll (plt0001)
respectively) However the ratio of chlorophyll ab showed non-significant values in
cropping irrigation interval and their interaction
ii Carotenoids
A significant increase (plt001) in carotinoids was observed in intercropped C cajan at 8th
day of irrigation Whereas non-significant increase was observed in sole crop at 4th day
irrigation interval with compare to intercrop However the irrigation intervals showed
significant (plt0001) difference Whereas interaction of cropping system with irrigation
time also showed significant correlation (plt0001)
22210 Electrolyte leakage percentage (EL)
Electrolyte leakage percentage (EL) of C cajan plant grown in two different cropping
system (sole and intercrop with Z mauritiana) in two different irrigation intervals has been
presented in Figure 25 Appendix-XIV A non-significant increase in EL percentage was
observed in sole crop compared to intercrop plants growing at 4th and 8th day of irrigation
No significant change was noticed between the irrigation times to C cajan The interaction
between cropping system (sole and intercropped) and irrigation interval (4th and 8th day)
also showed non-significant
22211 Phenols
Total phenolic contents in leaves of C cajan plant grown in two different cropping system
(sole and intercrop with Z mauritiana) in two different irrigation intervals has been
presented in Figure 25 Appendix-XIV A nonsignificant result was observed in total
phenolic contents of C cajan growing as sole and intercropped system at two different
irrigation intervals However the interaction between irrigation intervals with crop system
showed significant (p lt 005) results
77
22212 Proline
Total proline contents in leaves of C cajan plant grown in two different cropping system
(sole and intercrop with Z mauritiana) in two different irrigation intervals has been
presented in Figure 25 Appendix-XIV Proline contents in leaves of C cajan showed
nonsignificant increase at 4th day of irrigation interval in both sole and intercropped
system Whereas the interaction between irrigation intervals showed significant (Plt001)
results
22213 Protein and Sugars
Protein and sugar contents in leaves of C cajan plant grown in two different cropping
system (sole and intercrop with Z mauritiana) in two different irrigation intervals has been
presented in Figure 26 Appendix-XIV A less significant difference (plt005) was
observed in two different (4th and 8th day) irrigation intervals However there was
nonsignificant difference in two cropped system More decrease was observed at 4th day
intercropped plants Whereas nonsignificant increase in 8th day intercropped and 4th day
sole plants were observed However interaction between crop and time of irrigation
showed significant results (plt0001)
22214 Enzyme assay
Antioxidant enzymes like Catalase (CAT) Ascorbate peroxidase (APX) Guaiacol
peroxidase (GPX) Superoxide dismutase (SOD) and Nitrate reductase activity in leaf of
C Cajan plant grown in two different cropping system (sole and intercrop with Z
mauritiana) in two different irrigation intervals has been presented in Figure II27
Appendix-XIV
i Catalase (CAT)
A significant increase (plt001) in catalase activity was observed in intercropped C cajan
at 8th day of irrigation with compare to other irrigation time and cropped system Whereas
increase was observed in sole crop at 4th day irrigation interval with compare to 8th day
However the irrigation intervals and the interaction between cropping system with
irrigation interval also showed nonsignificant correlation
78
ii Ascorbate peroxidase (APX)
A non-significant increase in APX was observed in intercropped plant in 4th and 8th day
irrigation interval with compare to sole crops Sole crop at 8th day showed maximum
decline However the difference between cropping system and their interaction with
irrigation interval also showed nonsignificant results
iii Guaiacol peroxidase (GPX)
A significant increase (plt005) in GPX activity was observed in 8th day sole crop
However there was nonsignificant difference among intercropped at two time interval and
sole crop at 4th day irrigation Whereas interaction with time to irrigation interval also
showed less significant results
iv Superoxide dismutase (SOD)
A significant decrease (plt0001) in SOD activity was observed in intercropped at 8th day
irrigation interval with compare to 4th day Maximum decrease was observed in 8th day
intercropped Whereas sole crop at 8th day also showed better result to 4th day sole crop
However ANOVA showed significant correlation among crop system at two time interval
and 4th day irrigation
v Nitrate and Nitrate reductase
Nitrate content and activity of nitrate reductase was nonsignificant in both cropping
system using both irrigation intervals However nonsignificant increase was observed in
nitrate content and activity of nitrate reductase in intercropped Z mauritiana at 8th day
79
Sole IntercropSole Intercrop
No o
f le
aves
0
20
40
60
Len
gth
(cm
)
0
40
80
120
160
200
2404
th day
Cajanus cajan
a
RootShoot
ab
a
a
b
a
a
8th
day
Figure 21 Vegetative parameters of Z mauritiana and C cajan at grand period of growth under sole and
intercropping system at 4th and 8th day irrigation intervals (Bars represent means plusmn standard error
of each treatment and significance among the treatments was recorded at p lt 005)
Sole IntercropSole Intercrop
No of
leav
es
0
200
400
600
Len
gth
(cm
)
0
40
80
120
160
200
240
Ziziphus mauritiana
RootShoot
4th
day 8th
days
b b
a a
a
b
cc
80
Sole Intercrop
Dry
wei
ght
(g)
50
100
150
200
250
300
Fre
sh w
eight
(g)
100
200
300
400
500
Sole Intercrop
4th
day 8th
day
a
b
c
a
b b aa
b
b
c c
a
bc
a
c
ba
b
c
a
b
c
Leaf Stem Root
Ziziphus mauritiana
Sole Intercrop
Dry
wei
ght
(g)
2
4
6
8
10
12
Fre
ah w
eight
(g)
5
10
15
20
25
30
35
40
Sole Intercrop
4th
day 8th
day
aa
b
a
a
b
a
b
c
a
b
c
a
c
b
a a
b
a
b
c
a
b
c
Leaf Stem Root
Cajanus cajan
Figure 22 Fresh and dry weight of Z mauritiana and C cajan plants under sole and intercropping system
at 4th and 8th day irrigation intervals (Bars represent means plusmn standard error of each treatment
and significance among the treatments was recorded at p lt 005)
81
Figure 23 Leaf weight ratio (LWR) root weight ratio(RWR) shoot weight ratio(SWR)specific shoot
length (SSL) specific root length (SRL) plant moisture Succulence and relative growth rate (RGR) of
Zmauritiana and C cajan grow plants under sole and intercropping system at 4th and 8th
day irrigation
intervals (Bars represent means plusmn standard error of each treatment and significance among the treatments
was recorded at p lt 005)
Sole Intercrop
Mo
istu
re (
)
0
20
40
60
80
SS
L (
cm g
-1)
01
02
03
04
05
06
RW
R (
g g
-1 D
W)
005
010
015
020
LW
R (
g g
-1 D
W)
01
02
03
04
05
06
07
Sole Intercrop
Su
ccu
lan
ce
(g H
2O
g-1
DW
)00
05
10
15
20
25
RG
R
(g g
-1 d
ay-1
)
001
002
003
004
005
SR
L (
cm g
-1)
05
10
15
20
25
SW
R (
g g
-1 D
W)
02
04
06
08
10
Ziziphus mauritiana
a a
bb
b
a
bb
a
b
aa
a aa
b
a
bb
c
b
a
bb
b
aa a
ba
bc
4th day
8th day
82
(Figure 23 continuedhellip)
Sole Intercrop
Mo
istu
re (
)
0
20
40
60
80
SS
L (
cm g
-1)
2
4
6
8
10
12
RW
R (
g g
-1 D
W)
002
004
006
008
010
012
014
LW
R (
g g
-1 D
W)
01
02
03
04
05
06
07
08
Sole Intercrop
Su
ccu
lan
ce
(g H
2O
g-1
DW
)
00
05
10
15
20
25
RG
R
(g g
-1 d
ay-1
)
001
002
003
004
005
SR
L (
cm g
-1)
5
10
15
20
25
SW
R (
g g
-1 D
W)
02
04
06
08
10
Cajanus cajan
a aab
a aaa
a
bba
a
b b
c
a aab
a
bbb
abbb
aa
bc
8th day
4th day
83
Sole Intercrop
Car
oti
noid
s (m
g g
-1 F
W)
00
01
02
03
04
05
Ch
loro
phyll
(m
g g
-1 F
W)
00
03
06
09
12
15
Sole Intercrop
4th
day 8th
day
Ch
loro
phyll
ab
rat
io
00
05
10
15
20
25Chl ab
Ziziphus mauritiana
a a
bb
a
b
a
b
a ab
b
Chl aChl b
Figure 24 Leaf pigments of Zmauritiana and C cajan grow plants under sole and intercropping system at
4th and 8th
day irrigation intervals (Bars represent means plusmn standard error of each treatment and
significance among the treatments was recorded at p lt 005)
Sole Intercrop
Car
oti
noid
s (m
g g
-1 F
W)
00
01
02
03
04
05
Ch
loro
phyll
(m
g g
-1 F
W)
00
03
06
09
12
15
18
Sole Intercrop
4th
day 8th
day
ab r
atio
00
05
10
15ab
ab
Cajanus cajan
bb b
a
a
b
cc
bb b
a
84
Ele
ctro
lyte
lea
kag
e(
)
0
5
10
15
4th
day 8th
dayP
hen
ols
(m
g g
-1)
0
5
10
15
20
25
30
Sole Intercrop
Pro
line
( g g
-1)
0
10
20
30
40
Sole Intercrop
Ziziphus mauritiana
a a a
a
b b ba
a
b
c
d
Figure 25 Electrolyte leakage phenols and prolein of Z mauritiana and C cajan at grand period of growth
plants under sole and intercropping system at 4th and 8
th day irrigation intervals (Bars represent
means plusmn standard error of each treatment and significance among the treatments was recorded at
p lt 005)
85
(Figure 25 continuedhellip)
E
lect
roly
te l
eakag
e(
)
0
20
40
60
80
4th
day 8th
day
Phen
ols
(m
g g
-1)
0
2
4
6
8
10
12
Sole Intercrop
Pro
line
( g g
-1)
000
003
006
009
012
015
018
Sole Intercrop
Cajanus cajan
a aa
a
a a aa
aa a
a
86
Sole Intercrop
Sugar
s (m
g g
-1)
0
20
40
60
Sole Intercrop
Pro
tein
(m
g g
-1)
00
02
04
06
4th
day 8th
day
Ziziphus mauritiana
a aa a
a
a a a
Sole Intercrop
Sugar
s (m
g g
-1)
0
10
20
30
Sole Intercrop
Pro
tein
(m
g g
-1)
00
02
04
06
08
10
4th
day 8th
dayCajanus cajan
ab
a
c
a
b
cc
Figure 26 Total protein and sugars in leaves of Z mauritiana and C cajan plants under sole and
intercropping system at 4th and 8th
day irrigation intervals (Bars represent means plusmn standard
error of each treatment and significance among the treatments was recorded at p lt 005)
87
Sole Intercrop
SO
D (
Unit
s m
g-1
)
0
2
4
6
8
10
12
14
Sole Intercrop
Cat
alas
e (U
nit
s m
g-1
)
0
5
10
15
20
25
AP
X (
Unit
s m
g-1
)
0
20
40
60
80
GP
X (
Unit
s m
g-1
)
00
01
02
03
04
05
4th
day 8th
day
Ziziphus mauritiana
a
bc
c
a
b
cc
a
c
b
b
b bb
a
Figure 27 Enzymes activities in leaves of Z mauritiana and C cajan plants under sole and intercropping
system at 4th and 8th
day irrigation intervals (Bars represent means plusmn standard error of each
treatment and significance among the treatments was recorded at p lt 005)
88
(Figure 27 continuedhellip)
Sole Intercrop
SO
D (
Unit
s m
g-1
)
0
1
2
3
4
5
Sole Intercrop
Cat
alas
e (U
nit
s m
g-1
)
0
2
4
6
8
4th
day 8th
dayG
PX
(U
nit
s m
g-1
)
00
05
10
15
20
25
Cajanus cajan
aA
PX
(U
nit
s m
g-1
)
0
20
40
60
80
100
bb
b
aaa
b
a
bbb
a
c
a
b
89
Sole Intercrop
NO
3 (
mM
ol
g-1
)
00
02
04
06
08
10
12
14
8th
day
Sole Intercrop
Nit
rate
Red
uct
ase
(mM
ol
g-1
)
0
1
2
3
4
4th
day
Nitrate reductaseNO
3
Ziziphus mauritiana
a
b
c
cb
b
b
a
Sole Intercrop
NO
3 (
mM
ol
g-1
)
00
02
04
06
08
10
12
8th
day
Sole Intercrop
Nit
rate
Red
uct
ase
(mM
ol
g-1
)
0
2
4
6
8
10
12
4th
dayCajanas cajan
a
bb
b
aa
aa
Nitrate reductase NO3
Figure 28 Nitrate reductase activity and nitrate concentration in leaves of Z mauritiana and C cajan plants
under sole and intercropping system at 4th and 8th
dayirrigation intervals (Values represent means
plusmn standard error of each treatment and significance among the treatments was recorded at p lt
005)
90
23 Experiment No 8
Investigations of intercropping Ziziphus mauritiana with Cajanus cajan
on marginal land under field conditions
231 Materials and Methods
2311 Selection of plants
Ziziphus mautitiana and Cajanus cajan were selected for this study as described in chapter
1
2312 Experimental field
Field of Fiesta Water Park was selected to investigate intercropping of Z mauritiana with
Ccajan It is situated about 50 km from University of Karachi at super highway toward
HyderabadThe area of study has subtropical desert climate with average annual rain fall
is ~20 cmmost of which is received during the monsoon or summer seasonSince summer
temperature (April to October) are approx 30-35 degC and the winter months (November to
March) are ~20 degC Wind velocity is generally high all the year Topography of the area
was uneven with clay- loam soil having gravels Xerophytic plants are pre-dominantly
present in the area including Prosopis spp Acacia spp Euphorbia spp Caparus
deciduas etc
2313 Soil analysis
Before conducting experiment soil of Fiesta Water Park field was randomly sampled at
three locationsatone feet of depthusing soil augerThese soil samples were analyzed in
Biosaline Research Laboratory Department of Botany University of Karachi to
determine its physical and chemical properties
i Bulk density
Bulk density was determinedin accordance with Blake and Hartge (1986) by using the
following formula
Bulk density = Oven dried soil (g) volume of soil (cm3)
91
ii Soil porosity
Soil porosity was calculated in accordance with Brady and Weil (1996) by using the
following formula
Soil porosity = 1- (bulk density Particle density) times 100
Where particle density = 265 gcm3
iii Soil texture and particle size
Soil particle size was determined by Bouyoucos hydrometric method in accordance with
Gee and Or (1986)On the basis of clay silt and sand percentages soil texture was
determined by using soil texture triangle presented in Figure 31
iv Water holding capacity
Water holding capacity in percentages was calculatedaccording to George et al (2013)
v pH and Electrical conductivity of soil (ECe)
Soil saturated paste was made with de-ionized water and leave for 24 hours Soil solution
was extracted through Buckner funnel and suction pump (Rocker 300) pH of soil
solution was taken on Adwa AD1000 pHMV meter and ECe was taken on electrical
conductivity meter (4510 Jenway)
2314 Experimental design
Six months old grafted Ziziphus mauritiana saplings were carefully transported in field of
Fiesta Water Park
Three equal size plots of 100times10 sq ft were prepared for this experiment
Plot ldquoArdquo = Ziziphus mauritiana (Sole crop)
Plot ldquoBrdquo = Cajanus cajan (Sole crop)
Plot ldquoCrdquo = Ziziphus mauritiana + Cajanus cajan (intercropped)
In plot lsquoArsquo and lsquoCrsquo pits of two cubic feet depth were prepared in two parallel rows
at a distance of 10 feet (Yaragattikar amp Itnal 2003)so that the distance of pits within the
row and the distance of pits between the rows were same Each row bears nine pits
Eighteen healthy saplings of nearly equal height and vigor of Z mauritiana were
92
transplanted in the pits and were fertilized with cow-dong manure Plants were irrigated
with underground (pumped) water initially on alternate day for two weeks older leaves
fall down completely and new leaves appeared in this establishment period Later the
irrigation interval was kept fortnightly Electrical conductivity of irrigated water (ECiw)
was 24 plusmn 05 dSm-1
After establishment of Z mauritiana water soaked seeds of intercropping plant (C
cajan) were sown in plot lsquoCrsquo Three vertical lines (strips design) of equal distance were
made between the rows of Z mauritiana The distance between the line was one feet
Eleven C cajan were maintained in each line at a distance of one feet which constitute a
total of 33 C cajan in 3 lines There were 264 plants of C cajan arranged in strip pattern
as intercrop for eighteen Z mauritiana A sole crop of C cajan in plot lsquoBrsquo was arranged
with the same manner to serve as control Similarly plot lsquoArsquo was served as control of Z
mauritianaThe experiment was observed up to reproductive yield of each plant
Field diagram Theoritical model of intercropping system used in this study showing sole crop in Plot lsquoArsquo
(Z Mauritiana) and Plot lsquoBrsquo (C cajan) while Plot lsquoCrsquo represents intercropping of both
species at marginal land
Six Z mauritiana plants were randomly selected from their two rows of block lsquoCrsquo
which were facing two rows of C cajan on either sides Similarly ten plants of C cajan
facing Z mauritiana were randomly selected for further study At the same manner six Z
mauritiana from block lsquoArsquo and ten C cajan from block lsquoBrsquo grown as sole crop were
selected as control for further study
93
2315 Vegetative and reproductive growth
Vegetative growth of Z mauritiana plant was noted in terms of height volume of canopy
while height and number of branches in Ccajan bimonthly after establishment Fresh and
dry weightsof leaves stem and root were observed at final harvest in both plant species
growing as sole or intercropping
Reproductive growth of Z mauritiana such as number length and diameter fruit
weight per ten plant and average fruit yield was measured at termination of the experiment
Whereas reproductive growth in C cajan was monitored in terms of number of pods
number of seeds weight of pods and weight of seed
2316 Analyses on some biochemical parameters
Following biochemical analysis was conducted in Fully expended leavesof Z mauritiana
and C cajan growing as sole and as intercropped at grand period of growth Additionally
fruits of Z mauritiana were also analyzed for their protein soluble and insoluble sugars
and total phenolic contents
i Photosynthetic pigments
Photosynthetic pigments including chlorophyll a chlorophyll b and total chlorophyll were
estimated in leaves of Z mauritiana and C cajan according to procedure described in
chapter 1
ii Protein in leaves
Protein contents were estimated in leaves of Z mauritiana and C cajan according to
procedure described in chapter 1
iii Total soluble sugars in leaves
Total soluble sugars were estimated in leaves of Z mauritiana and C cajanaccording to
procedure described in chapter 1
94
iv Phenolic contents in leaves
Phenolic content were estimated in leaves of Z mauritiana and C cajan according to
procedure described in chapter 1
2317 Fruit analysis
i Protein in fruit
Protein content in fruit of Z mauritiana was estimated according to procedure described
in chapter 1
ii Total soluble sugars in fruits
Total soluble sugars in ripe fruits of Z mauritiana were estimated according to procedure
described in chapter 1
iii Phenolic contents in fruits
Phenolic contents in fruits of Z mauritiana were estimated according to procedure
described in chapter 1
2318 Nitrogen estimation
Nitrogen was also estimated in root zone soil as well as in fully expended leaves of Z
mauritiana and C cajan plants
Total nitrogen in leaves and soil was estimated through AOAC method 95504
(2005) One g of dried powdered sample in round bottle flask was digested in presence of
20 mL H2SO4 15 mL K2SO4 and 07g CuSO4 at 400oC heating mental After digestion 80
ml distilled water was added in digest Then distillation was done at 100oC by adding 100
mL of 45 NaOH (drop wise) in digested solution Steam was collected in 35 mL of 01M
HCl in a flask Three samples of 10 mL each steam collected solution were taken and 2-3
drops of methyl orange was added as indicator Titration was made with 01M NaOH
Changeappearance of color indicates the completion of reactionPercent nitrogen was
calculated through following equation
N = (mL of acid times molarity) ndash (mL of base times molarity) times 14007
95
2319 Land equivalent ratio and Land equivalent coefficient
The LER defined the total land area needed for sole crop system to give yield obtained
mixed crop It is mainly used to evaluate the performance of intercropping (Willey 1979)
Land equivalent ratio (LER) of two crops was estimated according to (Willey 1979) by
using formula
Whereas partial LER of Z mauritiana calculated according to
Similarly Partial LER of Ccajan were calculated as
Land equivalent coefficient (LEC) an assess of dealings the effectiveness of relationship
of two crops (Alhassan et al 2012) was calculated by using (Adetiloye et al 1983)
equation as
Yield was calculated in gram fresh weight LER and LEC of height and total chlorophyll
were also calculated by using above formula by substituting their values with yield (fruits
of Z mauritiana and seeds of C cajan) to height fruits and chlorophyll respectively
23110 Statistical analysis
Data were analyzed by using (ANOVA) and the significant differences between treatment
means wereexamined by least significant difference (Zar 2010) All statistical analysis
was performed using SPSS for windows version 14 and graphs were plotted using Sigma
plot 2000
LER= Yield of Z mauritiana + Yield of C cajan (in intercropped) + Yield of C cajan + Yield of Z mauritiana (in intercropped)
Yield of Z mauritiana (sole) Yield of C cajan (sole)
Partial LER = Yield of Z mauritiana + Yield of C cajan (in intercropped)
Yield of Z mauritiana (sole)
Partial LER = Yield of C cajan + Yield of Z mauritiana (in intercropped)
Yield of C cajan (sole)
LEC = Partial LER of Z mauritiana times Partial LER of C cajan
96
232 Observations and Results
2321 Vegetative parameters
Vegetative growth parameters of Z mauritiana include plant height volume of canopy
grown individually as well as intercropped with C cajan is presented in Figure 29
Appendix-XV A significant increase in height and canopy volume of Z mauritiana with
time (p lt 0001) and cropping system (p lt 005) was observed However the interaction
between time and cropping system showed non-significant results In general the
intercropped plants were showed higher values in all vegetative parameters than sole crop
and this increase was more pronounced after 60 days
Figure 29 Appendix-XVII showed the vegetative growth parameters of C cajan
including height and number of branches Height of C cajan was significantly increased
(plt0001) with increasing time in plants growing sole and as intercropped with Z
mauritiana The interaction with time to crop height also showed significant (plt0001)
results in both cropping systems However slight decline in height of intercropped C
cajan was noticed at 120 days compared to sole crop Number of branches was significant
increased (plt0001) in both crops with increasing time The interaction of time with
branches also showed significant (plt0001) results in both cropping systems However
number of branches was slightly increased in intercropped plants at 120 days compared to
sole crop
2322 Reproductive parameters
i Fruit number and weight (fresh and dry)
Reproductive parameters of Z mauritiana and C cajan at grand period of growth under
sole and intercropping system has been presented in Figure 210 Appendix-XVI XVIII
Individual and interactive effect of time (p lt0001) and treatment (plt001) on number and
fresh weight of fruits of Z mauritiana was showed significant results Similarly plants
grown with C cajan showed significant increase (p lt0001) in fresh weight of fruits (p
lt005) whereas fruit dry weight and circumference was non-significant in comparison to
sole crop
97
In C cajan flowers were appeared only at blooming phase (during 60 days of treatment)
and no difference in number of flowers was observed in both cropping systems (sole and
with Z mauritiana (Figure 210 XVII)
Leguminous pods were initiated soon after flowering period (during 60 days) and
last till end of the experiment (120 days) A significant increase (plt0001) in pod numbers
was observed with increasing time in both sole and intercropped system But non-
significant differences in number of pods of both cropping system and their interaction
with time were observed
Similarly number and weight of C cajan seeds were showed non-significant difference
in both cropping systems
2323 Study on some biochemical parameters
i Photosynthetic pigments
Leaf pigments of Zmauritiana and C cajan grow plants under sole and intercropping has
been presented in Figure 211 Appendix-XVI XVIII In Z muritiana leaves A significant
increase (plt005) in chlorophyll a chlorophyll b total chlorophyll and carotinoids was
observed when grown as intercrop whereas the effect on chlorophyll ab ratio was non-
significant as that of sole one
In C cajan a slight decrease (plt005) in chlorophyll lsquobrsquo and total chlorophyll
(plt001) was observed in intercropped plants compare to sole one Whereas chlorophyll
lsquoarsquo chlorophyll ab ratio and carotinoids showed nonsignificant difference between sole
and intercropped C cajan
ii Total proteins sugar phenols
Sugars protein and phenols in leaves of Z mauritianaand C cajan at grand period of
growth under sole and intercropping system is presented in Figure 212 Appendix-XVI
XVIII Total proteins and soluble and insoluble sugar content of Z mauritiana leaves was
unaffected throughout the experiment However an increase in total phenolic content
(plt001) was observed in intercropped Z mauritiana plants than grown individually
98
In C cajan total soluble sugars protein and phenols in leaves showed non-
significant differences between sole to intercropped plants
Sugars protein and phenols in fruits of Z mauritiana grown under sole and
intercropping system is presented in Figure 213 Appendix-XVI A non-significant
increase was observed in phenolic as well as in soluble insoluble and total sugar contents
in fruits of Z mauritiana plants grown with C cajan (intercrop) as compare to the fruits
of sole crop
2324 Nitrogen Contents
Nitrogen in leaves and in soil of Z mauritiana and C cajan growing under sole and
intercrop system is presented in Figure 214 Appendix-XVI XVIII ANOVA showed a
non significant effect on nitrogen content of leaf as well as root zone soil of Z mauritiana
and C cajan grown individually or as intercropping system
2225 Land equivalent ratio (LER) and land equivalent coefficient
(LEC)
Land equivalent ratio (LER) Land equivalent coefficient (LEC) of height chlorophyll and
yield of of Z 98auritiana and C cajan growing as sole and intercropping system in has
been presented in Table 22 The LER using height of both species was nearly 2 in which
PLER of Z mutitania was 48 and PLER of C cajan was 519 Whereas the calculated
values of the land equivalent coefficient (LEC) of Z mauritiana and C cajan remained
9994
The LER using yield of both species was above 2 in which PLER of Z mauritiana
was 46 Whereas PLER of C cajan was 543 However the calculated values of LEC
of both species were 100
The LER using total chlorophylls of both species were more than 25 in which
PLER of Z mauritiana was 344 and as that of PLER of C cajan was 655 Whereas
the calculated values of LEC was 999 of both the species
99
Table 21 Soil analysis data of Fiesta Water Park experimental field
Serial number Parameters Values
1 ECe (dSm-1) 4266plusmn0536
2 pH 8666plusmn0136
3 Bulk density (gcm3) 123plusmn0035
4 Porosity () 53666plusmn1333
5 Water holding capacity () 398plusmn2811
6 Soil texture Clay loam
7 Sand () 385plusmn426
8 Silt () 3096plusmn415
9 Clay () 305plusmn1
Ece is the electrical conductivity of saturated paste of soil sample
Figure 29 Soil texture triangle (Source USDA soil classification)
100
Ziziphus mauritiana
Days
0 60 120
Volu
me
(m3)
0
10
20
30
Days
0 60 120
Hei
ght
(cm
)
0
50
100
150
200
250
Sole Intercrop
a
a
bb
c c
aa
bb
c c
Cajanus cajan
Days
0 60 120
Bra
nch
es (
)
0
10
20
30
Days
0 60 120
Hei
ght
(cm
)
0
50
100
150
200
250
300
Sole Intercrop
aa
bb
c c
aa
bb
c c
Figure 210 Vegetative growth of Z mauritiana and C cajan growing under sole and intercropping
system (Bars represent means plusmn standard error of each treatment and significance among the
treatments was recorded at p lt 005)
101
Ziziphus mauritiana
Fresh Dry
Fru
it w
eig
ht
(g)
0
50
100
150
200
Days
0 60 120 180
Nu
mb
er o
f F
ruit
s
0
100
200
300
Sole Intercrop
a
b
a
b
c
c
dd
Cajanus cajan
0 60 120
Num
ber
of
Pods
0
50
100
150
200
Days
0 60 120
Num
ber
of
Flo
wer
s
0
50
100
150
Sole Intercrop
Days
aa
bb
c c
Sole Intercrop
Num
ber
of
See
ds
0
100
200
300
400
500
See
d W
eight
(g)
0
10
20
30
40
50
60Number of seedsSeed weight
Figure 211 Reproductive growth of Z mauritiana and C cajan growing under sole and intercropping
system (Bars represent means plusmn standard error of each treatment and significance among the
treatments was recorded at p lt 005)
102
Ziziphus mauritiana
Cajanus cajan
Figure 212 Leaf pigments of Zmauritiana and C cajan growing under sole and intercropping (Bars
represent means plusmn standard error of each treatment and significance among the treatments was
recorded at p lt 005)
Sole Intercrop
Car
ote
noid
s (m
g g
-1)
00
01
02
03C
hlo
rophyl
l (m
g g
-1)
00
02
04
06
08
ab r
atio
00
05
10
15
20
25
ab
ab
Sole Intercrop
Car
ote
no
ids
(mg
g-1
)
00
01
02
03
Ch
loro
ph
yll
(m
g g
-1)
00
02
04
06
08
10
ab
rat
io
0
1
2
3
4ab
ab
103
Ziziphus mauritiana
Sole Intercrop
Lea
f P
hen
ols
(m
g g
-1)
0
2
4
6
8
10
12
Lea
f P
rote
ins
(mg
g-1
)
0
2
4
6
8
Lea
f S
ug
ars
(mg
g-1
)
0
5
10
15
20
25
30
35SoluableInsoluable
Figure 213 Sugars protein and phenols in leaves of Z mauritiana and C cajan at grand period of growth under
sole and intercropping system (Bars represent means plusmn standard error of each treatment and
significance among the treatments was recorded at p lt 005)
104
(Figure 212 continuedhellip)
Cajanus cajan
Sole Intercrop
Lea
f P
hen
ols
(m
g g
-1)
0
2
4
6
8
Lea
f P
rote
ins
(mg g
-1)
00
05
10
15
20
Lea
f S
ugar
s (m
g g
-1)
0
2
4
6
8
105
Ziziphus mauritiana
Sole Intercrop
Fru
it P
hen
ols
(m
g g
-1)
0
2
4
6
8
10
12
14
Fru
it P
rote
ins
(mg g
-1)
00
02
04
06
08
10
Fru
it S
ugar
s (m
g g
-1)
0
5
10
15
20
25
30
35 SoluableInsoluable
Figure 214 Sugars protein and phenols in fruits of Z mauritiana grown under sole and intercropping
system (Bars represent means plusmn standard error of each treatment and significance among the
treatments was recorded at p lt 005)
106
Z mauritiana
Sole Intercrop
Nit
rogen
(
)
0
1
2
3
4
5
6
7 LeafSoil
Cajanus cajan
Sole Intercrop
Nit
rogen
(
)
0
1
2
3
4
5
6
7 LeafSoil
Figure 215 Nitrogen in leaves and in soil of Z mauritiana and C cajan growing under sole and intercrop
system (Bars represent means plusmn standard error of each treatment and significance among the
treatments was recorded at p lt 005)
107
Table 22 Land equivalent ratio (LER) and Land equivalent coefficient (LEC) with reference to height chlorophyll and yield of of Z mauritiana and C cajan growing
under sole and intercropping system
Plant species Parameters Formulated with
reference to Height
Formulated with
reference to Total
Chlorophyll
Formulated with reference to Yield
(fresh weight of Z mauritiana fruit
and seed of C cajan)
Z mauritiana Partial LER 1027 1666 1159
C cajan Partial LER 0950 0877 0993
Intercropped
Total LER 1977 2543 2152
Z mauritiana amp C cajan
(Sole and intercropped) LEC 0975 1461 1151
107
108
24 Discussion
Intercropping is a common practice used to obtain better yield on a limited area through
efficient utilization of given resources which may not be achieved by growing each crop
independently (Mucheru-Muna et al 2010) In this system selection of appropriate crops
planting rates and their spatial arrangement can reduce competition for light water and
nutrients (Olowe and Adeyemo 2009) In general increased growth (biomass height
volume circumference biomass succulence SSL SRL SSR LWR SWR RWR and
RGR) of each species is a good indicator of successful intercropping The SRL and SSL
measure the ratio between the lengths of root or shoot per unit dry weight of respective
tissues (Wright and Westoby 1999) The weight ratio of leaf stem and root to total plant
weight (LWR SWR and RWR) describes the allocation of biomass towards each organ to
maximize overall relative growth rate (RGR) which explains how plant responds to certain
type of condition (Reynolds and Antonio 1996) In this study height and canopy volume
of Z mauritiana and height and branches of C cajan were increased when grown together
in comparison to sole crop in field experiment (Figure 29) Whereas in drum pot culture
biomass generally the length of plant canopy volume number of leaves RGR LWR
SWR RWR SSL and SRL were either higher or unaffected in both species growing in
intercropping at 4th and 8th days intervals (Figure 21-23) Similar beneficial effects on
growth of other intercrops have also been reported under different conditions (Yamoah
1986 Atta-Krah 1990 Kass et al 1992 Singh et al 1997) Dhyani and Tripathi (1998)
observed increased height stem diameter crown width and timber volume of three
intercropped species than sole crop Bhat et al (2013) also revealed significant
improvement in annual extension height and spread in apple plants intercropped with
leguminous plants
The increased growth of both intercropped plants of this study was well reflected
by their biochemical parameters Leaf pigments like chlorophyll a chlorophyll b and total
chlorophyll were either higher or remained unaffected (Figure 211) in both intercropped
plants than sole crops of field experiments Whereas in drum pot culture chlorophyll
content (Figure 24) was higher only in intercropped C cajan (specially in 8th days) Bhatt
et al(2008) and Massimo and Mucciarelli (2003) also reported the increased accumulation
of chlorophyll a b and total chlorophylls in leaves of soybean and peppermint when
109
grown with their respective intercrops Our results are also in agreement with Liu et al
(2014) and Otusanya et al (2008) reported similar results in Lycopersican esculentum and
later in Capsicum annum as well Some other reports are also available which shows non-
significant effect on leaf pigments in both cropping systems (Shi-dan 2012 Luiz-Neto-
Neto et al 2014)The synthesis and activity of chlorophyll depends on severity and type
of applied stress it generally increase in low saline mediums (Locy et al 1996) or
remained unaffected however sometimes stimulated (Kurban et al 1999 Parida et al
2004 Rajesh et al 1998)
Proteins and carbohydrates (sugars) perform vast array of functions which are
necessary for plant growth and reproduction (Copeland and McDonald 2012) Variation
in their contents helps to predict plant health which is usually decreased with applied stress
(Arbona et al 2013) Both are also the compulsory factors of animals diet since they
cannot manufacture sugars and some of the components of proteins which must be
obtained from food (Bailey 2012) In our experiment protein content was either remained
unchanged or increased which indicated a good coordination of both intercrops in field
and drum pot experiments (Figure 26 and 212) Liu et al (2014) also found that protein
and sugars were not affected in tomatogarlic intercrops In another experiment similar
results were found when corn was grown with and without intercropping (Borghi et al
2013)
Reactive oxygen species (ROS) are produced as a spinoff of regular metabolism
however under stress the overproduction of ROS may lead to oxidative damage (Baxter et
al 2014) In low concentrations ROS worked as messengers to regulate several plant
processes and also helps to improve tolerance to various biotic and abiotic stresses (Miller
et al 2009 Nishimura and Dangl 2010 Suzuki et al 2011) but when the concentration
goes beyond the critical limit ROS would become self-threatening at every level of
organization (Foreman et al 2003) To maintain a proper workable redox state an
efficient scavenging system of enzymatic (SOD CAT GPX and APX) andor non-
enzymatic (polyphenols sugars glutathione and ascorbic acid) antioxidants is required
which would be of critical importance when plant undergoes stress (Sharma et al 2012)
Among these enzymes SOD is a first line of defense which converts dangerous superoxide
radicals into less toxic product (H2O2) In further CAT APX and GPX worked in
association to get rid off from the excessive load of other oxygen radicals or ions (H2O2
110
OH- ROO etc) In this study antioxidant enzymes (SOD CAT GPX and APX) were
found to work in harmony which was not affected during 4th day treatment in both species
in comparison to sole crop (Fig 27) showing strong antioxidant defense which was not
compromised by cropping system When comparing in 8th day treatment a significant
general increase in all enzyme activities were observed in both species except for SOD
and GPX of C cajan (Fig 27) These results displayed relatively better performance and
tight control over the excessive generation of ROS which would be predicted in this case
due to less availability of water than in 4th day treatment (Karatas et al 2014 Doupis et
al 2013) Similarly by coping oxidative burst and maintaining cellular redox equilibrium
plants were able to improve growth performance especially in Z mauritiana (Fig 21)
Water deficit affect stomatal conductance which could bring about changes in
photosynthetic performance hence overproduction of ROS is usually found among
different crops (Moriana et al 2002 Miller et al 2010) As a response tolerant plants
overcome this situation by increased activity of antioxidant enzymes which was evident in
Wheat Rice olive etc (Zhang and Kirkham 1994 Sharma and Dubey 2005 Guo et al
2006 Sofo et al 2005)
Phenolic compounds despite their role in physiological plant processes are
involved in adsorbing and neutralizing reactive oxygen species (ROS Ashraf and Harris
2004) The overproduction of ROS may cause several plant disorders Plants produce
secondary compounds like polyphenols to maintain balance between ROS generation and
detoxification (Posmyk et al 2009) Increased synthesis and accumulation of phenolic
compounds is reported to safeguard cellular structures and molecules especially under
biotic abiotic constraints (Ksouri et al 2007 Oueslati et al 2010) In this study
intercropped Z mauritiana of field and both species in drum pot culture showed higher
phenolic content than individual crop (Figure 25 and 212) which may be attributed to
adaptive mechanism for scavenging free radicals to prevent cellular damage (Rice-Evans
1996)
In terms of fruit yield we observed that Z mauritiana is suitable for intercropping
as suggested by Yang et al (1992) Number of flowers fruits and fruit fresh weight of
both species either increased considerably or no-affected in intercropped plants compared
to individual ones (Figure 210) Moreover fruit quality of Z mauritiana includes proteins
phenols and soluble extractable and total sugars were also higher in intercropped plants
111
(Figure 213) Results of this study are better than other experiments reported by
Sharma (2004) Kumar and Chaubey (2008) and Kumar et al (2013) who did not find
influence of other understory forage crops (like Aonla) on the yield of Z mauritiana
However in other case the yield of intercropped ber was some time higher (Liu 2002)
Singh et al 2013 found no adverse effects on the yield of pigeonpea when intercropped
with mungbean however it improved the grain yield of associated species
A leguminous plant C cajan is used in this experiment as secondary crop which
can supplement Z mauritiana by improving soil fertility Results of both experiments
showed that the nitrogen was higheror un-affected (Figure 214) in soils of intercropped
plants which supports our hypothesis that leguminous intercrop increase N supply This
can be achieved by acquisition of limited resources to manage rootrhizosphere
interactions which can improve resource-use efficiency (Zhang et al 2010
Shen et al 2013 White et al 2013b Ehrmann and Ritz 2014 Li et al 2014) As a
consequence it impact on overall plant performance which starts from high photosynthetic
activity by increasing chlorophyll results in more availability of photoassimilate for
growth and reproductive allocation (Eghball and Power 1999) Use of C cajan in tree
intercropping proved beneficial for producing high yield crops and for the environment
(Gilbert 2012 Glover et al 2012)
Land equivalent ratio (LER) is commonly used to evaluate the effectiveness of
intercropping by using the resources of same environment compared with sole crop
(Vandermeer 1992 Rao et al 1990 1991 Cao et al 2012) It is the ratio of area for sole
crop to intercrop required to produce the equal amount of yield at the same management
level (Mead and Willey 1980 Dhima et al 2007) On the other hand land equivalent
coefficient (LEC) describe an association that concern with the strength of relationship It
is the proportion of biomassyield of one crop explained by the presence of the other crop
The LER 1 or more indicate a beneficial effect of both species on each other which increase
the yield of both crops as compare to single one (Zada et al 1988) In this experiment all
LER values were about 2 or more than 2 while LEC values were around 1 or more than
one in ZizyphusCajnus intercropping Both LER and LEC values were in descending
order of chlorophylls gt yield gt height (Table 22) However the partial LER was higher in
Zizyphus than Cajanus in all cases These results describe the superiority of intercropping
over sole cropping where LER values are even gt2 Some other studies reported LER from
112
09-14 (Bests 1976) 12-15 (Cunard 1976) and up to 2 (Andrews and Kassam 1976)
Similar results were reported in poplarsoybean system (Rivest et al 2010) black
locustMedicago sativa (Gruenewald et al 2007) wheatjujube (Zhang et al 2013)
Acacia salignasorghum (Droppelmann et al 2000 Raddad and Luukkanen 2007) The
high LER values in our system indicating a harmony in resource utilization in both species
which was also corroborated with their respective LEC values The greater LEC values (gt
025) suggesting an inbuilt tendency of studied crops to give yield advantage (Kheroar and
Patra 2013) Experiments based on traditional practices of growing legumes with cereals
demonstrated greater and continuous cash returns than individual-crops (Baker 1978) In
addition the same authors found further increase in cash returns by increasing the
proportion of cereal and incorporating maize with sorghum and millet In agreement with
our findings similar reports are also available from different intercropping systems
including sesamegreengram (Mandal and Pramanick 2014) maizeurdbean (Naveena et
al 2014) and pegionpeasorghum (Egbe and Bar-Anyam 2010)
After detailed investigations of both species using two different experiment designs
(drum pot and field) it is evident that intercropping had beneficial effects on growth
physiology biochemisty and yield of both species Furthermore by using this system
higher outcome interms of edible biomass and green fodder using marginal lands can be
obtained in a same time using same land and water resources which can help to eliminate
poverty and uplift socio-economic conditions
113
3 Chapter 3
Investigations on rang of salt tolerance in Carissa carandas
(varn karonda) for determining possibility of growing at waste
saline land
31 Introduction
Carissa carandas commonly known as Karonda or lsquoChrist thornrsquo belonging to family
Apocynaceae shows capability of growing under haloxeric conditions It is an important
plant which has established well at tropical and subtropical arid zone under high
temperatures It is large evergreen shrub and having short stem It has fork thorn and hence
used as hedges or fence around fields The leaves are oval or elliptic 25 to 75 cm long
dark green leathery and secrete white milk if detached The fruits are oblong broad- ovoid
or round 125- 25 cm long It has thin but tough epicarp Fruits are in clusters of 3-10
Young fruits are pinkish white and become red or dark purple on maturation
The plant is propagated through seed in August and September Budding and cutting
could also be undertaken Planting is started after first shower of monsoon Plants raised
from seeds are able to flower within two years Flowering starts in March and fruit ripen
from July to September (Kumar et al 2007) The fruit possess good amount of pectin and
acidity hence used in prickle jelly jam squash syrup and in chutney by the commercial
name lsquoNakal cherryrsquo (Mandal et al 1992) They are rich in vitamin C and good source
of Anthocyanin (Lindsey et al 2000) Its fruits also are one of the richest source of iron
(391 mg 100gm) (Tyagi et al 1999) Juice of its root is also used to treat various
microbial diseases such as diarrhea dysentery and skin disease (Taylor et al 1996)
Hence its range of salt and suitability for cultivation at waste saline land or with saline
water irrigation is being undertaken for commercial exploitation by preparing jams jellies
and prickles (Kumar 2014) Investigations on its growth and development at higher range
of salinities are being undertaken with an interest to cultivate it if profitable at highly saline
waste land
114
32 Experiment No 9
Investigation on the effect of higher range of salinities on growth of
Carissa carandas (varn karonda) created by irrigation of different
dilutions of sea salt
321 Materials and methods
3211 Drum Pot Culture
Drum pot culture as recommended by Boyko (1966) and modified by Ahmed and
Abdullah (1982) was used for the present investigation which was been already described
in Chapter 1 earlier
3212 Plant material
About six months old sapling of Carissa carandas (varn Karonda) having almost equal
height and volume poted in polythene bag in 3kg of soil fertilized with cow-dong manure
were purchased from the Noor nursery Gulshan-e-Iqbal Karachi Sindh and were
transported to the Biosaline research field department of Botany University of Karachi
3213 Experimental setup
Plants were transplanted in drum pot (Homemade lysimeter) filled with sandy loam mixed
with cow dung manure (91) Each drum pot was irrigated weekly during summer and
fortnightly during winter months with 20 liters tap water (Eciw= 0 6 dSm-1) or water of
sea salt concentrations of various ie 03 (Eciw = 42 dSm-1) 04 (Eciw =61 dSm-1)
06 (Eciw = 99 dSm-1) and 08 (Eciw = 129 dSm-1) The plants were established initially
by irrigation with tap water for two weeks and later salinity was gradually increased till
desired percentage is achieved for different treatments by dessolving of sea salt in
irrigation water Three replicates were maintained for each treatment Urea DAP and
KNO3 were the source of NPK provided in the ratio 312 50g granules Osmocot (Scotts-
Sierra Horticulture Products) and 50g Mericle-Gro (Scotts Miracle-Gro Products Inc)
were dissolved in irrigation water per drum after six months at six monthly intervals
Height and volume of canopy of these plants were recorded prior to the starting the
experiment and then after every six months interval
115
Since the vegetative growth performance in plants irrigated with 03 sea salt (Eciw = 42
dSm-1) was found comparatively better than control and only 26 decrease was noticed
in volume of canopy at plant irrigated with 04 sea salt (Eciw = 61 dSm-1) (Table III41)
the onward investigations were focused at higher salinity levels and plants were irrigated
with 06 (Eciw = 99 dSm-1) and 08 (Eciw = 129 dSm-1) sea salt in rest of experiment
3214 Vegetative parameters
Vegetative growth on the basis of plant height and volume were recorded while
reproductive growth was observed on the basis of number of flowers and number and
weight of fruits per plant Length and diameter of fruit were also recorded in ten randomly
selected fruits
3215 Analysis on some biochemical parameters
Following biochemical analysis of leaves was performed at grand period of growth (onset
of flowers)
i Photosynthetic pigments
Fresh fully expended leaves (01g) was crushed in 80 chilled acetone Further procedure
was followed described in chapter 1
ii Soluble sugars
Dry leaf samples (01g) were milled in 5mL of 80 ethanol and were centrifuged at 4000
g for 10 minutes Same procedure was followed as described in chapter 1
iii Protein content
The protein contents were measured according to Bradford Assay reagent method against
Bovine Serum Albumin which was taken for standard (Bradford 1976) as described in
chapter 1
iv Soluble phenols
The dried leaf powder (01g) was milled in 3ml of 80 methanol and was centrifuged at
10000g for 15 min Further procedure has been described in chapter 2
116
3216 Mineral Analysis
Estimation of Na+ and K+ were made according to Chapman and Pratt (1961) Oven dried
grinded Leaves (1g) furnace at 550ordmC for 6 hours and were digested in 5 ml of 2N HCl
Diluted and filtered solution was used to estimated Na+ and K+ in flame photometer
(Petracourt PFP I) The concentration of these ions was calculated against the following
standard curve equations
Na+ (ppm) = 0016135x1879824
K+ (ppm) = 0244346x1314603
117
322 Observations and Result
3221 Vegetative parameters
Vegetative growth in terms of height and volume of canopy of C carandas growing under
salinities created by irrigation of different dilutions of sea salt is presented in Table 32
Appendix-XIX A significant increase (plt0001) in plant height and volume of canopy
was observed with increasing time but the increase was rapid at early period of growth
However there was significant (plt0001) reduction under salinity stress The interaction
of time and salinity also showed significant (plt001) effect on plant parameters but the
increase in height and volume of canopy at Eciw= 42dSm-1of sea salt salinity was more
than control Plants irrigated with Eciw= 61 dSm-1 and Eciw= 99 dSm-1sea salt solution
showed decrease in height with respect to control but the difference between their
treatments was insignificantly higher decrease was observed in Eciw= 129 dSm-1 sea salt
irrigated plants
3222 Reproductive parameters
Reproductive growth in terms of flowers and fruits numbers flower shedding percentage
fresh and dry weight of ten fruit their length and diameter under salinities created by
irrigation of different dilutions of sea salt is presented in Table 33 Appendix-XX Number
of flowers and fruits significantly (plt0001) decreased with increasing salinity treatment
Difference in flower initiation seems non-significant at early growth period in controls and
salinity treatments However drastic decrease was observed in plants irrigated beyond
Eciw= 99 dSm-1 with increase in salinity
Flowers shedding percentage (Table 33 Appendix-XX) show an increase directly
proportional with increase in salinity however the difference in number of flowers
between the plants irrigated with Eciw= 99 dSm-1 and Eciw= 129 dSm-1 sea salt solution
is of little significance level (plt001)
Fresh and dry weight of average fruits (plt001) and their diameter (plt001) showed
decrease with increasing salinity whereas diameter and length of fruits showed non-
significant difference
118
3224 Study on some biochemical parameters
i Photosynthetic Pigments
Photosynthetic Pigments including Chlorophyll a chlorophyll b total chlorophyll
chlorophyll a b ratio and carotenoids of C carandas growing under salinities created by
irrigation of different dilutions of sea salt is presented in Figure 31 Appendix-XX The
chlorophyll contents of leaves significantly decreased (plt0001) over control with
increasing salinity however Chlorophyll rsquobrsquo at Eciw= 99 dSm-1salinity shows significant
increase (plt0001) over control Similarly Carotenoids at Eciw= 99 dSm-1 salinity show a
bit less significant increase (plt001) compare to control while at higher salinity (Eciw=
129 dSm-1) the decline is observed at all above mentioned parameters
iii Protein Sugars and phenols
Some biochemical parameters including Protein sugars and phenolic contents of C
carandas growing under salinities created by irrigation of different dilutions of sea salt is
presented in Figure 31 Appendix-XX Soluble proteins in leaves show non-significant
decrease at Eciw= 99 dSm-1salinity as compared with controls but a significant decrease
(plt005) was noted at Eciw= 129 dSm-1 salinity Sugars also showed non-significant
decrease at both the salinity whereas on contrary soluble phenols showed significant
increase (plt0001) with increasing salinity
3225 Mineral analysis
Mineral analysis including Na and K ions performed in leaves of C carandas growing
under salinities created by irrigation of different dilutions of sea salt is presented in Figure
32 Appendix-XX Sodium significantly increased (plt0001) all the way with increasing
salinity of growth medium Whereas significant decrease (plt0001) was observed in
Potassium with increasing salinity K+Na+ ratio show continuous increase with increasing
salinity
119
Table 31 Electrical conductivities of different sea salt concentration used for determining
their effect on growth of C carandas
Treatment
Sea salt ()
ECiw of irrigation water (dSm-1) ECe of soil saturated paste
(dSm-1)
Non-saline control 06 09
03 42 48
04 61 68
06 99 112
08 129 142
Whereas ECiw and ECe are the electrical conductivities of irrigation water and soil saturated past measured in deci semen per meter
120
Table 32Vegetative growth in terms of height and volume of canopy of C carandas growing under salinities created by irrigation of different dilutions of
sea salt
Treatment
Sea salt
(ECiw dSm-1)
Initial values prior to
starting saline water
irrigation
Growth at different salinities after 06 months
Height Volume Height Volume of canopy
cm m3 cm
increase
over initial
values
increase
decrease over
control
m3 increase over
initial values
increase
decrease
over control
Control 3734plusmn455 0029plusmn0001 8227plusmn4919 5363plusmn830 - 014plusmn0015 7952plusmn269 -
42 3674plusmn1415 0026plusmn0003 9930plusmn6142 6280plusmn205 +1710 019plusmn0017 8593plusmn098 +806
61 3752plusmn1243 0026plusmn0001 6490plusmn5799 4132plusmn485 -2305 012plusmn0010 7740plusmn117 -282
99 3819plusmn4499 0028plusmn0005 5793plusmn5821 3123plusmn1446 -4185 009plusmn0008 6759plusmn377 -1499
129 3676plusmn3114 0026plusmn0008 5250plusmn4849 2775plusmn1276 -4836 006plusmn0005 5690plusmn1110 -2844
LSD0 05
Salinity
Time Fisherrsquos least significant difference
91
172
002
0005
Values represent means plusmn standard error of each treatment and significance among the treatments was recorded at p lt 005
120
121
Table 33 Vegetative growth in terms of height and volume of canopy of C carandas growing under salinities
created by irrigation of different dilutions of sea salt
Treatment
Sea salt
(ECiw dSm-1)
Growth at different salinities after 12 months
Height Volume of canopy
cm
increase
over initial
values
increase
decrease over
control
m3
increase
over initial
values
increase
decrease over
control
Control 16214 plusmn633 7674plusmn307 - 077plusmn012 9689plusmn449 -
99 9736plusmn1048 6056plusmn561 -2109 034plusmn006 9367plusmn412 -333
129 6942plusmn565 4741plusmn480 -3822 022plusmn002 9064plusmn623 -645
Table 33 continuedhellip
Treatment
Sea salt
(ECiw= dSm-1)
Growth at different salinities after 18 months
Height Volume of canopy
Cm
increase
over initial
values
increase
decrease over
control
m3
increase
over initial
values
increase
decrease over
control
Control 1676plusmn1135 7776plusmn756 - 094plusmn011 9701plusmn578 -
99 10547plusmn842 6351plusmn666 -1833 045plusmn010 9445plusmn1024 -264
129 7581plusmn593 5154plusmn716 -3372 030plusmn003 9318plusmn580 -395
Table 33 continuedhellip
122
Table 33 continuedhellip
Treatment
Sea salt
(ECiw= dSm-1)
Growth at different salinities after 24 months
Height Volume of canopy
Cm
increase
over initial
values
increase
decrease over
control
m3
increase
over initial
values
increase
decrease over
control
Control 1911plusmn6
05 8055plusmn941 - 121plusmn015 9837plusmn522 -
99 1110plusmn5
31 6557plusmn543 -1859 053plusmn002 9509plusmn1032 -334
129 8754plusmn10
67 5990plusmn801 -2564 040plusmn008 9287plusmn745 -560
Table 33 continuedhellip
Treatment
Sea salt
(ECiw= dSm-1)
Growth at different salinities after 30 months
Height Volume of canopy
Cm
increase
over initial
values
increase
decrease over
control
m3
increase
over initial
values
increase
decrease over
control
Control 2052plusmn1126 8182plusmn676 - 146plusmn029 9873plusmn729 -
99 11700plusmn816 6743plusmn610 -1759 070plusmn011 9565plusmn850 -312
129 9628plusmn552 6189plusmn573 -2436 050plusmn004 9417plusmn1011 -462
LSD0 05 Salinity 77 007
Time 168 016
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and significance among the treatments was recorded at p lt 005
123
Table 34 Reproductive growth in terms of flowers and fruits numbers flower shedding percentage fresh and dry weight of ten fruit and their totals
perplant fruit length and diameter of C carandas growing under salinities created by irrigation of different dilutions of sea salt
Treatment
Sea salt
(ECiw= dSm-1)
Flower Fruits Flower
shedding
Weight of
Ten
fruit(fresh)
Weight of
Ten
fruit(dry)
Weight of
total fruitplant
(fresh)
Weight of
total fruitplant
(dry)
length
fruit
diameter
fruit
Numbers Numbers g g g g mm mm
Control 19467plusmn203 16600plusmn231 1468plusmn208 2282plusmn022 605plusmn009 37891plusmn891 10047plusmn283 1800plusmn003 1423plusmn006
99 12050plusmn202 7267plusmn491 3980plusmn307 1880plusmn035 530plusmn029 13695plusmn1174 3880plusmn469 1732plusmn037 1297plusmn011
129 12567plusmn549 6967plusmn203 4449plusmn082 1541plusmn023 435plusmn026 10742plusmn470 3041plusmn268 1711plusmn015 1233plusmn038
LSD0 05 Salinity 1514 1417 929 115 097 3785 1494 0971 097
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and significance among the treatments was recorded at p lt 005
123
124
Sea Salt (ECiw
= dSm-1
)
Cont 99 129
Car
ote
nio
ds
(mg
g-1
)
00
01
02
03
04
Ch
loro
ph
yll
(m
g g
-1)
00
01
02
03
04
05
06
ab
rat
io
00
05
10
15
20
25
30
35
ab
Chl a Chl b
a
a
a a
b
bcbc
a
b
c
a a
b
Figure 31 Chlorophyll a chlorophyll b total chlorophyll chlorophyll a b ratio carotenoids contents of C
carandas growing under salinities created by irrigation of different dilutions of sea salt (Bars
represent means plusmn standard error of each treatment and significance among the treatments was
recorded at p lt 005)
125
Sea Salt (ECiw
= dSm-1
)
Cont 99 129
Ph
eno
ls (
mg
g-1
)
0
5
10
15
20
Pro
tein
s (m
g g
-1)
0
1
2
3
4
Su
gar
s (m
g g
-1)
0
30
60
90
120
150Soluble Insoluble
a
a
a
a
a
a
b
b
b
c
ab
a
a
b
Figure 32 Total protein sugars and phenolic contents of C carandas growing under salinities created by
irrigation of different dilutions of sea salt (Bars represent means plusmn standard error of each treatment
and significance among the treatments was recorded at p lt 005)
126
Sea Salt (ECiw
= dSm-1
)
Cont 99 129
Ions
(mg
g-1
DW
)
0
20
40
60
80
100
120
KN
a ra
tio
00
01
02
03
04
05
06
07
Na K KNa
c
a
b
b
a
c
a
b
c
Figure 33 Mineral analysis including Na and K ions was done on leaves of C carandas growing under salinities
created by irrigation of different dilutions of sea salt (Bars represent means plusmn standard error of each
treatment and significance among the treatments was recorded at p lt 005)
127
33 Discussion
The volume and height of plants were increased per unit time under saline conditions This
increase was observed after six months in 03 sea salt (ECiw = 42 dSm-1) treated plants in
comparison to control (Table 32) Slight decrease was observed at 04 sea salt
(ECiw=61dSm-1) irrigation after which (Eciw= 99 dSm-1 and Eciw = 129 dSm-1sea salt) the
growth was significantly inhibited (Table 33) Noble and Rogers (1994) also noticed a general
decrease in growth of some of the glycophytes Humaira and Ahmad (2004) and Rivelli et al
(2004) also reported a proportional decrease in height of canola with increasing salinity
Cotton plants irrigated with saline water or those grown at saline soil are reported to increase
Na+ content in leaves accompanied by significant reduction in vegetative biomass (Meloni et
al 2001) Bayuelo-Jimenez et al (2003) observed salt induced growth inhibition of tomato
plant which was higher in shoot than root
Reproductive growth in terms of number of flowers number of fruits fruit length and
diameter were decreased and percent flower shedding increased with increasing salinity
(Table 34) These effects were higher at Eciw= 99 dSm-1and then maintained with further
salinity increment However weight of fruits (fresh and dry) and total fruits per plant were
linearly decreased with increasing medium salt concentrations A decrease in different phases
of reproductive growth like flowering fertilization fruit setting yield and quality of seeds etc
are reported to be seriously affected at different level of salinity by various workers (Lumis et
al 1973 Waisel 1991 Shannon et al 1994 Tayyab et al 2016) Cole and Mclead (1985)
and Howie and Lloyd (1989) reported severe effects of different salinity treatments on
flowering intensity fruit setting and number of fruits of Citrus senensis Walker et al (1979)
also reported reduction in the fruit weight during early ripening stage of Psidium guajava
Decrease in fruit diameter of strawberries (Fragaria times ananassa) has been reported with
salinity (Ehlig and Bernstein 1958)
In this study photosynthetic pigments of C carandas were decreased with salinity and
this decrease was more sever at Eciw = 129 dSm-1sea salt salinity (Figure 31) Such a decline
in amount of leaf pigments across different salinity regimes was also reported in cotton
(Ahmed and Abdullah 1979) Pea (Hernandez et al 1995 and Hernandez et al 1999) Vicia
128
faba (Gadallah 1999) Mulberry genotype (Agastian et al 2000) and B parviflora (Parida et
al 2004)
Leaf sugars and protein were decreased in both salinity levels (Figure 32) which could
be attributed to inhibition in transport of photosynthetic product (Levit 1980) Decrease
synthesis and mobilization of glucose fructose and sucrose has been demonstrated in number
of plants growing under salt stress (Kerepesi and Galiba 2000) Inhibition in the protein and
nucleic acid synthesis in Pisum sativum and Tamarix tetragyna plants were also reported by
Bar-Nun and Poljahoff-Mayber (1977) Melander and Harvath (1977) suggested that salt
induced reduction in protein is due to increase in protein hydrolysis
A significant increase in leaves phenol with increase in salinity (Figure 32) was
observed in present investigation was also demonstrated previously in Achilleacollina (Giorgi
et al 2009) Lactuca sativa (Kim et al 2008) and B parviflora (Parida et al 2004)
Inspite of over irrigation of saline water and maintaining leaching fraction of about
40 in drum pots accumulation of salts in rhizosphere soil was not completely avoided which
was evident in the differences between ECiw and ECe values (Table 31) Deposition of salts
in rhizosphere soil interferer absorption of minerals in plants For instance leaf Na+ content
of C carandas was significantly increased while K+ decreased with increasing soil salinity
(Figure 33) Over accumulation of toxic ions disturbed plant water status which directly
affects plant growth (Flowers et al 1977 Greenway and Munns 1980) A negative
relationship between Na+ and K+ concentration in roots and leaves of guava was also reported
by Ferreira et al (2001) Increase in Na+ content decreased K+ availability and K+Na+ ratio
in Vicia taba (Gadallah 1999) and also affect the uptake of other essential minerals in
Casurina equsetifolia (Dutt et al 1991)
Carissa carandas found to be a good tolerant to salinity and drought and it can produce
edible fruits from marginal lands of arid areas Fruits of this species can be consumed in a raw
form as well as in industrial products like pickles jams jellies and marmalades
129
4 Conclusions
In the light of above mentioned investigations it appears that pre-soaking treatment of Cajanus
cajan seeds has initiated metabolic processes at faster rate earlier which has helped seeds to
start germinative metabolism prior to be effected by toxic Na+ ions at higher salinities Cajanus
cajan and Ziziphus mauritiana were found to be the good companions for intercropping These
species synergistically enhanced the growth and biochemical performance of each other by
improving fertility of marginal land and maintaining harmony among different physiological
parameters which was missing in their sole crop Their intercropping could produce fodder
and delicious fruits even from under moderately saline substrate up to profitable extant
Carissa carandas also tolerated low and moderately salinities well by adjusting proper
regulation of physiological and biochemical parameters of growth It can provide protein rich
edible fruits jams jellies and pickles of commercial importance for benefit of poor farmer
from moderately saline barren land
130
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Sudhakar C S Ramanjulu P Reddy and K Veeranjaneyulu (1997) Response of some
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Suzuki N G Miller J Morales V Shulaev MA Torres and R Mittler (2011) Respiratory
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Tavakkoli E F Fatehi S Coventry P Rengasamy and G K McDonald (2011) Additive
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168
6 THESIS APENDECES
Appendix-I One way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution of seed germination of pre-soaked seeds of C cajan in non-saline water prior to germination under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Mean
germination rate
(GR)
Salinity treatment 4422 20 221133 21015 0000
Error 441949 42 10522
Total 4864 62
Mean germination
velocity (GV)
Salinity treatment 418813 20 20941 51836 0000
Error 169671 42 40398
Total 588484 62
Mean
germination
time (GT)
Salinity treatment 0271 20 0013 8922 0000
Error 0064 42 0002
Total 0335 62
Mean germination
Index (GI)
Salinity treatment 4422 20 221133 21015 0000
Error 441949 42 10523
Total 4864607 62
Final
germination
(FG)
Salinity treatment 32107 20 1605397 25285 0000
Error 2666 42 63492
Total 34774 62
Appendix-II Two way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution of seed germination of pre-soaked seeds of C cajan in non-saline water prior to germination under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Germination percentage per
day
Salinity treatment 509583 20 25479 19187 0000
Time 53156 9 5906 4663 0002
Salinity treatment times time 251743 180 1398576 1053 ns
Error 531130 400 1327825
Total 1375283 629
Germination
rate per day
Salinity treatment
Time 761502 9 84611 83129 0000
Salinity treatment times time 442265 20 22113 24630 0000
Error 359117 400 0898
Total 2108622 629
Appendix-III One way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution of seed
germination of pre-soaked seeds of C cajan in respective saline water prior to germination under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Final mean germination
velocity (GV)
Salinity treatment 0538 6 0089 35585 0000
Error 0035 14 0003
Total 0573
Final mean
germination time (GT)
Salinity treatment 20862 6 3477 26256 0000
Error 1854 14 0132
Total 22716 20
Final mean germination
index (GI)
Salinity treatment 110514 6 18419 190215 0000
Error 1356 14 0097
Total 111869 20
Final
germination percentage (GP)
Salinity treatment 6857 6 1142857 40 0000
Error 400 14 28571
Total 7257 20
Appendix-IV Two way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution of seed
germination of pre-soaked seeds of C cajan in respective saline water prior to germination under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Germination percentage per
day
Salinity treatment 86644 6 14440816 505428 0000
Time 23378 6 3896 136373 0000
Salinity treatment times time 2717 36 75472 2641 0001
Error 2800 98 28571
Total 115540 146
Germination rate
per day
Salinity treatment 117386 6 19564 360762 0000
Time 128408 6 21401 394636 0000
Salinity treatment times time 58747 36 1632 30091 0000
Error 5314 98 0054
Total 309855 146
169
Appendix-V One way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution on seedling
emergence and height of germinating seeds of C cajan under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Seedling height of C cajan
Salinity treatment 200822 5 40056 169666 0000
Error 2833 12 0236
Total 203115 17
Seedling
emergence of C cajan
Salinity treatment 24805 6 4134 6381 000
Error 9070 14 647867
Total 33875 20
Appendix-VI Two way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution on growth and
development of C cajan in lysemeter (Drum pot) under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Plant height of
C cajan
Salinity treatment 261079 5 52215 720259 0000
Time 126015 8 15751 132488 0000
Salinity treatment times time 76778 40 1919 16144 0000
Error 11413 96 118893
Total 477028 161
Appendix-VII One way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution on growth
and development of C cajan in lysemeter (Drum pot) under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Number of
Flowers of C
cajan
Salinity treatment 3932 3 131075 39719 0000
Error 264 8 33
Total 419625 11
Number of pods
of C cajan
Salinity treatment 1473 3 491 23105 0000
Error 170 8 2125
Total 1643 11
Number of
seedspod of C cajan
Salinity treatment 3 3 1
Error 0 8 0
Total 3 11
Number of seeds plant of
C cajan
Salinity treatment 19332 3 6444 45621 0000
Error 1130 8 14125
Total 20462 11
Weight of
seeds plant of C cajan
Salinity treatment 592976 3 197658 85572 0000
Error 18478 8 2309
Total 611455 11
Chlorophyll a
of C cajan
Salinity treatment 0117 3 0039 81241 0000
Error 0004 8 0000
Total 0121 11
Chlorophyll b
of C cajan
Salinity treatment 0004 3 0001 15222 0001
Error 0001 8 0000
Total 0005 11
Total chlorophyll of
C cajan
Salinity treatment 0160 3 0053 164401 0000
Error 0002 8 0000
Total 0162 11
Chlorophyll a b
ratio of C cajan
Salinity treatment 242 3 0806 9327 0005
Error 0692 8 0086
Total 3112 11
Carotenoids of
C cajan
Salinity treatment 0015 3 0005 4510 0039
Error 0009 8 0001
Total 0025 11
Soluble sugars
of C cajan
Salinity treatment 0043 3 0014 6515 0015
Error 00178 8 0002
Total 0061 11
Insoluble
sugars of C
cajan
Salinity treatment 0118 3 0039 36262 0000
Error 0008 8 0001
Total 0127 11
Total sugars of
C cajan
Salinity treatment 0019 3 0006 4239 0045
Error 0012 8 0001
Total 0031 11
Protein of C cajan
Salinity treatment 0212 3 0070 15735 0001
Error 0036 8 0004
Total 0248 11
170
Appendix-VIII One way ANOVA for completely randomized design for range of salt tolerance of nitrogen fixing symbiotic bacteria
associated with root of C cajan
Variables Source Sum of Squares df Mean Square F-value P
Nodule
associated
Rhizobial colonies of C
cajan
Salinity treatment 35927 2 17963 229402 0000
Error 1409 18 0078
Total 37337 20
Appendix-IX Two way ANOVA for completely randomized design for growth and development of Z mauritiana in large size clay pot being irrigated with water of two different sea salt concentration
Variables Source Sum of Squares df Mean Square F-value P
Height of
Z mauritiana
Time 91030 2 45515 839 0000
Salinity treatment 3268 2 1634 10 0000
Time times Salinity treatment 1533 4 383 238 ns
Error 6751 42 161
Total 104554 71
Number of
branches of
Z mauritiana
Time 25525 2 127625 25333 0000
Salinity treatment 86333 2 43166 11038 0000
Time times Salinity treatment 27416 4 6854 1752 ns
Error 16425 42 3910
Total 6575 71
Number of
flowers of
Z mauritiana
Time 73506 2 36753 167777 0000
Salinity treatment 12133 2 6066 25061 0000
Time times Salinity treatment 27824 4 6956 28736 0000
Error 10166 42 242063
Total 127759 71
Fresh weight of
Shoot of
Z mauritiana
Time 3056862 2 1528431 340777 0000
Salinity treatment 107829 2 53914 12020 0000
Time times Salinity treatment 51303 4 12825 2859 0031
Error 251167 56 4485
Total 3515820 71
Dry weight of Shoot of
Z mauritiana
Time 784079 2 392039 338932 0000
Salinity treatment 26344 2 13172 11387 0000
Time times Salinity treatment 13042 4 3260 2818 0033
Error 64774 56 1156690
Total 913855 71
Succulence of
Z mauritiana
Time 0002 2 0001 0214 ns
Salinity treatment 0006 2 0003 0682 ns
Time times Salinity treatment 0007 4 0002 0406 ns
Error 0199 45 0004
Total 51705 54
Spacific shoot
length of Z mauritiana
Time 0000 2 914 0176 0000
Salinity treatment 0002 2 0001 2096 ns
Time times Salinity treatment 0003 4 0001 1445 ns
Error 0023 45 0001
Total 6413 54
Moisture
contents of Z mauritiana
Time 1264 2 0632 0243 ns
Salinity treatment 3603 2 1801 0691 ns
Time times Salinity treatment 4172 4 1043 0400 ns
Error 117146 45 2603
Total 131675 54
Relative growth
rate of Z mauritiana
Time 1584206 1 1584206 532968 ns
Salinity treatment 18921 2 9460 3183 ns
Time times Salinity treatment 61624 2 30812 10366 0000
Error 89172 30 2972
Total 4034 36
Appendix-X One way ANOVA for completely randomized design for growth and development of Z mauritiana in large size clay pot
being irrigated with water of two different sea salt concentration
Variables Source Sum of Squares df Mean Square F-value P
Chlorophyll a
of Z mauritiana
Salinity treatment 0004 2 0002 7546 0003
Error 0006 21 0000
Total 0010 23
Chlorophyll b of Z mauritiana
Salinity treatment 0037 2 0018 4892 0018
Error 0080 21 0003
Total 0117 23
171
Total
chlorophyll of
Z mauritiana
Salinity treatment 0144 2 0072 39317 0000
Error 0038 21 0002
Total 0182 23
Chlorophyll ab ratio of
Z mauritiana
Salinity treatment 1499 2 0749 33416 0000
Error 0471 21 0022
Total 1969 23
Total soluble
sugars of
Z mauritiana
Salinity treatment 378271 2 189135 36792 0000
Error 107952 21 5140
Total 486223 23
Total protein contents of
Z mauritiana
Salinity treatment 133006 2 66502 5861 0009
Error 238268 21 11346
Total 371274 23
Appendix-XI Three way ANOVA for split-split plot design for physiological investigations on growth of Z mauritiana and C cajan in
drum pot being irrigated with water of sea salt concentration at two irrigation intervals
Variables Source Sum of Squares df Mean Square F-value P
Height of
Z mauritiana
Time 4499 2 2249 28888 0004
Crop 448028 1 448028 2208 ns
Irrigation intervals 2523 1 2523 2774 ns
Time times Crop 928088 2 464044 2288 ns
Time times irrigation interval 1120400 2 560200 0615 ns
Crop times irrigation interval 2690151 1 2690 2957 ns
Time times Crop times irrigation interval 171927 2 85963 0094 ns
Error 10916 12 909732
Total 35
Canopy volume of Z mauritiana
Time 7943 2 3971 6554 ns
Crop 0382 1 0382 0579 ns
Irrigation intervals 0068 1 0069 0103 ns
Time times Crop 0265 2 0133 0201 ns
Time times irrigation interval 1142 2 0571 0852 ns
Crop times irrigation interval 0722 1 0722 1077 ns
Time times Crop times irrigation interval 1998 2 0999 1491 ns
Error 8043 12 0670
Total 29439 35
Appendix-XII Two way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C
cajan in drum pot being irrigated with water of sea salt concentration at two irrigation intervals
Variables Source Sum of Squares df Mean Square F-value P
Plant length of
Z mauritiana
Crop 2986 1 2986 75322 0000
Irrigation interval 2986 1 2986 75322 0000
Crop times Irrigation interval 15336 1 153367 3868 ns
Error 317166 8 39645
Total 292428 12
Shoot length of
Z mauritiana
Crop 1069741 1 1069741 30890 0000
Irrigation interval 1069741 1 1069741 30890 0000
Crop times Irrigation interval 253001 1 253001 73058 0026
Error 27704 8 3463
Total 103376 12
Root length of
Z mauritiana
Crop 19763 1 19763 2671 ns
Irrigation interval 481333 1 481333 65059 0000
Crop times Irrigation interval 800333 1 800333 108177 0000
Error 59186 8 7398
Total 49165 12
Main branches
of Z mauritiana
Crop 33333 1 33333 5797 0042
Irrigation interval 48 1 48 8347 0020
Crop times Irrigation interval 0333 1 0333 0057 ns
Error 46 8 575
Total 2888 12
Lateral
branches of Z mauritiana
Crop 1344083 1 1344083 41356 0000
Irrigation interval 54675 1 54675 16823 0000
Crop times Irrigation interval 784083 1 784083 24125 0000
Error 26 8 325
Total 22465 12
Leaf numbers of
Z mauritiana
Crop 22465 12 98283 96482 0000
Irrigation interval 25025 1 25025 24566 0001
Crop times Irrigation interval 11907 1 11907 11688 0009
Error 8149 8 1018667
172
Total 2037850 12
Shootroot ratio
of Z mauritiana
Crop 0027 1 0027 1842 ns
Irrigation interval 0001 1 0001 0097 ns
Crop times Irrigation interval 0825 1 0825 54909 0000
Error 0120 8 0015
Total 27776 12
Plant fresh
weight of Z mauritiana
Crop 398107 1 398107 577818 0000
Irrigation interval 139514 1 139514 20249 0000
Crop times Irrigation interval 146898 1 146898 21321 0000
Error 5511 8 688982
Total 7248659 12
Plant dry weight of Z mauritiana
Crop 87808 1 87808 471436 0000
Irrigation interval 57893 1 57893 31082 0000
Crop times Irrigation interval 61132 1 61132 32821 0000
Error 14900 8 186257
Total 1875710 12
Stem fresh
weight of
Z mauritiana
Crop 46687 1 46687 227539 0000
Irrigation interval 17933 1 17933 87402 0000
Crop times Irrigation interval 20180 1 20180 98351 0000
Error 16414 8 205185
Total 1718530 12
Root fresh weight of
Z mauritiana
Crop 58450 1 58450 2295 0000
Irrigation interval 42186 1 42186 165641 0000
Crop times Irrigation interval 37307 1 37307 146487 0000
Error 203746 8 25468
Total 357145 12
Leaf fresh weight of
Z mauritiana
Crop 29970 1 29970 19089 0000
Irrigation interval 117018 1 1170187 7453 0025
Crop times Irrigation interval 2310 1 2310 14714 0004
Error 125596 8 15699
Total 699711 12
Stem dry weight
of Z mauritiana
Crop 13587 1 13587 216591 0000
Irrigation interval 11856 1 11856 18899 0000
Crop times Irrigation interval 6787763 1 6787 108197 0000
Error 50188 8 62735
Total 4689795 12
Root dry weight
of Z mauritiana
Crop 1358787 1 13587 216591 0000
Irrigation interval 1497427 1 14974 118615 0000
Crop times Irrigation interval 128773 1 12877 1020052 0000
Error 100993 8 12624
Total 124421 12
Leaf dry weight
of Z mauritiana
Crop 2374 1 2374 135380 0000
Irrigation interval 8748 1 8748 4987 ns
Crop times Irrigation interval 26403 1 2640 150539 0000
Error 140313 8 17539
Total 127170 12
Plant moisture of Z mauritiana
Crop 22082 1 22082 5608 0045
Irrigation interval 38702 1 38702 9830 0013
Crop times Irrigation interval 44406 1 44406 11279 0009
Error 31496 8 3937
Total 29872 12
Stem moisture of Z mauritiana
Crop 0005 1 0005 0000 ns
Irrigation interval 110663 1 110663 12023 0008
Crop times Irrigation interval 0897 1 0897 0097 ns
Error 73633 8 9204
Total 28532 12
Root moisture of Z mauritiana
Crop 235266 1 235266 16502 0003
Irrigation interval 3923 1 3923 0275 ns
Crop times Irrigation interval 0856 1 0856 0060 ns
Error 114051 8 14256
Total 17572 12
Leaf moisture
of Z mauritiana
Crop 130413 1 130413 47746 0000
Irrigation interval 22256 1 22256 8148 0021
Crop times Irrigation interval 210662 1 210662 77127 0000
Error 21850 8 2731
Total 38888 12
173
Relative growth
rate of Z mauritiana
Crop 0000 1 0000 287467 0000
Irrigation interval 0000 1 0000 164217 0000
Crop times Irrigation interval 0000 1 0000 179626 0000
Error 0000 8 0000
Total 0009 12
Relative water
contents of Z
mauritiana
Crop 37381 1 37381 1380 ns
Irrigation interval 49871 1 49871 1841 ns
Crop times Irrigation interval 13496 1 13496 0498 ns
Error 216649 8 27081
Total 50855 12
Chlorophyll a of Z mauritiana
Crop 0103 1 0103 32466 0000
Irrigation interval 0003 1 0003 1075 ns
Crop times Irrigation interval 0000 1 0000 0187 ns
Error 0025 8 0003
Total 1498 12
Chlorophyll b
of Z mauritiana
Crop 0027 1 0027 196164 0000
Irrigation interval 0002 1 0002 15656 0004
Crop times Irrigation interval 0006 1 0006 45063 0000
Error 0001 8 0000
Total 0456 12
Total chlorophyll
of Z mauritiana
Crop 0257 1 0257 53469 0000
Irrigation interval 0001 1 0001 0315 ns
Crop times Irrigation interval 0002 1 0002 0442 ns
Error 0038 8 0004
Total 3736 12
Chlorophyll a b ratio of
Z mauritiana
Crop 0002 1 0002 0028 ns
Irrigation interval 0169 1 0169 1696 ns
Crop times Irrigation interval 1064 1 1064 10643 0011
Error 0799 8 0099
Total 43067 12
Carotenoids of
Z mauritiana
Crop 0018 1 0018 42747 0000
Irrigation interval 0002 1 0002 5298 0050
Crop times Irrigation interval 0003 1 0003 8118 0021
Error 0003 8 0000
Total 0451 12
Phenol of
Z mauritiana
Crop 24641 1 24641 13168 000
Irrigation interval 5078 1 5078 2714 ns
Crop times Irrigation interval 10339 1 10339 5525 0046
Error 14969 8 1871
Total 6289 12
Proline of Z mauritiana
Crop 0001 1 0001 52288 0000
Irrigation interval 0000 1 0000 6972 0029
Crop times Irrigation interval 0000 1 0000 0358 ns
Error 0000 8 0000
Total 0005 12
Protein of Z mauritiana
Crop 200001 1 200001 296 ns
Irrigation interval 69264 1 69264 102 ns
Crop times Irrigation interval 4453 1 4453 006 ns
Error 540367 8 67545
Total 814086 11
CAT enzyme of
Z mauritiana
Crop 74171 1 74171 11404 0009
Irrigation interval 299930 1 299930 46117 0000
Crop times Irrigation interval 15336 1 15336 2358 ns
Error 52029 8 65036
Total 441467 11
APX enzyme of
Z mauritiana
Crop 191918 1 191918 6693 0032
Irrigation interval 4665 1 4665 162723 0000
Crop times Irrigation interval 336912 1 336912 11750 0009
Error 229383 8 28672
Total 5423 11
GPX enzyme of
Z mauritiana
Crop 0000 1 0000 0020 ns
Irrigation interval 0103 1 0103 5893 0041
Crop times Irrigation interval 0109 1 0109 6220 0037
Error 0140 8 0017
Total 0353 11
SOD enzyme Crop 8471 1 8471 1364 ns
174
of
Z mauritiana
Irrigation interval 6220 1 6220 1001 ns
Crop times Irrigation interval 21142 1 21142 3405 ns
Error 49664 8 6208
Total 85498 11
NR enzyme of
Z mauritiana
Crop 7520 1 75208333333 37253364154 0003
Irrigation interval 1360 1 1360 6737 0318
Crop times Irrigation interval 0016 1 0016 0079 ns
Error 1615 8 0201
Total 10512 11
Nitrate of
Z mauritiana
Crop 003 1 003 3028 ns
Irrigation interval 0018 1 0018 1831 ns
Crop times Irrigation interval 0003 1 0003 0336 ns
Error 0079 8 0009
Total 0130 11
Appendix-XIII Three way ANOVA for split-split design for physiological investigations on growth of Z mauritiana and C cajan in drum
pot being irrigated with water of sea salt concentration at two irrigation intervals
Variables Source Sum of Squares df Mean Square F-value P
Height of
C cajan
Time 14990 2 7495 235059 0000
Crop 7848 1 7848 42235 0000
Irrigation intervals 749056 1 749056 9676 0009
Time times Crop 2638 2 1319140 7098 00262
Time times irrigation interval 309932 2 154966 2001 ns
Crop times irrigation interval 9127 1 9127 0117 ns
Time times Crop times irrigation interval 31974 2 15987 0206 ns
Error 928935 12 77411
Total 29065 35
Apendix-XIV Two way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C
cajan in drum pot being irrigated with water of sea salt concentration at two irrigation intervals
Variables Source Sum of Squares df Mean Square F-value P
Plant length of C cajan
Crop 1056563 1 1056563 12331 0007
Irrigation interval 21675 1 21675 2529 ns
Crop times Irrigation interval 137363 1 137363 1603 ns
Error 68544 8 8568
Total 334030 12
Shoot length of C cajan
Crop 808520 1 808520 36580 0000
Irrigation interval 165020 1 165020 7466 0025
Crop times Irrigation interval 285187 1 285187 12902 0007
Error 17682 8 22102
Total 224013 12
Root length of C cajan
Crop 16567 1 16567 0674 ns
Irrigation interval 3520 1 3520 0143 ns
Crop times Irrigation interval 26700 1 26700 1087 ns
Error 196453 8 24556
Total 11133 12
Main branches
of C cajan
Crop 80083 1 80083 64066 0000
Irrigation interval 10083 1 10083 8066 0021
Crop times Irrigation interval 075 1 075 06 ns
Error 10 8 125
Total 335 12
Letral branches
of C cajan
Crop 0 1 0
Irrigation interval 0 1 0
Crop times Irrigation interval 0 1 0
Error 0 8 0
Total 0 12
Leaf numbers
of C cajan
Crop 1776333 1 1776333 16679 0003
Irrigation interval 972 1 972 9126 0016
Crop times Irrigation interval 176333 1 17633 1655 0234
Error 852 8 1065
Total 22342 12
Shootroot ratio of C cajan
Crop 0385 1 0385 0638 0447
Irrigation interval 0007 1 0007 0011 0916
Crop times Irrigation interval 2669 1 2669 4424 0068
Error 4825 8 0603
Total 264061 12
Crop 76816 1 76816 7494853 0025
175
Plant fresh
weight of
C cajan
Irrigation interval 730236 1 730236 7124832 0028
Crop times Irrigation interval 266869 1 266869 2603812 0145
Error 81993 8 102491
Total 25941 12
Plant dry weight of C cajan
Crop 38270 1 38270 1150145 0009
Irrigation interval 53046 1 53046 15942 0003
Crop times Irrigation interval 20202 1 20202 6071 0039
Error 26619 8 3327
Total 4150 12
Stem fresh weight of
C cajan
Crop 16100 1 16100 1462 ns
Irrigation interval 9900 1 9900 0899 ns
Crop times Irrigation interval 00675 1 0067 0006 ns
Error 8806 8 11007
Total 3318 12
Root fresh weight of
C cajan
Crop 0190 1 0190 0248 ns
Irrigation interval 27331 1 27331 35753 0000
Crop times Irrigation interval 2698 1 2698 3529 0097
Error 6115 8 0764
Total 432050 12
Leaf fresh
weight of C cajan
Crop 541363 1 541363 13825 0005
Irrigation interval 347763 1 347763 8881 0017
Crop times Irrigation interval 208333 1 208333 5320 0049
Error 313246 8 39155
Total 7236 12
Stem dry weight
of C cajan
Crop 10323 1 10323 11530 0009
Irrigation interval 0452 1 0452 0505 ns
Crop times Irrigation interval 0232 1 0232 0259 ns
Error 7162 8 0895
Total 125151 12
Root dry weight
of C cajan
Crop 0007 1 0007 012 ns
Irrigation interval 0607 1 0607 972 0014
Crop times Irrigation interval 0367 1 0367 588 0041
Error 05 8 0062
Total 3515 12
Leaf dry weight
of C cajan
Crop 9363 1 9363 15649 0004
Irrigation interval 34003 1 3400 5683 0000
Crop times Irrigation interval 11603 1 11603 19392 0002
Error 4786 8 0598
Total 95072 12
Plant moisture of C cajan
Crop 199182 1 19918 6011 0039
Irrigation interval 272215 1 27221 8215 0020
Crop times Irrigation interval 76654 1 76654 2313 0166755
Error 265079 8 33134
Total 38272 12
Stem moisture
of C cajan
Crop 100814 1 10081 3290 0107246
Irrigation interval 53460 1 53460 1744 0223065
Crop times Irrigation interval 19778 1 1977 0645 0444938
Error 245119 8 30639
Total 31036 12
Root moisture
of C cajan
Crop 26266 1 26266 1389 ns
Irrigation interval 223809 1 223809 11836 0008
Crop times Irrigation interval 0097 1 0097 0005 ns
Error 151272 8 18909
Total 58346 12
Leaf moisture
of C cajan
Crop 2623 1 2623 39350 0000
Irrigation interval 1765 1 1765 26477 0000
Crop times Irrigation interval 1425 1 1425452 21378 0001
Error 533411 8 66676
Total 36263 12
Relative growth
rate of C cajan
Crop 0000 1 0000 17924 0002
Irrigation interval 0000 1 0000 21296 0001
Crop times Irrigation interval 0000 1 0000 88141 0017
Error 0000 8 0000
Total
Crop 256935 1 256935 1560 ns
Irrigation interval 268827 1 26882 1633 ns
176
Electrolyte
leakage of C
cajan
Crop times Irrigation interval 30379 1 30379 0184 ns
Error 1316923 8 16461
Total 50381 12
Chlorophyll a
of C cajan
Crop 0101 1 0101 7957 0022
Irrigation interval 0062 1 0062 4893 ns
Crop times Irrigation interval 0199 1 0199 15600 0004
Error 0102 8 0012
Total 5060 12
Chlorophyll b
of C cajan
Crop 0017 1 0017 7758 0023
Irrigation interval 0027 1 0027 12389 0007
Crop times Irrigation interval 0056 1 0056 25313 0001
Error 0017 8 0002
Total 1727 12
Total
chlorophyll of C cajan
Crop 0178 1 0178 14819 0004
Irrigation interval 0198 1 0198 16520 0003
Crop times Irrigation interval 0509 1 0509 42379 0000
Error 0096 8 0012
Total 13217 12
Chlorophyll a b
ratio of C cajan
Crop 0065 1 0065 0691 ns
Irrigation interval 0033 1 0033 0357 ns
Crop times Irrigation interval 0016 1 0016 0173 ns
Error 0756 8 0094
Total 35143 12
Carotenoids of C cajan
Crop 0021 1 0021 19599 0002
Irrigation interval 0028 1 0028 26616 0000
Crop times Irrigation interval 0041 1 0041 38531 0000
Error 0008 8 0001
Total 1443 12
Phenol of C cajan
Crop 0799 1 0799 3171 ns
Irrigation interval 0040 1 0040 0159 ns
Crop times Irrigation interval 0911 1 0911 3617 ns
Error 2016 8 0252
Total 970313 12
Proline of C cajan
Crop 0008 1 0008 14867 0004
Irrigation interval 0019 1 0019 34536 0000
Crop times Irrigation interval 0008 1 0008 14969 0004
Error 0004 8 0000
Total 0155 12
Protein of C
cajan
Crop 116376 1 116376 3990 ns
Irrigation interval 434523 1 434524 14899 0048
Crop times Irrigation interval 33166 1 33166 1137 ns
Error 233303 8 29163
Total 817371 11
CAT enzyme
of C cajan
Crop 0249 1 0249 0121 ns
Irrigation interval 2803 1 2803 13702 ns
Crop times Irrigation interval 92392 1 9239 4517 ns
Error 16362 8 2045
Total 28654 11
APX enzyme
of C cajan
Crop 855939 1 855939 4073 ns
Irrigation interval 1078226 1 1078226 5130 ns
Crop times Irrigation interval 13522 1 13522 64349 000
Error 1681112 8 210139
Total 17137 11
GPX enzyme
of C cajan
Crop 0965 1 0965 9265 0160
Irrigation interval 1167 1 1167 11195 0101
Crop times Irrigation interval 0887 1 0887 8514 0194
Error 0833 8 0104
Total 3854 11
SOD enzyme
of C cajan
Crop 4125 1 4125 9731 0142
Irrigation interval 4865 1 4865 11477 0095
Crop times Irrigation interval 20421 1 20421 48172 0001
Error 3391 8 0423
Total 32804 11
Nitrate
reductase
enzyme
Crop 0053 1 0053 0034 ns
Irrigation interval 0001 1 0001 0000 ns
Crop times Irrigation interval 10329 1 10329 6650 0327
177
of C cajan Error 12424 8 1553
Total 22808 11
Nitrate of
C cajan
Crop 0039 1 0039 0576 ns
Irrigation interval 0083 1 0083 1222 ns
Crop times Irrigation interval 0003 1 0003 0005 ns
Error 0545 8 0068
Total 0668 11
Appendix-XV Two way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C
cajan intercropped on marginal land under field condition
Variables Source Sum of Squares df Mean Square F-value P
Height of Z mauritiana
Time 79704 3 26568 77303 0000
Treatment 979209 1 979209 4702 0455
Time times Treatment 756019 3 252006 1210 3381 ns
Error 3332 16 208259
Total 90366 39
Canopy volume of Z mauritiana
Time 1049 3 3498 115444 0000
Treatment 3509 1 3509 5966 0266
Time times Treatment 3374 3 1124 1911 1684 ns
Error 9413 16 5883
Total 1284 39
flowers numbers of Z
mauritiana
Time 1794893 3 598297 770043 0000
Treatment 19980 1 19980 10152 0057
Time times Treatment 21017 3 7005 3559 0381
Error 31488 16 1968
Total 1882468 39
Fruits numbers
of Z mauritiana
Time 324096 3 108032 297941 0000
Treatment 10824 1 10824 64081 0000
Time times Treatment 7141 3 2380 14093 0001
Error 2702 16 168913
Total 351833 39
Appendix-XVI One way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C cajan intercropped on marginal land under field condition
Variables Source Sum of Squares df Mean Square F-value P
Weight of ten
fruits (FW) of
Z mauritiana
Treatment 557113 1 557113 6663 0032
Error 668923 8 83615
Total 1226036 9
Weight of ten fruits (DW) of
Z mauritiana
Treatment 4356 1 4356 0321 ns
Error 10862 8 13577
Total 112976 9
diameter of fruit of Zmauritiana
Treatment 0534 1 0534 0946 ns
Error 4514 8 0564
Total 5048 9
Fruit weight per plant of
Z mauritiana
Treatment 0739 1 0739 4022 ns
Error 1471 8 0184
Total 2211 9
Fruit sugar
(soluble) of
Z mauritiana
Treatment 5041 1 5041 0081 ns
Error 497328 8 62166
Total 502369 9
Fruit sugar (extractable) of
Z mauritiana
Treatment 32041 1 32041 0424 ns
Error 604384 8 75548
Total 636425 9
Total fruit
sugars of Z mauritiana
Treatment 16 1 16 0780 ns
Error 164 8 205
Total 18 9
Chlorophyll a of
Z mauritiana
Treatment 0082 1 0082 1384 0020
Error 0024 4 0006
Total 0105 5
Chlorophyll b
of Z mauritiana
Treatment 0011 1 0011 8469 0043
Error 0005 4 0001
Total 0016 5
Total chlorophyll of
Z mauritiana
Treatment 0152 1 0152 11927 0025
Error 0051 4 0013
Total 0203 5
Treatment 0015 1 0015 0867 ns
Error 0067 4 0017
178
Chlorophyll a b
ratio of Z mauritiana
Total 0082 5
Carotinoids of Z mauritiana
Treatment 0011 1 0011 9719 0035
Error 0004 4 0001
Total 0015 5
Leaf protein of
Z mauritiana
Treatment 0106 1 0106 4 ns
Error 0106 4 0027
Total 0213 5
Leaf sugars
(soluble) of
Z mauritiana
Treatment 054 1 054 0025 ns
Error 848 4 212
Total 8534 5
Leaf sugars
(Extractable) of Z mauritiana
Treatment 486 1 486 8055 0046
Error 2413 4 0603
Total 7273 5
Total sugars in
leaf of Z
mauritiana
Treatment 216 1 216 0104 ns
Error 83333 4 20833
Total 85493 5
Leaf phenols of
Z mauritiana
Treatment 8166 1 8166 5665 ns
Error 5766 4 1442
Total 13933 5
Leaf nitrogen of Z mauritiana
Treatment 15 1 15 1939 ns
Error 3093 4 0773333
Total 4593 5
Soil nitrogen of
Z mauritiana
Treatment 0375 1 0375 21634 ns
Error 0693 4 0173
Total 1069 5
Appendix-XVII Two way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C
cajan intercropped on marginal land under field condition
Variables Source Sum of Squares df Mean Square F-value P
Height of Ccajan
Time 700196 2 350098 2716 0000
Treatment 594405 1 594405 16017 0000
Time times Treatment 488829 2 244415 6586 0004
Error 1001996 27 37111
Total 705495 59
Number of branches of
Ccajan
Time 8353 2 4176 1050050 0000
Treatment 24066 1 24066 18672 0000
Time times Treatment 24133 2 12066 9362 0000
Error 348 27 1288
Total 8572 59
Number of flowers of
Ccajan
Time 289297 2 144648 301277 0000
Treatment 365066 1 365066 0701 ns
Time times Treatment 730133 2 365066 0701 ns
Error 14059 27 520733
Total 317415 59
Number of pods
of Ccajan
Time 347682 2 173841 70559 0000
Treatment 159135 1 159135 1558 ns
Time times Treatment 8167 2 40835 0399 ns
Error 27574 27 1021276
Total 447407 59
Appendix-XVIII One way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C
cajan intercropped on marginal land under field condition
Variables Source Sum of Squares df Mean Square F-value P
Shoot weight
(FW) of
Ccajan
Treatment 0 1 0 0 ns
Error 87444 4 21861
Total 87444 5
Shoot weight
(RW) of Ccajan
Treatment 0 1 0 0 ns
Error 13808 4 3452
Total 13808 5
Number of
seeds of
Ccajan
Treatment 245 1 245 0005 ns
Error 940182 18 52232
Total 940427 19
Weight of seeds
of Ccajan
Treatment 02 1 02 0000 ns
Error 7585 18 421406
Total 7585 19
179
Chlorophyll a of
Ccajan
Treatment 0001 1 0001 5442 ns
Error 0001 4 0000
Total 0002 5
Chlorophyll b
of Ccajan
Treatment 0006 1 0006 9079 0039
Error 0002 4 0001
Total 0008 5
Total
chlorophyll of
Ccajan
Treatment 0017 1 0017 51558 0001
Error 0001 4 0000
Total 0019 5
Chlorophyll a b ratio of
Ccajan
Treatment 0183 1 0183 5532 ns
Error 0132 4 0033
Total 0316 5
Leaf protein of Ccajan
Treatment 0001 1 0001 0017 ns
Error 0228 4 0057
Total 0228 5
Leaf sugars of
Ccajan
Treatment 0015 1 0015 0003 ns
Error 1624 4 406
Total 16255 5
Leaf phenols of
Ccajan
Treatment 0201 1 0201 0140 ns
Error 5746 4 1436
Total 5948 5
Leaf nitrogen
of Ccajan
Treatment 1306 1 1306 3062 ns
Error 1706 4 04266
Total 3013 5
Appendix-XIX Two way ANOVA for completely randomized design for investigations on determining range of salt tolerance in Carissa
carandas
Variables Source Sum of Squares df Mean Square F-value P
Height of C carandas
Time 72042 5 14408 55957 0000
Salinity treatment 49345 2 24672 196775 0000
Time times Salinity treatment 16679 10 1667920 13302 000
Error 3009 24 125385
Total 143777 53
Volume of
canopy of
C carandas
Time 3329 4 0832 38126 000
Salinity treatment 1393 2 0696 67129 000
Time times Salinity treatment 0813 8 0102 9792 000
Error 0207 20 0010
Total 5969 44
Appendix-XX One way ANOVA for completely randomized design for investigations on determining range of salt tolerance in Carissa carandas
Variables Source Sum of Squares df Mean Square F-value P
Number of
flowers of C carandas
Salinity treatment 10288 2 5144194 1342937 0000
Error 229833 6 38305
Total 10518 8
Number of fruits of
C carandas
Salinity treatment 18000 2 9000 268215 0000
Error 201333 6 33555
Total 18201 8
Flower shedding
percentage of C carandas
Salinity treatment 1541647 2 770823 53455 0000
Error 86519 6 144199
Total 1628166 8
Weight of ten fruits (FW) of
C carandas
Salinity treatment 82632 2 41316 187678 0000
Error 1321 6 0220
Total 83953 8
Weight of ten
fruits (DW) of
C carandas
Salinity treatment 4355 2 2177 13753 0005
Error 095 6 0158
Total 5305 8
Fruits per plant
(FW) of
C carandas
Salinity treatment 133127 2 66563 278148 0000
Error 1435861 6 239310
Total 134563 8
Fruits per plant
(DW) of C carandas
Salinity treatment 8782 2 439117 117790 0000
Error 223677 6 37279
Total 9006 8
Size of fruits of C carandas
Salinity treatment 1301 2 0651 4125 ns
Error 0946 6 0158
Total 2248 8
Salinity treatment 5607 2 2804 17592 0003
180
Diameter of fruit
of C carandas
Error 0956 6 0159
Total 6563 8
Chlorophyll a of C carandas
Salinity treatment 0112 2 0056 119786 0000
Error 0003 6 0000
Total 0115 8
Chlorophyll b of
C carandas
Salinity treatment 0005 2 0002 434 0000
Error 0000 6 0000
Total 0005 8
Total chlorophyll of C carandas
Salinity treatment 0159 2 0079 104188 0000
Error 0005 6 0001
Total 0164 8
Chlorophyll a b
ratio of C carandas
Salinity treatment 9661 2 4831 324691 0000
Error 0089 6 0015
Total 9751 8
Carotenoids of C carandas
Salinity treatment 0029 2 0014 28822 0000
Error 0003 6 0001
Total 0032 8
Leaf Protein of
C carandas
Salinity treatment 2722 2 1361 98 0012
Error 0833 6 0138
Total 3555 8
Soluble sugar of
C carandas
Salinity treatment 234889 2 117444 12735 0006
Error 55333 6 9222
Total 290222 8
In soluble sugars
of C carandas
Salinity treatment 595395 2 297698 39094 0000
Error 45689 6 7615
Total 641085 8
Total sugar of
C carandas
Salinity treatment 1576898 2 788448 39201 0000
Error 120676 6 20113
Total 1697574 8
Phenols of C carandas
Salinity treatment 14675 2 7338 74202 0000
Error 0593 6 0099
Total 15268 8
Leaf Na+ of
C carandas
Salinity treatment 1346 2 673 673 0000
Error 6 6 1
Total 1352 8
Leaf K+ of C carandas
Salinity treatment 798 2 399 133 0000
Error 18 6 3
Total 816 8
Leaf K+ Na+
ratio of C carandas
Salinity treatment 0305 2 0153 654333 0000
Error 0001 6 0000
Total 0307 8
181
7 Publications
vi
ACKNOWLEDGMENTS
All the praises for almighty Allah and all respects for Prophet Muhammad (Peace be Upon
Him) who has shown me the straight path
I am grateful to my supervisor Prof Dr Rafiq Ahmad for his keen interest
patronage and guidance during this research work which made successful submission of
this thesis
I also obliged to Prof Dr Ehtesham Ul Haque and Prof Dr Javed Zaki (Present
and Former Chairmen Department of Botany respectively) for providing me all the
necessary facilities and administrative support
Being employed as lecturer in Department of Botany Govt Islamia Science
College Karachi I am also thankful to Education and literacy Department Govt of Sindh
(Pakistan) for providing me facilities to perform this study
Thanks are due to Dr D Khan in assessing statistical data analysis and colleague
of Biosaline lab Dr M Azeem Dr Naeem Ahmed and M Wajahat Ali Khan for their
cooperation throughout the course of study
I am also gratefully acknowledged to Mr Noushad Raheem and Mr Noor Uddin
of Fiesta Water Park for providing field plot and facilities to perform this study I am also
thankful to Pakistan Metrological Department for providing environmental data
I am also obliged to Dr M Qasim and Dr M Waseem Abbasi for their suggestions
and support in writing this thesis
Assistance of Abbul Hassan (Lab attendant) Tajwar Khan (Biosaline field
Attendant) and Mr Wahid (Plant Physiology Lab Assistant) is also acknowledged
Thanks are also due to my friends Dr Rafat Saeed Dr Kabir Ahmad Dr Zia Ur
Rehman Farooqi Dr Noor Dr M Yousuf Adnan Asif Bashir Dr A Rauf A Hai Faiz
Ahmed MA Rasheed Jallal Uddin Saadi Ahsan Shaikh Saima Fehmi A Mubeen
Khan Dr Noor Ul Haq Saima Ahmad S Safder Raza SM Akber and my college
colleagues for giving me encouragement during this research work
vii
I can never forget the support and encouragement and good wishes of Mr M
Wilayat Ali Khan Mrs Shahnaz Rukhsana Mr Mansoor Mrs Rabia Mansoor Mrs
Chand Bibi and Mrs Saeeda Anwar
In the last I am highly grateful to my beloved father Muhammad Hanif my loving
mother Arifa (when she alive) my caring wife Shaheen and sweet childrenrsquos Sara and
Sarim my supportive brothers and sisters and all family members for their prayers love
sacrifices and encouragements provided during course of this research work
viii
TABLE OF CONTENTS
No Title Page no
Acknowledgement vi
Summary xix
Urdu translation of summary xxi
General introduction 1
Layout of thesis 11
1 Chapter 1 13
11 Introduction 13
12 Experiment No 1 15
121 Materials and methods 15
1211 Seed collection 15
1212 Experimental Design 15
122 Observations and Results 17
13 Experiment No 2 22
131 Materials and methods 22
1311 Seed germination 22
132 Observations and Results 23
14 Experiment No 3 28
141 Materials and methods 28
1411 Seedling establishment 28
142 Observations and Results 29
1421 Seedling establishment 29
1422 Shoot height 29
15 Experiment No 4 31
151 Materials and methods 31
1511 Drum pot culture 31
1512 Experimental design 31
1513 Vegetative and Reproductive growth 32
1514 Analysis on some biochemical parameters 32
152 Observations and Results 34
1521 Vegetative and Reproductive growth 34
ix
No Title Page no
1522 Study on some biochemical parameters 34
16 Experiment No 5 41
161 Materials and methods 41
1611 Isolation Identification and purification of bacteria 41
1612 Preparation of bacterial cell suspension 41
1613 Study of salt tolerance of Rhizobium isolated from root
nodules of C cajan
41
162 Observations and Results 42
17 Experiment No 6 44
171 Materials and methods 44
1711 Experimental design 44
1712 Vegetative and reproductive growth 45
1713 Analysis on some biochemical parameters 45
172 Observations and Results 46
1721 Vegetative and Reproductive growth 46
1722 Study on some biochemical parameters 46
18 Discussion (Chapter 1) 51
2 Chapter 2 59
21 Introduction 59
22 Experiment No 7 60
221 Materials and Methods 60
2211 Growth and Development 60
2212 Drum pot culture 60
2213 Experimental Design 60
2214 Irrigation Intervals 61
2215 Estimation of Nitrate content 62
2216 Relative Water content (RWC) 62
2217 Electrolyte leakage percentage (EL) 62
2218 Photosynthetic pigments 63
2219 Total soluble sugars 63
22110 Proline content 63
22111 Soluble phenols 64
x
No Title Page no
22112 Total soluble proteins 64
22113 Enzymes Assay 64
222 Observations and Results 67
2221 Vegetative growth 67
2222 Photosynthetic pigments 70
2223 Electrolyte leakage percentage (EL) 70
2224 Phenols 70
2225 Proline 71
2226 Protein and sugars 71
2227 Enzyme essays 71
2228 Vegetative growth 73
2229 Photosynthetic pigments 75
22210 Electrolyte leakage percentage (EL) 76
22211 Phenols 76
22212 Proline 77
22213 Protein and Sugars 77
22214 Enzyme assay 77
23 Experiment No8 90
231 Materials and Methods 90
2311 Selection of plants 90
2312 Experimental field 90
2313 Soil analysis 90
2314 Experimental design 91
2315 Vegetative and reproductive growth 93
2316 Analysis on some biochemical parameters 93
2317 Fruit analysis 94
2318 Nitrogen estimation 94
2319 Land equivalent ratio and Land equivalent coefficient 95
23110 Statistical analysis 95
232 Observations and Results 96
2321 Vegetative parameters 96
2322 Reproductive parameters 96
xi
No Title Page no
2323 Study on some biochemical parameters 97
2324 Nitrogen Contents 98
2325 Land equivalent ratio land equivalent coefficient 98
24 Discussion (Chapter 2) 108
3 Chapter 3 113
31 Introduction 113
32 Experiment No 9 114
321 Materials and methods 114
3211 Drum Pot Culture 114
3212 Plant material 114
3213 Experimental setup 114
3214 Vegetative parameters 115
3215 Analysis on some biochemical parameters 115
3216 Mineral Analysis 116
322 Observations and Result 117
3221 Vegetative parameters 117
3222 Reproductive parameters 117
3223 Study on some biochemical parameters 118
3224 Mineral analysis 118
33 Discussion (Chapter 3) 127
4 Conclusion 129
5 References 130
6 Appendices 168
7 Publications 181
xii
LIST OF FIGURES
Figure Title Page no
11 Effect of irrigation water of different sea salt solutions on seed
germination indices of C cajan
27
12 Effect of irrigating water of different sea salt solutions on
seedling emergence (A) and shoot length (B) of C cajan
30
13 Environmental data of study area during experimental period
(July-November 2009)
36
14 Effect of salinity using irrigation water of different sea salt
concentrations on height of C cajan during 18 weeks treatment
36
15 Effect of salinity using irrigation water of different sea salt
concentrations on initial and final biomass (fresh and dry) of C
cajan
37
16 Percent change in moisture succulence relative growth rate
(RGR) and specific shoot length (SSL) of C cajan under
increasing salinity using irrigating water of different sea salt
concentrations
37
17 Effect of irrigating water of different sea salt solutions on
reproductive growth parameters including number of flowers
pod seeds and seed weight of C cajan
38
18 Effect of irrigating water of different sea salt solutions on leaf
pigments including chlorophyll a chlorophyll b total
chlorophyll and carotenoids of C cajan
39
19 Effect of irrigating water of different sea salt solutions on total
proteins soluble insoluble and total sugars in leaves of C cajan
40
110 Growth of nitrogen fixing bacteria associated with root of C
cajan under different NaCl concentrations
42
111 Photographs showing growth of Rhizobium isolated from the
nodules of C cajan in vitro on YEM agar supplemented with
different concentrations of NaCl
43
xiii
Figure Title Page no
112 Effect of salinity using irrigation water of different sea salt
concentrations on height number of branches fresh weight and
dry weight of shoot of Z mauritiana after 60 and 120 days of
treatment
47
113 Effect of salinity using irrigation water of different sea salt
concentrations on succulence specific shoot length (SSL)
moisture and relative growth rate (RGR) of Z mauritiana
48
114 Effect of salinity using irrigation water of different sea salt
concentrations on number of flowers of Z mauritiana
49
115 Effect of salinity using irrigation water of different sea salt
concentrations on leaf pigments including chlorophyll a
chlorophyll b total chlorophyll and chlorophyll ab ratio of Z
mauritiana
49
116 Effect of salinity using irrigation water of different sea salt
concentrations on total sugars and protein in leaves of Z
mauritiana
50
21 Vegetative parameters of Z mauritiana and C cajan at grand
period of growth under sole and intercropping system at two
irrigation intervals
79
22 Fresh and dry weight of Z mauritiana and C cajan plants under
sole and intercropping system at 4th and 8th day irrigation
intervals
80
23 Leaf weight ratio (LWR) root weight ratio (RWR) shoot weight
ratio (SWR)specific shoot length (SSL) specific root length
(SRL) plant moisture Succulence and relative growth rate
(RGR) of Z mauritiana and C cajan grow plants under sole and
intercropping system at 4th and 8th day irrigation intervals
81
24 Leaf pigments of Z mauritiana and C cajan grow plants under
sole and intercropping system at 4th and 8th day irrigation
intervals
83
xiv
Figure Title Page no
25 Electrolyte leakage phenols and proline of Z mauritiana and C
cajan at grand period of growth plants under sole and
intercropping system at 4th and 8th day irrigation intervals
84
26 Total protein in leaves of Z mauritiana and C cajan plants
under sole and intercropping system at 4th and 8th day irrigation
intervals
86
27 Enzymes activities in leaves of Z mauritiana and C cajan plants
under sole and intercropping system at 4th and 8th day irrigation
intervals
87
28 Nitrate reductase activity and nitrate concentration in leaves of
Z mauritiana and C cajan plants under sole and intercropping
system at 4th and 8th day irrigation intervals
89
29 Soil texture triangle (Source USDA soil classification) 99
210 Vegetative growth of Z mauritiana and C cajan growing under
sole and intercropping system
100
211 Reproductive growth of Z mauritiana and C cajan growing
under sole and intercropping system
101
212 Leaf pigments of Z mauritiana and C cajan growing under sole
and intercropping
102
213 Sugars protein and phenols in leaves of Z mauritiana and C
cajan at grand period of growth under sole and intercropping
system
103
214 Sugars protein and phenols in fruits of Z mauritiana grown
under sole and intercropping system
105
215 Nitrogen in leaves and in soil of Z mauritiana and C cajan
growing under sole and intercrop system
106
31 Chlorophyll a chlorophyll b total chlorophyll chlorophyll a b
ratio carotenoids contents of C carandas growing under
salinities created by irrigation of different dilutions of sea salt
124
xv
Figure Title Page no
32 Total protein sugars and phenolic contents of C carandas
growing under salinities created by irrigation of different
dilutions of sea salt
125
33 Mineral analysis including Na and K ions was done on leaves of
C carandas growing under salinities created by irrigation of
different dilutions of sea salt
126
xvi
LIST OF TABLES
Table Title Page no
11 Electrical conductivities of different sea salt solutions
used in germination of C cajan
18
12 Effect of irrigation water of different sea salt solutions
on germination percentage (GP) per day of C cajan
seeds pre-soaked in non-saline water prior to
germination with duration of time under various salinity
regimes
19
13 Effect of irrigation water of different sea salt solutions
on germination rate (GR) per day of seeds C cajan pre-
soaked in non-saline water prior to germination with
duration of time under various salinity regimes
20
14 Effect of irrigation water of different sea salt solutions
on mean germination rate (GR) coefficient of
germination velocity (GV) mean germination time
(GT) mean germination index (GI) and final
germination (FG) of C cajan seeds pre-soaked in non-
saline water prior to germination under various salinity
regimes
21
15 Electrical conductivities of different sea salt solutions
used in germination of C cajan
24
16 Effect of irrigation water of different sea salt solutions
on germination percentage (GP) per day of C cajan
seeds pre-soaked in respective sea salt concentrations
with duration of time
25
17 Effect of irrigation water of different sea salt solutions
on germination rate (GR) per day of C cajan seeds pre-
soaked in respective sea salt concentrations with
duration of time
26
xvii
Table Title Page no
18 Electrical conductivities of different Sea salt
concentrations and ECe of soil saturated paste at the end
of experiment
30
21 Soil analysis data of Fiesta Water Park experimental
field
99
22 Land equivalent ratio (LER) and Land equivalent
coefficient (LEC) with reference to height chlorophyll
and yield of Z mauritiana and C cajan growing under
sole and intercropping system
107
31 Electrical conductivities of different sea salt
concentration used for determining their effect on
growth of C carandas
119
32 Vegetative growth in terms of height and volume of
canopy of C carandas growing under salinities created
by irrigation of different dilutions of sea salt
120
33 Vegetative growth in terms of height and volume of
canopy of C carandas growing under salinities created
by irrigation of different dilutions of sea salt
121
34 Reproductive growth in terms of flowers and fruits
numbers flower shedding percentage fresh and dry
weight of ten fruit and their totals per plant fruit length
and diameter of C carandas growing under salinities
created by irrigation of different dilutions of sea salt
123
xviii
LIST OF ABBREVIATIONS
APX Ascorbate peroxidase
CAT Catalase
DAP Diammonium Phosphate (fertilizer)
dSm-1 Deci Siemens per meter
ECe Electrical conductivity of the Soil saturated extract
ECiw Electrical conductivity of the irrigation water
GPX Guaiacol Peroxidase
GR Glutathione reductase
GSH Reduced glutathione
LEC Land equivalent coefficient
LER Land equivalent ratio
NPK Nitrogen Phosphate Potash (fertilizer)
NR Nitrate reductase
RGR Relative growth rate
ROS Reactive oxygen species
RWR Root weight ratio
SOD Superoxide dismutase
SRL Specific Root Length
SSL Specific Shoot Length
SWR Shoot weight ratio
xix
Summary
Salinity is a growing threat to crop production which affects sustainability of agriculture
in aridsemiarid areas Growth responses of plant to salinity vary considerably among
species Cajanus cajan Ziziphus mauritiana and Carissa carandas are sub-tropical crops
grown worldwide particularly in Asian subcontinent for edible and fodder purposes but
not much is known about their salinity tolerance and intercropping
Effect of salinity has been initially studied in present work at germination of C cajan
under different sea salt salinities using presoaked seeds with water and respective salt
solutions Seed germination decreased with increasing salinity and it was more sever in
presoaking under water of different salinities The 50 threshold reduction started at
ECiw= 35 dSm-1 sea salt in presoaking treatments However this threshold was decreased
up to ECiw= 168 dSm-1 sea salt at further seedling establishment stage Growth experiment
of C cajan in drum pot culture (Lysimeter) also showed a salt induced growth reduction
in which plant tolerate salinity up to 42 dSm-1 At this salinity leaf pigments (chlorophylls
and carotenoids) proteins and insoluble sugars decreased up to 50 whereas soluble
sugars were increased (~25) Reproductive growth was also affected at this salinity in
which at least 70 reduction in flowers pods and seeds were observed
Salt tolerance of symbiotic nitrogen fixing bacteria associated with root of C cajan
showed salinity tolerance up to ECw= 366 dSm-1 NaCl salinity invitro environment For
intercropping experiments Ziziphus mauritiana (grafted variety) was selected with C
cajan Preliminary investigations showed a growth promotion in Z mauritiana at low
salinity (ECe= 72 dSm-1) and growth was remained unaffected up to ECe= 111 dSm-1
Intercropping of C cajan with Z mauritiana was primarily done in drum pot (Lysimeter)
culture Result showed better growth responses of both species when growing together as
intercrops than sole in which encouraging results were found in 8th day irrigation interval
rather than of 4th day Biochemical parameters eg photosynthetic pigments protein
phenols electrolyte leakage and sugars of these species displayed increase or decrease
according to their growth responses Increased activity of antioxidant enzymes and that of
nitrate reductase and its substrate (NO3) also contributed in enhancement of growth
Field experiment of intercropping of above mentioned plants at marginal land
irrigated with underground water (Eciw= 28 dSm-1) showed better vegetative growth of
xx
both species than sole crop The overall reproductive growth remained unaffected
although the numbers size and weight of fruit were better in intercropping system
Photosynthetic pigments were mostly increased whereas leaf protein and sugars remained
unchanged In addition higher values of LER and LEC (gt 1) indicated the success of
intercropping system
Experiment on salinity tolerance of Carissa carandas (varn karonda) using drum
pots culture showed improvement at low salinity (up to ECiw= 42 dSm-1 sea salt) whereas
higher salinity (ECiw= 129 dSm-1 sea salt) adversely affected vegetative and reproductive
growth Plant managed to tolerate up to ECiw= 99 dSm-1 sea salt Salinity severely affected
biochemical parameters including photosynthetic pigments proteins and sugars whereas
leaf phenolics were increased Leaf accumulated high amount of Na+ whereas affect
absorption of essential minerals like K+ was decreased
In the light of above mentioned investigations it appears that C cajan can be
propagated in saline soils with good presoaking techniques in non-saline water which
would helped to grow at moderately saline conditions It could be a good option used as
intercrop species because of its ability to improve soil fertility even under water deficit
conditions The proposed Cajanus-Ziziphus intercropping system could help poor farmers
to generate income from unproductive soils by obtaining sufficient fodder from C cajan
for their cattle and producing delicious edible fruits from Z mauritiana for commercial
purposes Carissa carandas could also be introduced as new crop for producing fruits from
moderate saline waste lands and used for preparing prickle jam and jelly for industrial
purposes
xxi
لاصہ خ
کا عمل ے ں ب ڑھئ لف پ ودوں می ی ےمخ طرہ ہ
وا خ ا ہ ے ب ڑھی لئ داوار کے ی ں زرعی ب وں می
ر علاق ج
ن ی م ب
ر و ب ج ن کھاری پ ن کھاری پ ن ب
دا کروت ی ر اور ر ب ے ارہ ا ہ وت لف ہ ی ی مخ کاف ں ودگی می اص Subtropical کی موج ا اور خ ی و پ وری دب ں ج ی ں ہ صلی
کی ف طے
خ
وراک و ں ج می
ی ملکوں
ائ ی ش کھاکر ای کی ی ان پ ودوں کم لوگ ہ ہت کن ب ں لی ی ی ہ
وئ عمال ہ
ارے کے طور ب ر است ری پ ن سے خ
ں ی ے ہ ں علم رکھئ ارے می ے عمل کے ت گئ ے گائ
کر ا ھ ملا
ی سات ک ہ رواداری اور ات
وں ج ن ر کےب ے ارہ
ھگوئ ہلے سے ت ں ب کاز والے محلول می لف ارت ی
مک کے مخ
دری ں ں سمی ی مطالعہ می
دائ ی کھاری اب کا
کہ پ ن کے و ی ج وئ ع ہ
کمی واف ں ی ت می ب
کی طن وں ج ن
ھ ب ہ کے سات
اف ں اض کھاری پ ن می ا گی ا کی دہ اہ کا مش رات
iwEC =اب
1-35 dSm می خ ی کہ ت ی ج مک کے ب راب ررہ
دری ں زی سمی کا
ہ ارت ں ی ام می ی ت صدی dSm= iwEC 168-1پ ودوں کے ق
ق
ی ک رہ ں Lysemeterت ے والے پ ودوں می ڑھئ ں ب روان چ می 1-dSm 24 ں جوضلہ مک محلول می
دری ں زی سمی کا
ارت
ں کر می ر خل ب زب ر س ی
ات اور غ روز مادوں لمخی
گ اف الت ف کے رت ی ت
ائ ی ں ض کھاری پ ن می ی اس
گئ کھی
ت ت د زا ب رداش
ت صدی 05اف
ق
ی ش کم وب ں کر می ی کہ خل ب زب ر س ں 50کمی ج وں می ج ن
ھلی اورب ھول ت ں ت ن می ری ج دی ب ڑھوب ولی
ا پ ا رہ مات
ہ ں اف ت صدی اض
05ق
ی گئ کھی
ت ح طور د
کمی واض ت صدی
ق
ی وی شلک سہب ڑ سے می کی چ ر مک (Symbiotic)ارہ
کی ں ا رت ی
کٹ ی ے والے ب
کرئ مد خ
ن من روج ی
اب سے (NaCl)ت
ی ر کے سا dSmwEC 366 =-1رواداری ں ب ری ہ می ج ے عمل کے ت گئ ے
گائ
کر ا ھ ملا
ی سات ک ہ یات
گئ کھی
ت ک د ر ت ھ ارہ
ت
بی ق کے ب
حق ی ت دائ ی ا اب گی ا ی
کھاری پ ن کو ج کم ں ے می ج ں dSme (Ec 72 =-1(ن ی کہ می ری ج ں ب ڑھوب ی ر می e (Ec =ب
)1-111 dSm ہل ہلے ب ے عمل ب گئ ے
گائ
کر ا ھ ملا
ی سات ک ہ کو ات ر ی ر اور ب ی ارہ
ر رہ اب ر می ی
ک غ کی خد ت
Lysemeter ج ب رآم ت ا ی زا ب ی کے جوضلہ اف
اش ی ے سے آب
ف ف ھ دن کے و
سی ت آت
کی ی ار دن ی خ
گئ کی ں ں دمی ن می ے ج
وئ ہ
ے عمل گئ ے گائ
کر ا ھ ملا
ی سات ک ہ سی ت ات
کی ی ے پ ودوں
گائ
ن ہا ا کی پ ودوں ب شام
وں اق
ے دوپ ج گئ
ت ا ی زا ب ادہ جوضلہ اف ں زت می
ی ول ب ات ف روزمادوں لخمی
گ اف الت ف کے رت ی ت
ائ ی ضلاات می درخ ی می
ائ کی می ی
ائ ےجی
وئ Electrolyteب رآمد ہ
Leakage کی کر ں س ی وں می ب ی ان پ ودوںاور ب
ی ش کمی ب ں دار می ی دپ ں مق
ں دکھائ ر می
اظ ی ری کے ب
کے ب ڑھوب
xxii
Antioxidant ی ظرح سے ہ اور اس ہ اف ں اض کی سرگرمی وں می امروں
اور اس کے Nitrate Reeducatesخ
Substrate )3(NO ا ی کا سی ب ب ہ اف ں اض ما می وں
ش ھی ی
ت
ےdSmiw(Ec 28 =-1(معمولی گئ ے ئ کب راب ں سی ی می ائ ہ ت والے ت درج ں می ری ہ می ج
ی ت ئ ن ہا زمب کی ب الا پ ودوں
ے عمل گئ ے گائ
کر ا ھ ملا
ی سات ک ہ سی ت ات
کی ی ے پ ودوں
ادوں ب ر لگائ ی
ب ما ب وں
ش دی ی ولی
ے پ
وئ ج خاضل ہ
ت ا ی ر بہی ادہ ب ں زت می
ےض ر رہ ہی ں ب ام می ط ے ت گئ ے
گائ
کر ا ھ ملا
ی سات ک ہ شامت اور وزن ات عداد ج
کی ت ھلوں ی کہ ت ی ج ر رہ اب ر می ی
الت ف ی غ ی ت
ائ
ی وئ ں ہ ہی
ع ب ی دت لی واف ی ب
کوئ ں دار می کی مق کر
ات اور س ں لمخی ی وں می ب ی کہ ب ہ ج
اف ا اض مات
ں ں روزمادوں می
گ اف د کے رت LER مزت
ے LEC (gt1)اور ی ہ کرئ ارہ کی ظرف اس ی ائ کامی کی ام
ط ے ت گئ ے
گائ
کر ا ھ ملا
ی سات ری ات ک ہ
کی ب ڑھوب
ک دا کروت ں ری ہ می ج کھاری پ ن ) Lysemeterو کھاری پ ن روداری کے ت ا کم گی ا ں اگات iwEC = 142می
1-dSm ( کھاری پ ن ادہ ی کہ زت ی ج وئ ری ہ ہی ں ب مک( می
دری ں زی سمی کا
زی dSm= iwEC 129-1 ارت کا دری ارت سمی
ی وئ ر ہ
اب ری ب ری ظرح می
دی ب ڑھوب ولی
ی اور پ
ائ علی
ں ف مک( می
ی کہ ں ک dSm9= iw(Ec 9-1(ج مک ت
دری ں زی سمی کا
ارت
ت کب رداش ات اور س روز مادوں لخمی گ اف الت ف کے رت ی ت
ائ ی ضلاات می درخ ی می
ائ کی می ی
ائ ےجی اب رہ کامی ں ےمی
ر ب ری ظرح کرئ
ں ی وں می ب وا ب ہ ہ
اف ں اض ی ول می ب
ں ف ی وں می
ب ی کہ ب ں ج ی
وب ر ہ اب می
+Na ہ سے کی وج مع ی ج اف رلز کے K+اض روری می
ی سے ض ج
ی وئ ر ہ
اب کی ضلاجی ت می ے
کرئ زب چ
ا ت ق حق الا ت ہ ت درج ے ظر می
وئ ےہ
ھگوئ ں ت ی می
ائ ہلے سے ت کہ ب ی
ے آئ مئ ں ی ہ ت ات سا ی می
ئ کی روش ر ت ہ سے ارہ کی وج ے
ت ف
ھی مدد دے س ں ت ے می گئ ں ا ن می ن زمی مکی دل ں وکہ معی ے ج ا ہ اسکی ا خ ھی لگات
ں ت ن خالات می مکی کو ں وں ج ن
وزہ کے ب ے مج ا ہ کی
داواری ی ر ب ی ے عمل غ گئ ے
گائ
کر ا ھ ملا
ی سات ک ہ ی ر ات ر اور ب ی ضلاجی ت والی ارہ
اف ے اض لئ وروں کے
اپ کی صور ت خ ر ن ارہ زمی
ھی دا ت کروت ے ا ہ وسکی ت ہ اب کا ذرت عہ ت ے ی ب ڑھائ
کی آمدئ وں
کشاپ ی صورت
ارئ ح کی ت ل
ھ ی ت وردئ دار ج ی ر سے مزت ارہ اور ب ی خ
عئصت
صل کے طور ب ی ف ئے ب لئ ے کے
کرئ دا ی ھل ب ن سے ت کارآمد زمی ر ی
ن اور غ مکی
دل ں ے معی
لئ اضد کے ے رمق ا ہ اسکی ا خ کی ی ش ب
1
General Introduction
Intercropping is a major resource conservation technique for sustainable agriculture under
various climatic conditions (Zhang et al 2010 Li et al 2014) It can reduced operational
cost for the production of multiple crops with maintained or even higher level of
productivity (Vandermeer 2010 Perfecto and Vandermeer 2010) It can enhance the
water use efficiency by saving 20 to 40 irrigation water with improved fertilizer
management (Fahong et al 2004 Jat et al 2005 Jani et al 2008) Intercropping system
is more suitable in marginal areas with lower mechanization and cultivation input by
farmers on small tracts of farmlands (Ngwira et al 2012) It can enhance the cumulative
production per unit area and protect the small farmers against market fluctuations or crop
failure ensure the income improve soil fertility and food demands (Rusinamhodzi et al
2012) In this system dominating more compatible and productive species are selected or
replaced in which complementarity effects and beneficial interactions resulting enhanced
yield as compared to monoculture (Huston 1997 Loreau and Hector 2001) It was
estimated that in species diverse systems biomass production is 17 times higher as
compared to monoculture (Cardinale et al 2007)
It is suggested that intercropping is the best suitable cropping system which can
improve the resource-use efficiency by procurement of limiting resources enhanced
phyto-availability and effective plants interactions (Marschner 2012 White and
Greenwood 2013 Ehrmann and Ritz 2014) It is widespread in many areas of world
particularly in latin America it is estimated about 70-90 by small farmers which mainly
grow maiz potatoes beans and other crops under this system whereas intercropping of
maiz with different crops is estimated about 60 (Francis 1986) Additionally
agroforestry is more than 1 billion ha in this area (Zomer et al 2009) The land used for
intercropping system of various crops is greatly varied from 17 in India to 98 in Africa
(Vandermeer 1989 1992 Dupraz and Liagre 2011)
In intercropping system two or more crops or genotypes coexist and growing
together at a same time on a similar habitat (Li et al 2013) It may be divided into various
types such as in mixed intercropping system two or more crops simultaneously growing
without or with limited distinct arrangements whereas in relay intercropping system
second crop is planted when the first is matured while in strip intercropping both the crops
2
are simultaneously growing in strips which can facilitate the cultivation and crop
interactions (Ram et al 2005 Sayre and Hobbs 2004)
Several less-conventional fruit tress including Manilkara zapota (Chicko)
Ziziphus mauritiana (Jujubar) Carissa carndas (Karanda) Annona squamosa (Sugar
apple) and Grewia asiatica (Falsa) has been reported with high nutritional value with
capability to grow at marginal lands (Mass and hoffman 1997) Qureshi and Barrett-
Lennard (1998) suggested few grafted plants that can widely use to improve the quality
and productivity of fruits Grafting is also used to induce stress tolerance in plants against
various abiotic and biotic stresses including salinity stress (Rivero et al 2003) Both root
stocks and shoot stocks contribute to increase the tolerance level of plants Root stocks
represent the first part of defense to control the uptake and translocation of nutrients and
salts throughout the plant (Munns 2002 Santa-cruz et al 2002 Zrig et al 2011) while
shoot stocks develops physiological and biochemical changes to promote plant growth
under stress conditions (Moya et al 2002 Chen et al 2003)
Ziziphus mauritiana Lamk (varn grafted ber) belongs to the family Rhamnaceae
grows widely in most of the dry tropical and subtropical regions around the world Various
grafting methods are used for their propagation including wedge and whip or tongue
methods (Nerd and Mizrahi 1998) Intercropping of these grafted fruit trees with various
leguminous crops is also being successfully practiced in many countries thought the world
Leguminous crops are considered excellent symbiotic nitrogen fixing crops It can
effectively improve soil fertility and offset the critical problems of sub-tropical areas to
fight against desertification and soil degradation These plants are considered as an
excellent source of proteins for humans and animals They can fix the 90 of atmospheric
nitrogen and contribute 40 nitrogen to the soil thus increase the soil fertility (Peoples et
al 1995) However most of the leguminous plants are not salt tolerant while some
species are better drought tolerant and effectively contribute in marginal lands (Zahran
1999)
Among the leguminous plants Pigeon pea (Cajanus cajan (L) Millspaugh) of the
family Fabaceae is widely grown for food fodder and fuel production particularly in
semiarid areas The salinity tolerance of this specie is not well documented both at
germination and seedling stages This crop is still underexploited due to its edible and
3
economic importance While limited investigations has been made to uncover its
nutritional quality medicinal uses and drought tolerance
The identical physiological traits are important in both the mono and intercropping
systems to maximize the resource acquisition The exploitation of best possible
combination of traits of different plants in intercropping system is very important to
maximize the overall performance in intercropping system It depends on the above ground
beneficial plant interactions for light space and optimal temperatures (Wojtkowski 2006
Zhang et al 2010 Shen et al 2013 Ehrmann and Ritz 2014) as well as the
complementary below ground plant interactions with soil biotic factors (Bennett et al
2013 Li et al 2014)
Water is also a major limiting factor intercropping can enhanced the acquisition
of water by root architecture and distribution in the soil profile for effective utilization of
rainfall (Zegada-Lizarazu et al 2006 De Barros et al 2007) and enhanced the water use
efficiency for effective hydraulic redistribution by deep rooted crops and water stored in
the soil profile (Morris and Garrity 1993 Xu et al 2008) Mycorrhizal networks around
the roots of intercrop plants also enhanced the availability of water and available resources
and reduced the surface runoff (Caldwell et al 1998 Van-Duivenbooden et al 2000
Prieto et al 2012)
Intercropping with leguminous plants can enhanced the agricultural productivity in
less productive soils due to enhanced nitrogen availability and also improved the soil
fertility by effective nitrogen fixation (Seran and Brintha 2010 Altieri et al 2012) Due
to weaker soil nitrogen competition intercropping with legumes enhanced the nitrogen
availability to the non-leguminous intercrop which also absorbs the additional nitrogen
released in the soil or root nodules of the leguminous plant (Li et al 2013 White et al
2013a) The use of legumes in many intercropping systems is pivotal According to the
listing of Hauggaard-Nielsen and Jensen (2005) seven out of ten are the legumes among
the most frequently used intercrops around the world
The ecological range of adaptability of legumes reaches from the inner tropics to
arctic regions with individual species expressing tolerance to drought temperature
nutrient deficiency in soil water logging salinity and other environmental conditions
(Craig et al 1990 Hansen 1996) The woody perennial leguminous plants have a number
4
of purposes they can be used to reclaim degraded wastelands retard erosion and provide
shade fuel wood timber and green manure (Giller and Wilson 1991)
Trees with nitrogen fixing capability play an important role to offset the critical
problems of tropical and sub-tropical regions in their fight against desert encroachment
and soil impoverishment These plants are capable to live in N-poor soils through their
association with Rhizobium that fix atmospheric nitrogen Nitrogen fixing activity in the
field depends both on their N2-fixing potential and on their tolerance to existing
environmental stresses (Galiana et al 2002) Symbiotic N2 fixation in leguminous plants
can mainly be considered an excellent source of protein supply for human and animal
consumption They range from extensive pasture legumes to intensive grain legumes and
are estimated to contribution up to 40 of their nitrogen to the soil (Simpson 1987)
The traits in the monocropping system in the selected crop extensively exploit the
acquisition of limiting resources in the environment and continuously focused on the
availably of similar resources for the successful crop production (White et al 2013 ab)
whereas in intercropping with different crops cycling of resources can be optimized to
the complementarity or facilitation traits (Costanzo and Barberi 2014) to overcome
resource limitations during the growing season (Hill 1996 George et al 2014)
For the long term sustainable agriculture and food production in resource limiting
areas with lower input Intercropping systems have the potential to increase the
productivity With efficient mechanization cultural practices and optimized nutrient
management rapid improvements are also possible through this system In future
perspective intercrops with higher resource use efficiency through plant breeding and
genetics is likely to be the most effective option for sustainable agriculture and
development
Increase of world population and demand of additional food production
The demand and production gap of food fodder fuel wood and livestock products is
increasing day by day due to global population which will increase from about 7 billion
(FAO 2014) to 9 billion by 2050 (Haub 2013) The increasing urbanization further
intensifies the problem which will increase from 54 to 66 expected in 2050 (UN
2014) Majority of this rise in urbanization will occur in developing countries around the
5
globe The major problem is to meet the challenge of increasing food demand for this ever
growing population up to 70 more food crops to feed the additional 23 billion population
worldwide by 2050 (FAO 2010 2011) Hence there is great need to increase the re-
vegetation for fuel wood and fodder production (Thomson 1987) An increase in
production could be envisaged through increasing the yield of already productive land or
through more extensive use of unproductive land The high concentration of salts in soil
or water does not let the conventional crops grow and give feasible economic return
Hence it is necessary to search for unconventional crops for foods fodder and fuel which
could give profitable yield under saline conditions (Ahmad and Ismail 1993) Reclamation
of this land through chemical and engineering treatments is very expensive The most
appropriate use of saline wasteland is the production of high yielding salt tolerance fuel
wood timber and forage species (Qureshi et al 1993) Therefore the most attractive
option is to screen a range of species and identify those which have potential of being
commercially valuable for the degraded environments (Ismail et al 1993)
Pakistan is in semi-arid region and the 6th most populated county of the world
Population drastically increased in Pakistan which was 80 million in 1980 and annual
increase in population is about 4 million (UNDES 2011) This is continuously
overburdened and it is estimated that in 2025 it will reach to 250 million and 335 million
in 2050 which decrease the available water per capita to less than 600 m3 resulting 32
shortfall of water requirements causing an alarming condition particularly for Pakistan
Furthermore this shortfall in 2050 leading to severe food shortage upto 70 million tones
which indicates the further development and serious measures for the new resources
(ADB 2002) Subsequent severe food and fodder crises along with all the resource
limitations with continuous increase in urbanization from the current 35 to 52 in 2025
will further intensity the agriculture production and demand
Shortage of good quality irrigation water
On earth surface the major resources of available fresh water is deposited in the form of
ponds lakes rivers ice sheets and caps streams and glaciers whereas underground water
as underground streams and aquifers With the drastic increase in population the water
consumption rise as the twice of the speed of population growth The scarcity of water is
widespread to many countries of different regions Majority of population in developing
countries suffering from seasonal or year round water shortage which will increase with
6
expected climatic changes Currently almost 50 countries around the globe are facing
moderate to severe shortage of water
Due to the greenhouse effect it is estimated that since the start of 20th century 14
degF temperature is already risen which will likely rise at least another 2degF and over the next
100 years it is estimated about more than 11degF due to the consequences of biogenic gases
(El-Sharkawy 2014) This is mainly due to the product of human activities including
industrial malpractices excess fossil fuel consumption deforestation poor land use and
cultural practices
Rising in atmospheric CO2 concentration which probably reached 700 μmol (CO2)
molminus1 resulting severe climatic changes It will accelerate the melting of ice and glacier
resulting the rising rainfall and storms in tropics and high latitude consequently 06 to 1
meter rise in sea level on the expense of costal lowlands across the continents After this
initial high flows the decrease in inflow was very terrifying Due to these climatic changes
humans suffering from socioeconomic changes including degradation of lands with lower
agricultural output and degradation of natural resources will further enhanced the poverty
and hunger resulting dislocation and human migrations (Randalls 2010)
In the mean while scarcity of good quality water is increasing day by day with the
demands of water for domestic agricultural and industrial utilization which will further
increase up to 10 of the total available resources as estimated by 2025 which needs
serious water managements (Bhutta 1999) It is very challenging for the modern
agriculture to ensure the increasing demand of more arable and overburdened population
with the limiting resources including the unavailability of good quality water and
deterioration of even previously productive land (Du et al 2015)
In Pakistan Indus River basin is the back bone of agriculture and socioeconomic
development which contributes 65 of the total river flows and 90 for the food
production with a share of 25 to the GDP It is estimated that about 30-40 of its surface
storage capacity will reduce by 2025 due to siltation of reservoirs and climatic changes It
will impose serious threat to irrigated agriculture in near future consequently with
decreases in groundwater resources resulting shortage of fresh water and 15-20
reduction in grain yield in Pakistan (World Bank 2006)
7
Spread of saline soil and reduction in agricultural yield
Along with scarcity of water soil salinity is one of the major environmental stresses which
severely threaten the agriculture The damages of salinity is widespread around the world
which is so far effected the more than 800 million hectare (more than 6) of land
worldwide including 397 million ha by salinity associated with 434 million ha by sodicity
(FAO 2010) The out of total 230 million hactares of irrigated land more than 45 million
hactares (20) is so far effected by salinity which is about the 15 of total cultivated land
(Munns and Tester 2008)
In Pakistan out of 2036 million hectares of cultivated land more than 6 million
hectares is affected by salinity and water logging of various degrees (Qureshi et al 2004)
About 16 million hectares of tropical arid plains which have been put under crop
cultivation depend extensively on canal irrigation network This area (about 60) is now
seriously affected by water logging and salinity (Qureshi et al 2004) The rise of subsoil
water levels accompanied by its subsequent decline due to irrigation combined with
insufficient drainage has led to salinization of valuable agricultural land in arid zones all
over the world (Ahmad and Abdullah 1982) The dominated cation in salt-affected soil is
Na+ followed by Ca2+ and Mg2+ while the anions Cl and SO4 are almost equal in
occurrence (Qureshi et al 1993) Salt content varies in different regions of the salt-
affected areas but at certain sites could reach up to an ECe of 90-102 dSm-1 (Ahmad and
Ismail 1993)
Salinity is a chief anxiety to meet the ever growing demands of food crops Salinity
adversely affects the plant growth and productivity Plants differentially respond to salt
stress and categories into four classes Salt sensitive moderately salt sensitive moderately
salt tolerant and highly salt tolerant plants on the basis of their tolerance limits Whereas
mainly plants are divided into halophytes (salt tolerant) and glycophytes (salt sensitive) on
the basis of adaptive evolution (Flowers 2004 Munns and Tester 2008) Unfortunately
majority of cultivated crops are not able to withstand in higher salinity regimes and
eventually die under higher saline conditions which proposed serious attentions to manage
the dissemination of salinity (James et al 2011 Rozema and Flowers 2008)
Excessive accumulation of salts in rhizosphere initially reduced the water
absorption capacity of roots leading to hyperosmotic stress followed by specific ion
8
toxicity (Munns 2008 Rahnama et al 2010) Plants initially manage the overloaded salt
by various excluding and avoidance mechanisms depending on their tolerance levels The
management of salt inside the cytosol is depends on the compartmentalization capacity of
plants followed by osmotic adjustments and efficient antioxidant defense mechanisms
Whereas higher salt beyond the tolerance impose injurious effects on various
physiological mechanisms These are including disruption of membrane integrity
increased membrane injuries nutrient ion imbalances osmotic disturbance
overproduction of reactive oxygen species (ROS) compromised photosynthesis and
respiration due to stomatal closure and damages of enzymatic machinery (Munns and
Tester 2008) In specific ion toxicity Na+ and Cl- are the chief contributors in
physiological disorders Excessive Na+ in rhizosphere antagonize the uptake of K+
resulting lower growth and productivity (James et al 2011) Salt load in the cytosol trigger
the overproduction of ROS including H2O2 OH- super oxides and singlet oxygen They
are involved in sever oxidative damages to various vital cellular components including
DNA RNA lipids and proteins (Apel and Hirt 2004 Ahmad and Umar 2011)
Strategies to cope up the salinity problem
The development and cultivation of highly salt tolerant crop varieties for salt affected areas
is the major necessity to meet the future demands of food production whereas the majority
of available food crops are glycophytes Therefore it is an emergent need of crop
improvement methods which are more efficient cost effective and grow on limiting
resource The use of poor quality water for irrigation is also very important under the
proposed shortage of fresh water in near future For the development of salt tolerant
varieties more understanding of stress mechanisms are required at whole plant molecular
and cellular levels
The variability in stress tolerance of salt sensitive genotypes (glycophytes) and
highly salt tolerant plants (halophytes) showed genetic basis of salt tolerance It indicate
that salt tolerance is a multigenic trait which involves variety of gene expressions and
related mechanisms Salt stress induces both the qualitative and quantitative changes in
gene expression (Manchanda and Garg 2008) These multigenetic expressions play a key
role in upregulation of various proteins and metabolites responsible for the management
of anti-stress mechanisms (Bhatnagar-Mathur et al 2008) Plant breeding and transgenic
strategies are intensively used for decades to improve the crop performance under salinity
9
and aridity conditions Few stress tolerant varieties are so far released for commercial
production whereas in natural condition where plant exposed to variety of climatic
conditions the overall performance of plant have changed as compared to controlled in
invitro conditions (Schubert et al 2009 and Dodd and Perez-Alfocea 2012) The success
stories about transgenic approaches for crop improvement under stressful environments
are still very scanty because of the insufficient understanding about the sophisticated
mechanisms of stress tolerance (Joseph and Jini 2010) It indicates that there is less
correlation between the assessment of stress tolerance in invitro and invivo conditions
Although there have been some achievement in this connection in some model plants
including rice tobacco and Arabidopsis (Grover et al 2003) which proposed the
possibilities of success in other crops in future Variety of technicalities and associated
financial challenges are still associated with this strategy
In conventional cultivation practices continuous irrigation with poor quality water
can enhanced the salinization due to evapotranspiration leading to increased saline andor
sodic soils This problem can be cope up by intercropping system in which high salt
tolerant or salt accumulator plants are intercropped with salt sensitive crops which can
accumulate salt thus can reduce the risk of salt increment in soil Additionally better
cultivation practices including the micro-jet or drip irrigation and partial root zone drying
technique is also very fruitful to optimize the water requirements and avoid the risks
associated with conventional flooding irrigation system
In dry land agriculture plantation of deep rooted perennials during off season or
annuals can reduced the risk of salinization They continuously grown and utilize excess
amount of water create a balance between water utilization and rail fall Thus prevent the
chance of salt accumulation on soil surface due to increased water table and
evapotranspiration (Manchanda and Garg 2008) The efficient irrigation and
intercropping strategy is seemed quite attractive cost effective and very beneficial in less
mechanized poor marginal areas It can ameliorate the injurious effects of salinity and
increased production per unit area thus ensure the sustainable agriculture in semi-arid or
marginal lands (Venkateswarlu and Shanker 2009)
A number of plant species are available that are highly compatible with saline
sodic and marginal lands The cultivation of these species with proposed intercropping
system is economically feasible to grow in marginal soil Some plants including Carissa
10
carandus Ziziphus mauritiana and Cajanus cajan was selected to revealed their potential
for intercropping under saline marginal lands These are important plants which can
established well at tropical and subtropical arid zone under high temperatures Hence their
range of salt tolerance and suitability for cultivation at waste saline land or with saline
water irrigation is being undertaken for commercial exploitation
Objective of present investigation
The plan of present investigation has been worked out to look into possibility of increasing
production of an unconventional salt tolerant fruit tree (Z mauritiana) by intercropping
with a legume ( C cajan) which apart from increasing fertility of soil could be able to
provide fodder for grazing animals from salt effected waste land Possibility of making
use of saline water for irrigation has also been considered for growing leguminous plant
(C cajan) and salt tolerant unconventional fruit tree (Crissa carandas) under saline
condition
11
LAYOUT OF THESIS
Chapter 1 Monoculture of Cajanus cajan (Vern Arhar) and Ziziphus mauritiana
(Varn Ber) under different range of salinities created by irrigation of
various sea salt concentrations
A Experiments on Cajanus cajan
Following experiments were performed under A
Experiment No 1 Effect of Pre-soaked seeds of C cajan in distilled water for
germination in water of different sea salt concentrations
Experiment No 2 Effect of Pre-soaked seeds of C cajan in various dilutions of sea salt
for germination in water of respective sea salt concentrations
Experiment No 3 Seedling establishment experiment of C cajan on soil irrigated with
sea salt of different concentrations
Experiment No 4 Growth and development of C cajan in Lysimeter (Drum pot culture)
being irrigated with water of different sea salt concentrations
Experiment No 5 Range of salt tolerance of nitrogen fixing symbiotic bacteria
associated with root of C cajan
B Experiments on Ziziphus mauritiana
Experiment No 6 Growth and development of Z mauritiana in large size clay pot being
irrigated with water of two different sea salt concentrations
Discussion (Chapter 1)
Chapter 2 Intercropping of Ziziphus mauritiana with Cajanus cajan
Experiment No 7 Physiological investigations on Growth of Ziziphus mauritiana and
Cajanus cajan intercropped in drum pot (Lysimeter) culture being
irrigated with water of sea salt concentration at two irrigation
intervals
Experiment No 8 Investigations of intercropping Ziziphus mauritiana with Cajanus
cajan on marginal land under field conditions
12
Discussion (Chapter 2)
Chapter 3 Investigations on rang of salt tolerance in Carissa carandas (varn
karonda) for determining possibility of growing at waste saline land
Experiment No 9 Investigation on the effect of higher range of salinities on growth of
Carissa carandas (varn karonda) created by irrigation of different
dilutions of sea salt
Discussion (Chapter 3)
13
1 Chapter 1
Monoculture of Cajanus cajan (Vern Arhar) and Ziziphus mauritiana
(Varn Ber) under different range of salinity created by irrigation of
various sea salt concentrations
11 Introduction
Scarcity of good quality water enforced the growers to irrigate the crops with
lowmoderately saline water at marginal lands which ultimately enhance soil salinity due
to high evapo-transpiration (Azeem and Ahmad 2011) To overcome this situation people
are now focusing on less-conventional plants which can grow on resource limited areas
and can produce edible biomass for human and animal consumption
Ziziphus mauritiana (varn grafted ber) is salt and drought tolerant plant which can
grow on marginal and degraded land (Morton 1987) It has wide spread crown and a short
bole fast growing tree with average bearing life of 25 years The ripe fruit (drupe) is juicy
hard or soft sweet-tasting pulp has high sugar content vitamins A amp C carotene
phosphorus and calcium (Nyanga et al 2013 2008 Pareek 2013) The leaves contain 6
digestible crude protein and an excellent source of ascorbic acid and carotenoids The
leaves are used as forage for cattlesheepgoats and also palatable for human consumption
(Sharma et al 1982 Bal and Mann 1978 Agrawal et al 2013) The timber is very hard
can be worked to make boats charcoal and poles for house building Roots bark leaves
wood seeds and fruits are reputed to have medicinal properties The tree also used as a
source of tannins dyes silk (via silkworm fodder) shellac and nectar (Dahiru et al 2006
Chrovatia et al 1993 Gupta 1993)
Some atmospherics nitrogen fixing bacterial associated deep rooted drought
tolerent leguminious plants like Cajanus cajan can fix up to 200 Kg nitrogen ha-1 year-1
due to symbiotic association of Rhizobium with its deep penetrating roots (Bhattacharyya
et al 1995) Total cultivated area of Pigeon pea is about 622 million hectare and global
annual crop production is around 474 million tonnes whereas total seed production of
this crop is about 015 million tonnes (FAOSTAT 2013) Its seeds are an excellent source
of good quality protein (up to 24) and foliage is used as animal fodder with high
nutritional value (Pandey et al 2014) Besides being used as food and fodder this plant
14
also have therapeutic value and it is used against diabetes fever dysentery hepatitis and
measles (Grover et al 2002) It also use traditionally as a laxative and was identified as
an anti-malarial remedy beside other medicinal species (Ajaiyeoba et al 2013 Qasim et
al 2010 2011 2014)
Following experiments were conducted to evaluate the seed germination seedling
establishment and growth of C cajan as well as grafted sapling of Z mauritiana under
various salinity regimes Investigations were also undertaken to find-out of their
intercropping has any beneficial effect on growth at marginal saline land saline
environment
15
12 Experiment No 1
Effect of Pre-soaked seeds of Cajanus cajan in distilled water for
germination in water of different sea salt concentrations
121 Materials and methods
1211 Seed collection
Seeds of C cajan were purchased from local seed market Mirpurkhas Sindh and were
tested to determine the effect of salinity on germination at the biosaline laboratory Botany
department Karachi University Karachi The best lot of healthy seeds having 100
germination was selected for further experiments
1212 Experimental Design
Seeds of C cajan were surface sterilized with 01 sodium hypochlorite solution for 2-3
minutes washed in running tap water then soaked in sterilized distilled water for one hour
(Saeed et al 2014) Sterilized glass petri plates (9cm) lined with filter paper were moist
with 10 ml of distilled water at different saline water of different sea salt concentrations
and their germination percentage was observed Their electrical conductivities on these
sea salt dilutions are mentioned in Table 11 Three replicates were used for each treatment
Ten seed were placed in each petri plate which were kept in temperature controlled
incubator (EYELA LTI-1000 Japan) at 28 plusmn 1ordmC in dark Experiment was continued for 7
days Data were recorded on daily bases Analyses of varience by using repeated measures
and the significant differences between treatment means were examined by least
significant difference (Zar 2010) All statistical analysis was performed using SPSS for
windows version 14 and graphs were plotted using Sigma plot 2000
Germination percentage of C cajan was recorded every 24 hours per seedling
evaluation procedure up to 07 days The final percent germination related with salinity in
accordance with Maas and Hoffman (1977) The percent germination was calculated using
the following formula (Cokkizgin and Cokkizgin 2010)
16
Germination index for C cajan was recorded according to AOSA (1990) by using
following formula
Where Gt is the number of germinated seed on day t and Dt is the total number of
days (1 - 7)
Coefficient of germination velocity of C cajan was calculated described by Maguire
(1962)
Where G represents the number of germinated seeds counted per day till the end of
experiment
Mean germination time of C cajan was calculated by Ellis and Roberts (1981) by
using following formula
Where lsquonrsquo is the number of germinated seeds in day d whereas Σn is the total
germinated seeds during experimental period
Germination rate was of C cajan determined according to following formula
(Shipley and Parent 1991)
Where numbers of germinated seeds were recorded from 1 to 7
17
122 Observations and Results
Cajanus cajan (imbibed in distilled water) grown at different salinity regimes showed 50
reduction at 16 salt concentration corresponding ECiw 168 dSm-1 (Table 1 2 Appendix
I)
Rate of germination was inversely correlated with sea salt concentration It was
significantly (p lt 0001) decreased from first day to final (day 7) of observation Higher
germination rate was recorded in control and at lower concentrations of sea salt in early
days of seed incubation with contrast to higher concentrations of sea salt which was
reduced with increasing day of incubation (Table 13 Appendix I)
A significant decrease (p lt 0001) in coefficient of germination velocity was
observed with increasing salinity (Table 14 Appendix I)
A significantly increase (p lt 0001) in mean germination time of seeds was observed
with increasing sea salt concentrations However the difference was insignificant at lower
salinities (Table 14 Appendix I)
A significant decrease (p lt 0001) in mean germination index was observed with
increasing salt concentrations except lower salinities More reduction was observed
byhond 16 and onward sea salt concentration (Table 14 Appendix I)
18
Table 11 Electrical conductivities of different sea salt solutions used in germination of C cajan
Sea salt () ECiw (dSm-1)
Non saline control 06
01 09
02 16
03 35
04 42
05 58
06 62
07 79
08 88
09 99
10 101
11 112
12 128
13 131
14 145
15 159
16 168
ECiw is the electrical conductivity of irrigation water measured in deci semen per meter
19
Table 12 Effect of irrigation water of different sea salt solutions on germination percentage (GP) per day
of C cajan seeds pre-soaked in non-saline water prior to germination with duration of time under
various salinity regimes
Sea Salt
(ECiw= dSm-1)
GP
1st day
GP
2nd day
GP
3rd day
GP
4th day
GP
5th day
GP
6th day
GP
7th day
Control 8333plusmn667 90plusmn00 9333plusmn333 9333plusmn333 9333plusmn333 9333plusmn333 9333plusmn333
09 8667plusmn333 9333plusmn333 9667plusmn333 9667plusmn333 100plusmn00 100plusmn00 100plusmn00
16 7667plusmn667 80plusmn10 8333plusmn882 8333plusmn882 8333plusmn882 8333plusmn882 8667plusmn667
35 6667plusmn333 9333plusmn333 9333plusmn333 9333plusmn333 9333plusmn333 9333plusmn333 9333plusmn333
42 70plusmn00 8667plusmn333 90 plusmn00 90 plusmn00 90 plusmn00 90 plusmn00 90 plusmn00
58 6333plusmn667 7333plusmn333 8333plusmn333 90 plusmn00 90 plusmn00 90 plusmn00 90 plusmn00
62 5667plusmn667 80plusmn577 90 plusmn00 90plusmn00 90 plusmn00 90 plusmn00 90plusmn00
79 5333plusmn333 70plusmn00 8333plusmn333 8333plusmn333 8333plusmn333 8333plusmn333 8333plusmn333
88 4000plusmn00 6667plusmn667 8333plusmn333 8333plusmn333 8333plusmn333 8333plusmn333 8333plusmn333
99 2667plusmn333 60 plusmn00 90 plusmn00 90plusmn00 90 plusmn00 90 plusmn00 90 plusmn00
101 2333plusmn333 70plusmn577 7333plusmn333 7333plusmn333 7333plusmn333 7333plusmn333 7333plusmn333
112 70plusmn577 7667plusmn333 80plusmn00 8333plusmn333 8333plusmn333 8333plusmn333 8333plusmn333
128 6667plusmn333 6667plusmn333 6667plusmn333 6667plusmn333 6667plusmn333 6667plusmn333 6667plusmn333
131 3333plusmn882 50plusmn00 5333plusmn333 5333plusmn333 5333plusmn333 5333plusmn333 5667plusmn333
145 3333plusmn667 40 plusmn00 50 plusmn577 50plusmn577 50 plusmn577 5333plusmn333 5333plusmn333
156 3667plusmn667 40plusmn577 4667plusmn882 4667plusmn882 50plusmn577 50plusmn577 5333plusmn667
168 1667plusmn882 3333plusmn333 3333plusmn333 3333plusmn333 3667plusmn333 3667plusmn333 4333plusmn333
LSD 005 Salinity 18496
Time (days) 13322
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and LSD among the treatments is recorded at p lt 005
20
Table 13 Effect of irrigation water of different sea salt solutions on germination rate (GR) per day
of seeds C cajan pre-soaked in non-saline water prior to germination with duration of
time under various salinity regimes
Sea Salt
(ECiw= dSm-1)
GR
1st day
GR
2nd day
GR
3rd day
GR
4th day
GR
5th day
GR
6th day
GR
7th day
Control 833plusmn067 450plusmn00 311plusmn011 233plusmn008 187plusmn007 156plusmn006 133plusmn005
09 867plusmn033 467plusmn017 322plusmn011 242plusmn008 200plusmn00 167plusmn00 143plusmn00
16 767plusmn067 400plusmn050 278plusmn029 208plusmn022 167plusmn018 139plusmn015 124plusmn010
35 667plusmn033 467plusmn017 311plusmn011 233plusmn008 187plusmn007 156plusmn006 133plusmn005
42 700plusmn00 433plusmn017 300plusmn00 975plusmn750 180plusmn00 150plusmn00 129plusmn00
58 633plusmn067 367plusmn017 278plusmn011 225plusmn00 180plusmn00 150plusmn00 129plusmn00
62 567plusmn067 400plusmn029 300plusmn00 225plusmn00 180plusmn00 150plusmn00 129plusmn00
79 533plusmn033 350plusmn00 278plusmn011 208plusmn008 167plusmn007 139plusmn006 119plusmn005
88 400plusmn00 333plusmn033 278plusmn011 208plusmn008 167plusmn007 139plusmn006 119plusmn005
99 267plusmn033 300plusmn00 300plusmn00 225plusmn00 180plusmn00 150plusmn00 129plusmn00
101 233plusmn033 350plusmn029 244plusmn011 183plusmn008 147plusmn007 122plusmn006 105plusmn005
112 700plusmn058 383plusmn017 267plusmn00 208plusmn008 167plusmn007 139plusmn006 119plusmn005
128 667plusmn033 333plusmn017 222plusmn011 167plusmn008 133plusmn007 111plusmn006 095plusmn005
131 333plusmn088 250plusmn00 178plusmn011 133plusmn008 107plusmn007 089plusmn006 081plusmn005
145 333plusmn067 200plusmn00 167plusmn019 125plusmn014 100plusmn012 089plusmn006 076plusmn005
156 367plusmn067 200plusmn029 156plusmn029 117plusmn022 100plusmn012 083plusmn010 076plusmn010
168 167plusmn088 167plusmn017 111plusmn011 083plusmn008 073plusmn007 061plusmn006 062plusmn005
LSD 005 Salinity 0481
Time (days) 0378
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and LSD among the treatments is recorded at p lt 005
21
Table 14 Effect of irrigation water of different sea salt solutions on mean germination rate (GR)
coefficient of germination velocity (GV) mean germination time (GT) mean
germination index (GI) and final germination (FG) of C cajan seeds pre-soaked in non-
saline water prior to germination under various salinity regimes
Sea Salt
(ECiw= dSm-1) GR GV GT GI FG
Control 2624plusmn100 369plusmn005 027plusmn00 2624plusmn100 9667plusmn333
09 2743plusmn063 365plusmn009 027plusmn001 2743plusmn063 100plusmn00
16 2398plusmn218 423plusmn036 024plusmn002 2398plusmn218 8333plusmn882
35 2467plusmn086 378plusmn005 026plusmn00 2467plusmn086 9333plusmn333
42 3169plusmn733 311plusmn058 035plusmn008 3169plusmn733 9333plusmn333
58 2264plusmn081 399plusmn015 025plusmn001 2264plusmn081 90plusmn00
62 2253plusmn073 400plusmn013 025plusmn001 2253plusmn073 9333plusmn333
79 2074plusmn081 402plusmn00 025plusmn00 2074plusmn081 8333plusmn333
88 1927plusmn043 449plusmn008 022plusmn00 1927plusmn043 90plusmn577
99 1853plusmn033 486plusmn009 021plusmn00 1853plusmn033 90plusmn00
101 1635plusmn056 470plusmn022 021plusmn001 1635plusmn056 8667plusmn882
112 2263plusmn042 369plusmn020 027plusmn001 2263plusmn042 9667plusmn333
128 1953plusmn098 341plusmn00 029plusmn00 1953plusmn098 9667plusmn333
131 1368plusmn059 440plusmn018 023plusmn001 1368plusmn059 6667plusmn333
145 1276plusmn099 446plusmn019 023plusmn001 1276plusmn099 60plusmn577
156 1289plusmn153 447plusmn030 023plusmn002 1289plusmn153 8000plusmn100
168 876plusmn104 589plusmn078 018plusmn002 876plusmn104 8667plusmn333
LSD005 5344 3312 0064 5344 1313
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and LSD among the treatments is recorded at p lt 005
22
13 Experiment No 2
Effect of Pre-soaked seeds of Cajanus cajan in various dilutions of sea
salt for germination in water of respective sea salt concentrations
131 Materials and methods
1311 Seed germination
Procedure of seed germination has been mentioned in Experiment No 1 earlier The seeds
were pre-soaked in various sea salt concentrations instead of non-saline water and
germinated in respective sea salt concentrations Their electrical conductivities mentioned
in Table 15 Data were calculated and analysed according to formulas given in Experiment
No 1
Since these pre-soaked seeds in different sea salt concentration showed 50
germination at 03 equivalent to ECiw= 42dSm-1 sea salt solution any further work
beyond ECiw= 42dSm-1was not continued
132 Observations and Results
The final percent germination related with salinity in accordance with Maas and
Hoffman (1977) linear relative threshold response model as follows
Relative Final Germination = 100-200 (Ke ndash 005)
Where threshold salt concentration was 005 and Ke is the concentration of salts
at which relative final germination may be predicted This model indicated 50
declined in final germination at 030 salt concentration corresponding to ECiw= 42
dSm-1 (Table 16 Appendix II)
Rate of germination was significantly decreased (p lt 0001) from first day to final
(day 07) of observation and it was inversely correlated with sea salt concentration High
germination rate was recorded in control and low sea salt concentrations in early days of
seed incubation compared to higher sea salt concentrations but the difference in rate was
reduced (Table 17 Appendix II)
23
A progressive decline (p lt 0001) in coefficient of germination velocity was
observed with increasing salinity and fifty percent reduction was observed at 021 sea
salt concentration (ECiw = 319 dSm-1 Figure 11 Appendix II)
Final germination percentage was decreased significantly with increasing sea salt
concentrations However the difference was insignificant at lower (ECiw = 16 dSm-1)
salinity (Figure 11 Appendix II)
Mean germination time of seeds was increased significantly (p lt 0001) with
increasing sea salt concentrations However the difference was insignificant at lowest
(ECiw = 09 dSm-1) salinity (Figure 11 Appendix II)
Mean germination index was also significantly decreased (plt0001) with
increasing salt concentrations except for ECiw = 09 dSm-1 salinity Fifty percent reduction
in mean germination index was observed at 0188 sea salt concentration (ECiw = 289
dSm-1 Figure 11 Appendix II)
24
Table 15 Electrical conductivities of different sea salt solutions used in germination of C cajan
Sea salt () ECiw (dSm-1)
0 04
005 09
01 16
015 24
02 32
025 39
03 42
ECiw is the electrical conductivity of irrigation water measured in deci semen per meter
25
Table 16 Effect of irrigation water of different sea salt solutions on germination percentage (GP) per day of C cajan seeds pre-soaked in respective sea salt concentrations
with duration of time
Sea salt
ECiw (dSm-1)
GP
1st day
GP
2nd day
GP
3rd day
GP
4th day
GP
5th day
GP
6th day
GP
7th day
Control 6667plusmn333 8667plusmn333 10000plusmn000 10000plusmn000 10000plusmn000 10000plusmn000 10000plusmn000
09 7000plusmn000 7667plusmn333 9000plusmn000 10000plusmn000 10000plusmn000 10000plusmn000 10000plusmn000
16 4667plusmn333 6000plusmn000 7333plusmn333 8000plusmn000 8667plusmn333 8667plusmn333 9000plusmn577
24 4333plusmn333 5000plusmn000 6000plusmn577 6667plusmn333 7333plusmn333 7333plusmn333 8000plusmn000
32 3000plusmn000 3333plusmn333 3667plusmn333 4333plusmn333 5000plusmn577 6000plusmn577 7000plusmn577
39 1667plusmn333 2333plusmn333 2333plusmn333 4000plusmn577 4333plusmn333 5000plusmn000 6000plusmn000
42 667plusmn333 1333plusmn333 2333plusmn333 2333plusmn333 3333plusmn333 3667plusmn333 5000plusmn000
LSD 005 Salinity 327 Time 327
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and LSD among the treatments was recorded at p lt 005
25
26
Table 17 Effect of irrigation water of different sea salt solutions on germination rate (GR) per day of Ccajan
seeds pre-soaked in respective sea salt concentrations with duration of time
Sea salt
(ECiw= dSm-1)
GR
1st day
GR
2nd day
GR
3rd day
GR
4th day
GR
5th day
GR
6th day
GR
7th day
Control 667plusmn033 433plusmn017 333plusmn000 250plusmn000 200plusmn000 167plusmn000 143plusmn000
09 700plusmn000 383plusmn017 300plusmn000 250plusmn000 200plusmn000 167plusmn000 143plusmn000
16 467plusmn033 300plusmn000 244plusmn011 200plusmn000 173plusmn007 144plusmn006 129plusmn008
24 433plusmn033 250plusmn000 200plusmn019 167plusmn008 147plusmn007 122plusmn006 114plusmn000
32 300plusmn000 167plusmn017 122plusmn011 108plusmn008 100plusmn012 100plusmn010 100plusmn008
39 167plusmn033 117plusmn017 078plusmn011 100plusmn014 087plusmn007 083plusmn000 086plusmn000
42 067plusmn033 067plusmn017 078plusmn011 058plusmn008 067plusmn007 061plusmn006 071plusmn000
LSD 005 Salinity 014
Time 014 Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and LSD among the treatments is recorded at p lt 005)
27
Sea salt (ECiw = dSm-1
)
Contr
ol
09
16
24
32
39
42
Germ
ination Index(s
eedd
ays
-1)
0
2
4
6
8
Fin
al germ
ination (
)
0
20
40
60
80
100
Coeff
icie
nt of
germ
ination v
elo
city
(seedd
ays
-1)
00
01
02
03
04
05
06
07
Sea salt (ECiw = dSm-1
)
Contr
ol
09
16
24
32
39
42G
erm
ination tim
e (
Days
)
0
1
2
3
4
LSD005 = 0086
a = 0664 b = 1572
R2 = 0905 n =21
LSD005 = 062
a = 1239
b = 9836
R2 = 0894 n=21
LSD005 = 053
a = 8560b = -2272
R2 = 0969 n=21
RGF = 100-200 (Ke -005) Ke = 030
Figure 11 Effect of irrigation water of different sea salt solutions on seed germination indices of C cajan
(Bars represent means plusmn standard error of each treatment and significance among the treatments
was recorded at p lt 005)
28
14 Experiment No 3
Seedling establishment experiment of Cajanus cajan on soil irrigated with
sea salt of different concentrations
141 Materials and methods
1411 Seedling establishment
Seedling establishment experiment was carried out in Biosaline research field Department
of Botany University of Karachi Surface sterilized seeds pre-soaked were sown in small
plastic pots filled with 15 Kg sandy loam soil provided with farm manure at 91 ratio (30
water holding capacity) Sea salt solutions of different concentrations mentioned above
were used for irrigation The electrical conductivity of soil saturated paste (ECe) was also
determined at the end of the experiment (Table 18) Data on seedlings emergence was
recorded and their height were measured after 14 days of salinity treatment EC of the soil
(ECe) was initially 054 dSm-1 Statistical analyses were done according to the procedures
given in Experiment No 1
Since germination percentage of seeds pre-soaked in non-saline water was found
better under different concentrations of sea salt the seeds sown in soil for taking for
seedling establishment were pre-soaked in distilled water
29
142 Observations and Results
1421 Seedling establishment
Seedling emergence from soil was reduced significantly (p lt 0001) with increasing salt
concentration of irrigation water Not a single seedling emerged from soil in ge ECiw= 39
dSm-1 saline water irrigation However lower salinities (ECiw= 09 16 dSm-1) showed
slight decrease in seedling emergence with respect to controls Seedling emergence related
with salinity in accordance with a quadratic model as follows
Equation for seedling emergence () = 977751+ 44344 salt ndash 22215238 (salt)2 plusmn
6578 r = 09810 F = 15358 (p lt 00001)
Fifty percent reduction in seedling emergence was noticed at 016 sea salt
concentration (ECiw = 241 dSm-1 Figure 12 Appendix III)
1422 Shoot height
Shoot height was measured after fourteen days of irrigation Shoot length was
significantly decreased (p lt 0001) with increasing salinity A lower decrease was
observed in low sea salt salinity (ECiw= 09 and 16 dSm-1) compared to controls while
higher decrease in shoot height was noticed from ECiw= 2 dSm-1sea salt concentration
Shoot height related with salinity as follows
Equation for shoot height (cm) = 9116714 ndash 3420286 salt plusmn 09221 r = 0968 F =
128893 (p lt 0001)
Fifty percent reduction in shoot height was estimated at 013 sea salt concentration
(ECiw = 210 dSm-1) (Figure 12 Appendix III)
30
Table 18 Electrical conductivities of different Sea salt concentrations and ECe of soil saturated paste at the
end of experiment (ECe = 0447 + 1204 (salt ) plusmn 02797 R = 0987 F = 72301 (p lt
000001)
Sea salt () ECiw (dSm-1) ECe (dSm-1)
0 04 05
005 09 161
01 16 278
015 24 354
02 32 433
025 39 483
03 42 552
Electrical conductivity of soil saturated paste determined after 14 days of saline water irrigation in pots
Figure 12 Effect of irrigating water of different sea salt solutions on seedling emergence (A) and shoot
length (B) of C cajan (Bars represent means plusmn standard error of each treatment where similar
letters are not significantly different at p lt 005)
e f
Sea salt (ECiw = dSm-1
)
Contr
ol
16
27
8
35
4
43
3
48
3
Shoot le
ngth
(cm
)
0
2
4
6
8
10ab
c
de
Contr
ol
16
27
8
35
4
43
3
48
3Seedlin
g e
merg
ence (
)
0
20
40
60
80
100a
bb
c
d
A B
31
15 Experiment No 4
Growth and development of Cajanus cajan in Lysimeter (Drum pot
culture) being irrigated with water of different sea salt concentrations
151 Materials and methods
1511 Drum pot culture
A modified drum pot culture (lysimeter) installed by Ahmad amp Abdullah (1982) at
Biosaline research field (Department of Botany University of Karachi) was used in
present experiment Each drum pot (60 cm diameter 90 cm depth) was filled with 200 kg
of sandy loam mixed with cow-dung manure (91) having 28 water holding capacity
They are fixed at cemented platform at slanting position with basal hole to ensure rapid
drain Over irrigation was practiced to avoid the accumulation of salt in the root zone
1511 Experimental design
Growth and development of C cajan in drum pots was carried out in six different drum
pot sets (each in triplicate) and irrigated with sea salt of following concentrations
Drum pot Sets Sea salt
()
ECiw ( dSm-1) of
irrigation water
Resultant ECe (dSm-1) after
end of experiment
Set I Non saline (C) 04 05
Set II 005 sea salt 09 16
Set III 001 sea salt 16 28
Set IV 015 sea salt 24 35
Set V 02 sea salt 28 38
Set VI 025 sea salt 34 43
Note ECiw is the electrical conductivity of irrigation water and ECe is the electrical conductivity of the saturated soil extract taken after
eighteen weeks at the end of experiment
Ten surface sterilized seeds with 01 sodium hypochlorite were sowed in each
drum pot and were thinned to three healthy and equal size seedlings after two weeks of
establishment in their respective sea salt concentration Each drum pot was irrigated with
15 liters non-saline or respective sea salt solution at weekly intervals Electrical
conductivity of soil was measured by EC meter (Jenway 4510) using saturated soil paste
32
at the end of experiment Experiment was conducted for a period of 18 weeks (July to
November 2009) during which environmental data which includes average humidity
(midnight 76 and noon 54) temperature (low 23oC and high 33oC) wind velocity (14
kmph) and rainfall (~4 cm) was recorded (Pakistan Metrological Department Karachi) is
given in Figure 13Statistics were analysed according to the procedures given in
Experiment No 1
1512 Vegetative and Reproductive growth
Shoot height was measured at every two week interval after seedling establishment Fresh
and dry weight of shoot was recorded at final harvest (18th week when pods were fully
matured) Leaf succulence (dry weight basis Abideen et al 2014) Specific shoot length
(SSL Panuccio et al 2014) and relative growth rate (RGR Moinuddin et al 2014) were
measured using following equations
Succulence (g H2O gminus1 DW) = (FW minus DW) DW
SSL = shoot length shoot dry weight
RGR (g gminus1 dayminus1) = (lnW2 - lnW1) (t2 - t1)
Whereas FW fresh weight DW dry weight W1 and W2 initial and final dry weights and
t1 and t2 initial and final time of harvest in days
Reproductive data in terms of number of flowers number of pods number of seeds
and seed weight per plants was recorded during reproductive period
1513 Analysis on some biochemical parameters
Biochemical analysis of leaves was carried out at grand period of growth Following
investigations was undertaken at different biochemical parameters
i Photosynthetic pigments
Fresh and fully expended leaves (at 2nd3rd nodal part) samples (01g) were crushed in 80
chilled acetone and were centrifuged at 3000rpm for 10 minutes Supernatant were
separated and adjusted to 5ml final volume The absorbance was recorded at 663nm and
645 nm on spectrophotometer (Janway 6305 UVVis) for chlorophyll content while 480
33
and 510 nm for carotenoids Chlorophyll ab ratio was calculated after the amount
estimated The chlorophyll and carotenoid contents were determined according to Strain
et al (1971) and Duxbury and Yentsch (1956) respectively
Chlorophyll a (microgml) = 1163 (A665) ndash 239 (A649)
Chlorophyll b (microgml) = 2011 (A649) ndash 518 (A665)
Total Chlorophylls (microgml) = 645 (A665) + 1772 (A649)
Carotenoids (microgml) = 76 (A480) ndash 263 (A510)
ii Total soluble sugars
Dry leaf samples (01g) were homogenized in 5mL of 80 ethanol and were centrifuged
at 4000 g for 10 minutes 10 mL diluted supernatant in 5mL Anthronrsquos reagent was kept
to boil in 100oC water bath for 30 minutes and were cooled in running tap water Optical
density was taken at 620nm for the determination of soluble carbohydrates according to
Fales (1951)Total soluble carbohydrates was estimated against glucose as standard and
was calculated from the equation mentioned and expressed in mgg-1 dry weight
Total carbohydrates (microgmL-1) = 228462 OD 097275 plusmn004455
iii Protein content
Fresh and fully expended leaves at 2nd3rd nodal part were taken for protein estimation
The protein contents were measured according to Bradford Assay reagent method against
Bovine Serum Albumin as standards (Bradford 1976) Dye stock was made to dissolved
50mg comassie blue in 25 ml methanol The solution is added to 50ml of 85 phosphoric
acid and diluted to 100 ml with distilled water 02g fresh leaf samples were mills in 5 ml
phosphate buffer pH7 5ml of assay reagent (diluting 1 volume of dye stock with 4 volume
distilled water) were added in 01 ml leaf extract used for enzyme assay Absorbance was
recorded at 590nm and was expressed in mgg-1 fresh weight Proteins were calculated
from the following best fit standard curve equation
Protein (microgml-1) = -329196 + 1142755 plusmn 53436
34
152 Observations and Results
1521 Vegetative and Reproductive growth
Effect of sea salt on vegetative growth including height fresh and dry weight of Cajanus
cajan is presented in (Figure 14 and 15 Appendix-VI) Comparative analysis showed
that plant growth (all three parameters) was significantly increased with time (plt 0001)
however it was linearly decreased (plt 0001) with increasing salinity (Figure 16
Appendix-VI) shows the water content succulence relative growth rate (RGR) and
specific shoot length (SSL) of Cajanus cajan Under saline conditions all parameters were
significantly reduced in comparison to control however SSL showed decline after ECe38
dSm-1 Salt induced growth reduction was more pronounced at ECe 38 and 43 dSm-1 in
which plants died before reaching the reproductive maturity after 12 and 14 weeks at sea
salt treatments respectively Therefore further analysis was carried out in plant grown up
to ECe= 35 dSm-1 sea salt concentrations
Salinity significantly reduced (plt 0001) reproductive parameters including
number of flowers pods seeds and seed weight (Figure 17 Appendix-VII) Among all
treatments highest reduction was observed in 315 dSm-1 in which number of flowers and
pods reduced up to 7187 and 70 respectively Similar trend was observed in total
number and weight of seeds which showed 80 and 8793 reduction respectively
1522 Study on some biochemical parameters
i Photosynthetic pigments
Figure 18 Appendix-VII shows the effect of salinity on pigments (chlorophyll a b ab
ratio and carotenoids) of C cajan leaves A slight increase in total chlorophyll contents
(1828) and chlorophyll ab ratio (1215) was observed at low salinity (ECe= 16 dSm-
1) however they were significantly reduced (4125 and 3630 respectively) in high salt
treatment (plt 0001) Chlorophyll a was higher than chlorophyll b in all treatments
however chlorophyll b was un-affected by salinity whereas total chlorophyll content and
ab ratio was disturbed due to change in chlorophyll a This reduction was more
pronounced at high salinity (ECe= 35 dSm-1) in which chlorophyll a total chlorophylls
and ab ratio was decreased by 505 412 and 3630 respectively Carotenoid content
was maintained at ECe= 16 dSm-1 and decreased with further increase in salinity
35
ii Total soluble sugars
Total soluble sugars in leaves of C cajan is presented in Figure 19 Appendix-VII Total
leaf sugars in C cajan were remained un-affected at 16 dSm-1 and subsequently decreased
with further increase in medium salinity Although total sugars were decreased at ECe 28
and 35 dSm-1 a significant increase (~25) of soluble sugars was observed at higher
salinities However this increment was accounted for decrease (504 ) in insoluble sugar
content at that salinity levels
iii Protein
Total protein in leaves of C cajan is presented in Figure 19 Appendix-VII An increase
in leaf protein content in C cajan was found at lower salinity regime (ECe= 16 dSm-1)
which was followed by significant reduction with further increase in salinity This decline
was 2040 at 28 which was more pronounced (5646 ) at high salinity level (ECe=
35dSm-1)
36
Months (2009)
Jun Jul Aug Sep Oct Nov Dec
Valu
es
0
10
20
30
40
50
60
70
80
90
Rainfall (cm)Low Temp (
oC)
High Temp (oC)
Humidity at noon () Wind (kmph)
Humidity at midnight ()
Figure 13 Environmental data of study area during experimental period (July-November 2009)
Time (Weeks)
2 4 6 8 10 12 14 16 18
Pla
nt heig
ht (c
m)
0
30
60
90
120
150
180
210
43 38 35 28 16 Control
Figure 14 Effect of salinity using irrigation water of different sea salt concentrations on height of C cajan
during 18 weeks treatment (Lines represent means plusmn standard error of each treatment represents
significant differences at p lt 005)
37
Sea salt (ECe= dSm
-1)
Cont 16 28 35 38 43
Sea salt (ECe= dSm
-1)
Cont 16 28 35 38 43
Fre
sh w
eig
ht (g
)
0
5
10
15
20
25
30
35Initial Final
a
b b
c c cab b
c c cC 16 28 35 38 43
Fre
sh w
eig
ht
(g)
012345 a
bb
bc ca a ab b c c
Dry weightMoisture
Figure 15 Effect of salinity using irrigation water of different sea salt concentrations on initial and final
biomass (fresh and dry) of C cajan (Bars represent means plusmn standard error of each treatment Different
letters represent significant differences at p lt 005)
Mo
istu
re (
)
0
20
40
60
80
100
Succu
lance
(
)
0
20
40
60
80
100
Sea salt (ECe= dSm
-1)
Co
nt
16
28
35
38
43
RG
R (
)
0
20
40
60
80
100
Co
nt
16
28
35
38
43
SS
L (
)
0
20
40
60
80
100
Sea salt (ECe= dSm
-1)
ab
b b
c c
a
b bc c c
a
b b
c c c
a a a ab
c
Figure 16 Percent change (to control) in moisture succulence relative growth rate (RGR) and specific
shoot length (SSL) of C cajan under increasing salinity using irrigating water of different sea
salt concentrations (Bars represent means plusmn standard error of each treatment Different letters
represent significant differences at p lt 005)
38
Sea salt (ECe= dSm-1)
Control 16 28 35
Tota
l seeds (
Pla
nt-1
)
0
20
40
60
80
100
120
140 Seed w
eig
ht (g
pla
nt -1
)
0
5
10
15
20
25
Num
ber
10
20
30
40
50
60
70 a
b
cc
a
a
b
b
b c
c
a
b
a
c c
Flowers
Pods
Seed weightTotal seeds
Figure 17 Effect of irrigating water of different sea salt solutions on reproductive growth parameters
including number of flowers pod seeds and seed weight of C cajan (Values represent means
plusmn standard error of each treatment Different letters represent significant differences at p lt
005)
39
Sea salt (ECe=dSm-1
)
Control 16 28 35
Caro
tinoid
s (
mg g
-1 F
W)
000
005
010
015
020
025
030
Chlo
rophyll
(mg g
-1 F
W)
00
02
04
06
08
ab
ratio
00
05
10
15
20
25
ab
ab
b
a
cd
b
a
c
d
a
b
c
d
a
a
ab
b
Figure 18 Effect of irrigating water of different sea salt solutions on leaf pigments including chlorophyll a
chlorophyll b total chlorophyll and carotenoids of C cajan (Bars represent means plusmn standard
error of each treatment Different letters represent significant differences at p lt 005)
40
Figure 19 Effect of irrigating water of different sea salt solutions on total proteins soluble insoluble and
total sugars in leaves of C cajan (Bars represent means plusmn standard error of each treatment
Different letters represent significant differences at p lt 005)
Sea salt (ECe= dSm
-1)
C 16 28 35
Pro
tein
(m
g g
-1 F
W)
00
01
02
03
04
05
06
Su
gar
s (m
g g
-1 F
W)
00
02
04
06
08
a ab b
a a
b b
a ab b
a
b
ab
c
SoluableInsoluable
41
16 Experiment No 5
Range of salt tolerance of nitrogen fixing symbiotic bacteria associated
with root of Cajanus cajan
161 Materials and methods
1611 Isolation Identification and purification of bacteria
Nodules of C cajan grow in large clay pots and irrigated with running tap water at
biosaline agriculture research field were collected from the lateral roots (about 15 cm soil
depth) Nodules were surface sterilized with sodium hypochloride (2) for 5 min and
vigorously washed with sterilized distilled water Each nodule was crushed with sterilized
rod in 5 ml distilled water The bacterial suspension was streaked on yeast extract mannitol
agar (YEM) (K2HPO4 05 g MgSO 4 025g Na Cl 01 g Manitol 10g Yeast Extract 1g
Agar 20 g in 1000 ml of Distilled water) with the help of sterilized wire lope Colonies
were identified by studying different phenotypic characters as Rhizobium fredii
(Cappuccino and Sherman 1992 Sawada et al 2003) Pure culture of Rhizobium species
was stored at -20oC temperature
1612 Preparation of bacterial cell suspension
Bacteria were multiplied by growing in YEM broth for 48 hrs on shaking incubator (140
rpm) at 37oC in dark The culture in broth was centrifuged at 4000 rpm for 10 min to
obtained bacterial cell pellet Pellet was washed and centrifuged twice with sterilized
distilled water Pellet then re-suspended in sterilized distilled water before use
1613 Study of salt tolerance of Rhizobium isolated from root nodules of
C cajan
Assessment for salinity tolerance of Rhizobium species was assessed on YEM agar
Salinity levels of 0 05 10 15 20 25 and 30 having electrical conductivity 06 90
188 242 306 366 and 423 dSm-1 respectively were maintained with NaCl Bacterial
cell suspension of 01 ml (5times 103 colony forming unitsml) was poured in each sterilized
Petri dish 10 ml of molten YEM agar was poured immediately and shake well before
solidification of agar Petri plates were incubated at 37deg C in dark Colonies were observed
and counted in colony counter after 48 h and photographed (Dubey et al 2012 Singh and
42
Lal 2015) There were three replicates of each treatment and data were transformed to
log10 before analysis
162 Observations and Results
Colonies of Rhizobium on YEM agar at different salinity levels is presented in Figure 110
and 111 Appendix-VIII A significant decrease (plt0001) in rhizobial colonies was
observed with increasing salinity However the difference between non saline control and
90 dSm-1 and as that of 242 dSm-1 and 302 dSm-1 salt (NaCl) concentration showed
nonsignificant difference in rizobial colonies Whereas drastic decreased was observed on
further salinity levels Rhizobial colonies were not found at 423 dSm-1salt concentration
NaCl (ECw= dSm
-1)
06 9 188 242 306 366 423
Rh
izo
bia
l co
lonie
s (l
og
10)
0
1
2
3
4 a a
b
c c
d
e
Figure 110 Growth of nitrogen fixing bacteria associated with root of C cajan under different NaCl
concentrations (Bars represent means plusmn standard error of each treatment among the treatments
is recorded at p lt 005)
43
Figure 111 Photographs showing growth of Rhizobium isolated from the nodules of C cajan invitro on
YEM agar supplemented with different concentrations of NaCl (ECw)
188
423 90
Control
366
306 242
44
17 Experiment No 6
Growth and development of Ziziphus mauritiana in large size clay pot
being irrigated with water of two different sea salt concentrations
171 Materials and methods
1711 Experimental design
The grafted plants obtained from the local nursery of Mirpurkhas Sindh were transported
to the Biosaline Agriculture Research field Department of Botany University of Karachi
and were transplanted carefully in large earthen pots containing 20 Kg sandy loam soil
mixed with cow dung manure at 91 ratio having about 5 liters of water holding capacity
with a basal hole for drainage of excess salts to avoid accumulation in the rhizosphere
Over irrigation with about 15 liters of non-saline saline water was kept weekly in summer
and biweekly in winter to avoid accumulation of salts in rhizosphere Plants were irrigated
to start with non-saline tap water for about two weeks for establishment All the older
leaves were fallen and new leaves were developed during establishment period Following
irrigation schedule of non-saline (control) and saline water was selected in view of Z
mauritiana being moderately salt tolerant plant which includes both low and as well as
higher concentrations of the salt in irrigation
Sea salt () ECiw (dSm-1)
of irrigation water
Average resultant ECe (dSm-1) of soil
with some fluctuation often over
irrigation
Non saline (Control) 06 12
04 63 72
06 101 111
ECiw = Electrical conductivity of irrigation water ECe = Electrical conductivity of saturated soil
Healthy and well established plants were selected of nearly equal height and
divided into three sets each contain three replicates (total nine pots) Salinity was provided
through irrigation water of different sea salt concentrations All pots except non-saline
control were initially irrigated with 01 sea salt solution and then sea salt concentration
45
in irrigation medium was increased gradually upto the required salinity level The salinity
level of soil was monitored by taken the electrical conductivity of saturated soil paste the
end of experiment The electrical conductivity of soil (ECe) maintained at the level of 12
72 and 111 dSm-1 respectively as described by Mass and Hoffman (1977)
1712 Vegetative and reproductive growth
Vegetative growth in terms of shoot height fresh and dry weight of shoot and number of
branches were noted at destructive harvesting at initial (establishment) 60 and 120 days
of growth For dry weight shoots were dried in oven at 70˚C for three days Shoot
succulence specific shoot length (SSL) moisture percentage and relative growth rate
(RGR) was calculated at final harvest by using formulas given in Experiment No 4
Whereas number of flowers in reproductive data were recorded at onset of reproductive
period
As regard of fruit formation the duration of experiment was not sufficient for fruit
setting and furthermore the amount of sol in pots was not sufficient for healthy growth of
this plant Secondly flowering and fruiting is reported to be poor at the time of 1st initiation
of reproductive period (Azam-Ali 2006) Furthermore statistical significance of flower
and fruit count also become far less due to their excess dropping at early stage Hence it
was decided to proceed with study of fruit formation in forthcoming field trials of their
intercropping culture
1713 Analysis on some biochemical parameters
Biochemical analyses were performed at the grand period (at the time of flower initiation)
in fully expended fresh leaves Chlorophyll contents soluble sugar contents and soluble
proteins were analyzed Leaves samples taken from 3rd 4th node below the apex according
to the procedures given in Experiment No 4
46
172 Observations and Results
1721 Vegetative and Reproductive growth
Effect of sea salt on vegetative growth of Z mauritiana including height fresh and dry
weight is presented in (Figure 112 Appendix-IX) Comparative analysis showed that
plant growth (all three parameters) was significantly increased with time (plt 0001)
however number of branches was decreased (plt 0001) with increasing salinity
Figure 113 shows the moisture content succulence relative growth rate (RGR)
and specific shoot length (SSL) of Z mauritiana A non-significant difference in shoot
succulence SSL and moisture content was observed with time salinity and interaction of
both factors However RGR showed decline Salt induced growth reduction was more
pronounced at higher salinities
In Z mauritiana plants number of flowers showed significant decrease (plt0001)
with increasing salinity treatment Flower initiation seems non-significant at early growth
(60 days) period in controls and salinity treatments However drastic decrease was
observed with increasing salinity in 120 days of observation (Figure 114 Appendix-IX)
1722 Study on some biochemical parameters
i Photosynthetic pigments
The effect of Z mauritiana leaves pigments (chlorophyll a b ab ratio) on salinity shower
a slight difference in chlorophyll lsquoarsquo over control However chlorophyll lsquobrsquo contents
showed increase over control in both salinity treatments due to which the total chlorophylls
were also enhanced compared to controls Chlorophyll ab ratio was significantly
(plt0001) decreased in both salinities as compared to control (Figure 115 Appendix-IX)
ii Sugars and protein
In Z mauritiana plant soluble sugars were significantly decreased (plt0001) over controls
whereas proteins showed little decrease under salinity treatments compared to controls
(Figure 116 Appendix-IX)
47
Control 72 111
Fre
sh w
eig
ht (g
)
0
150
300
450
600
750
900
Sea salt (ECe= dSm
-1)
Control 72 111
Dry
weig
ht (g
)
0
150
300
450
600
750
900
Num
ber
of bra
nches
3
6
9
12
15
18
Heig
ht (c
m)
20
40
60
80
100
120
140
160
Initial 60 days 120 days
AcBb
Ba
AcBb Ba
AcBb Ba
Ac
BbBa
Figure 112 Effect of salinity using irrigation water of different sea salt concentrations on height number of
branches fresh weight and dry weight of shoot of Zmauritiana after 60 and 120 days of
treatment (Bars represent means plusmn standard error of each treatment Different letters represent
significant differences at p lt 005)
48
120 days 60 days InitialS
uccula
nce (
g g
-1 D
W)
00
03
06
09
12
Sea salt (ECe= dSm
-1)
SS
L (
cm
g-1
)
00
01
02
03
04
05
Control 72 111
Mois
ture
(
)
0
10
20
30
40
50
60
Control 72 111
RG
R (
mg g
-1 d
ay
-1)
0
5
10
15
20
a a aa a a a a a a
a aa a a a a a
a a aa a a a a a a a
b
b b
c
Figure 113 Effect of salinity using irrigation water of different sea salt concentrations on succulence
specific shoot length (SSL) moisture and relative growth rate (RGR) of Z maritiana (Bars
represent means plusmn standard error of each treatment Different letters represent significant
differences at p lt 005)
49
Sea salt (ECe= dSm
-1)
Control 72 111
Num
ber
of flow
ers
0
20
40
60
80
100
120
140 60 days120 days
Ac
BbBa
Figure 114 Effect of salinity using irrigation water of different sea salt concentrations on number of flowers
of Z mauritiana (Bars represent means plusmn standard error of each treatment Different letters
represent significant differences at p lt 005)
Sea salt (ECe= dSm
-1)
Control 72 111
Ch
loro
ph
yll
(mg g
-1)
00
03
06
09
12
15
18
bba
bba
bb
a
chl b chl a ab
ab
ra
tio
00
05
10
15
20
Figure 115 Effect of salinity using irrigation water of different sea salt concentrations on leaf pigments
including chlorophyll a chlorophyll b total chlorophyll and chlorophyll ab ratio of Z mauritiana (Values
represent means plusmn standard error of each treatment Different letters represent significant differences at p lt
005)
50
Figure 116 Effect of salinity using irrigation water of different sea salt concentrations on total sugars and
protein in leaves of Z mauritiana (Bars represent means plusmn standard error of each treatment
Different letters represent significant differences at p lt 005)
Sea salt (ECe= dSm
-1)
C 04 06
Pro
tein
s (m
g g
-1)
0
10
20
30
40
50
60
70
80
Solu
ble
sugar
s (m
g g
-1)
0
3
6
9
12
15
18a
a
bb
b b
Control 72 111
51
18 Discussion
Seed germination is the protrusion of radicle from the seed which is adversely affected by
salinity stress (Kaymakanova 2009) Salinity imposes the osmotic stress by accumulation
of Na+ and Cl- which decrease soil water potential that ultimately inhibits the imbibition
process (Othman 2005) Effect of seed germination against salinity is reported in linear
threshold response model of Maas and Hoffman (1977) The germination of a salt tolerant
desert legume Indigofera oblongifolia and a desert graminoid Pennisetum divisum are
also reported to behave to salinity in similar manner (Khan and Ahmad 1998 2007) Many
workers used chemical (organic inorganic) salt temperature biological and soil matrix
priming techniques to enhance seed germination percentage and especially germination
rate in saline medium (Ashraf et al 2008 Ashraf and Foolad 2005)Encouraging results
in most of the species of glycophytes and hydrophytes were found by presoaking in pure
water prior to germinating under saline condition Our study supports this finding and
seeds soaked in distilled water prior to germination performed better than those which
were presoaked in sea salt solutions Salinity adversely affects at all germination
parameters (germination percentage germination rate coefficient of germination velocity
and germination index) directly proportional with increasing salinity (Tayyab et al 2015)
With increase in time a delayed germination at higher salinity was found Higher sea salt
(168 dSm-1 for pure water presoaking and 35 dSm-1 for presoaking in respective
salinities) showed 50 or more reduction in all germination indices as compared to control
(Table 13-16 Figure 11)Our results are parallel with the finding of other workers such
as Kafi and Goldani (2001) who found the same trend in chickpea at higher salinities Pujol
et al (2000) reported that increased salinity inhibit the seed germination as well as delays
germination initiation in various halophyte species as well Similar response was also
found in some other crops such as pepper (Khan et al 2009) sunflower (Vashisth and
Nagarjan 2010) and eggplant (Saeed et al 2014) Salt tolerance within species may vary
at germination and other growth phases (Khan and Ahmad 1998)
According to our results C cajan appeared to be a salt sensitive in initial growth
phase specially when presoaked in saline medium (Figure 12) however at later growth
stages it proved relatively salt tolerant Salt stress delays or either seize the metabolic
activities during seed germination in salt sensitive and even in salt tolerant plants (Khan
and Ahmad 1998 Ali et al 2013b) Salinity also imposes the oxidative stress due to
52
overproduction of reactive oxygen species which may alter metabolic activities during
germination growth and developmental stages (Zhu 2001 Munns 2005
Lauchli and Grattan 2007)
In our study seeds of pigeon pea were unable to emerge beyond ECe39 dSm-1 sea
salt concentration Height of seedling was significantly affected by increasing salinity
(Figure 12) Similar results are also reported in Indian mustered (B juncea Almansouri
et al 2001) some Brassica species (Sharma et al 2013) and tomato cultivars (Jamil et
al 2005) Growth retardation with increasing salinity may be due to reduced
photosynthetic efficiency and inhibition of enzymatic and non-enzymatic proteins
(Tavakkoli et al 2011) Furthermore salt stress also limit the DNA and RNA synthesis
leads to reduced cell division and elongation during germination growth and
developmental stage
Khan and Sahito (2014) found variation in salt tolerance within species subspecies
and provenance level Furthermore the salt tolerance of a species may also vary at
germination and growth phases (Khan and Ahmad 1998 Ali et al 2013a) Srivastava et
al (2006) suggested that the genetic variability influences salinity tolerance eg wild
species like Cajanus platycarpus C scaraboides and C sericea showed better salt
tolerance than C cajan In this connection Wardill et al (2006) has also reported genetic
diversity in Acacia nilotica C cajan in this study appeared to be a salt sensitive at
germination in compression with later stages of growth Seedling establishment at saline
solution faces adverse effects when emerging radicle and plumule come in contact with
salt effected soil particle or saline water hence percent seedling establishment remains
less than germination percentage observed at petri plate Ashraf (1994) found that salinity
tolerance of different varieties of C cajan do not much differ at germination and early
growth stages whereas at adult growth stage show improvement in salt tolerance
Soil salinity is a major limiting factor for plant growth and yield production
particularly in leguminous plants (Guasch-Vidal et al 2013 Tayyab et al 2016) In
present study Plant height RGR fresh and dry biomass were severely reduced with
increasing salinity and plant was unable to grow after ECe= 43 dSm-1(Figure 14-16)
This growth inhibition of C cajan may be accounted for individual and synergistic effect
of water stress nutrient imbalances and specific ions toxicities (Hasegawa et al 2000
Silvera et al 2001) Salt induced ion imbalance results in lower osmotic potential which
53
alter physiological biochemical and other metabolic processes leading to overall growth
reduction (Del-Amor et al 2001) Excessive amount of salt in cytoplasm challenge the
compartmentalization capacity of vacuole and disrupts cell division cell elongation and
other cellular processes (Munns 2005 Munns et al 2006) Our results are parallel with
some other studies in which significant growth inhibition of peas chickpea and faba beans
have been reported against salt stress (El-Sheikh and Wood 1990 Delgado et al 1994)
Singla and Garg (2005) also observed a similar salt sensitive growth response in Cicer
arietinum In our study the fresh and dry biomass of C cajan also showed inhibitory
behavior to salt stress (Figure 15) Hernandez et al (1999) also found significant reduction
in dry biomass of pea plant and common bean (40 and 84 respectively) when grown
in saline medium Mehmood et al (2008) also found similar results in Susbania sasban
Salinity also has imposed deleterious effects on reproductive growth of C cajan
Production of flowers and pods are significantly decreased in response to salinity (Figure
19) Increase in flower shedding leads to decreased number of pods indicating salt
sensitivity of plant at reproductive phase which was more pronounced at high salinity
(Vadez et al 2007) Furthermore seed production and weight of seed per plant was also
linearly decreased Salt induced reduction of reproductive growth has also been found in
mung bean in which 60 and 12 less pods and seeds were produced respectively at 06
saline solution (Qados 2010) Similar results are reported in faba bean (De-Pascale and
Barbieri 1997) tomato (Scholberg and Locascio 1999) maiz sunflower (Katerji et al
1996) and watermelon (Colla et al 2006) Salinity reduces reproductive growth by
inhibiting growth of flowers pollen grains and embryo which leads to inappropriate ovule
fertilization and less number of seeds and fruits (Torabi et al 2013)
On biochemical parameters total chlorophyll and chlorophyll ab ratio has
increased in low salinity in contrast the adverse effect at higher salinity could be due to
high Na+ dependent breakdown of these pigments (Li et al 2010 Yang et al 2011)
Chlorophyll a is usually more prone to Na+ concentration and decrease in total chlorophyll
is mainly attributed to the destruction of chlorophyll a (Fang et al 1998 Eckardt 2009)
This diminution could be due to the destruction of enzymes responsible for green pigments
synthesis (Strogonov et al 1973) and increased chlorophyllase activity (Sudhakar et al
1997) Thus insipid of leaf was a visible indicator of salt induced chlorophyll damage
which was well correlated with quantified values as reported in other legume species
54
(Soussi et al 1998 Al-Khanjari et al 2002) In this study chlorophyll a was found to be
more sensitive than chlorophyll b (Figure 18) Garg (2004) also found similar reduction
in chlorophyll pigments (a b and total chlorophyll) in chickpea cultivars under salinity
stress
At low salinity (16 dSm-1) total carotenoids remained unaffected along with
increased total chlorophyll (Figure 18) which may suggest a role of carotenoids in
protection of photosynthetic machinery (Sharma et al 2012) Similar response was found
in Cajanus indicus and Sesamum indicum (Rao and Rao 1981) however
Sivasankaramoorthy (2013) and Ramanjulu et al (1993) reported slight increase of leaf
carotenoids in Zea maiz and mulberry when exposed to NaCl High salinity was destructive
for both leaf pigments (chlorophyll and carotenoids) of C cajan which was in accordance
with Reddy and Vora (1985) who found similar decrease in some other salt sensitive crops
Salinity led to the conversion of beta-carotene to Zeaxanthin which protect plants against
photo-inhibition (Sharma and Hall 1991)
In present study with increasing salinity water content and succulence of C cajan
were significantly reduced which indicated loss of turgor (Figure 16) Our data suggest
that decreased succulence by lowering water content may help in lowering leaf osmotic
potential when exposed to increasing salinity which is in agreement with findings of Parida
and Das (2005) and Abideen et al (2014) In addition increased production and
accumulation of organic substances is also necessary to sustain osmotic pressure which
provide osmotic gradient to absorb water from saline medium (Hasegawa et al 2000
Cha-um et al 2004) Compatible solutes including carbohydrates amino acids proteins
and ammonium compounds play important roles in water relations and cell stabilization
(Ashraf and Harris 2004) In this study C cajan produce more soluble sugars (Figure 18)
which is considered as a typical plant response under saline conditions (Murakeozy et al
2003) Sugars serve as organic osmotica and their available concentration is related to the
degree of salt stress and plantrsquos tolerance (Ashraf 1994 Murakeozy et al 2003) Sugars
are involved in osmoprotection osmoregulations carbon storage and radical scavenging
activities (Pervaiz and Satyawati 2008) On the other hand insoluble and total sugars were
reduced in higher salinity which is also supported by Parida et al (2002) and Gadallah
(1999) who found similar results in Bruguiera parviflora and Vicia faba
55
Total soluble proteins of C cajan were reduced due to deleterious effects of salinity
(Figure 18) The accumulation of Na+ in cytosol disrupts the protein and nucleic acid
synthesis (Bewley and Black 1985) Gill and Sharma (1993) and Muthukumarasamy and
Panneerselvam (1997) also reported decreased protein content with increasing salinity in
Cajanus cajan seedlings Similar results were found when tomato (Azeem and Ahmad
2011) Zingiber officinale (Ahmad et al 2009) and Sorghum bicolor (Ali et al 2013a)
were grown under variable salt concentrations (Figure 19)
Nodule formation of Rhizobium in Legume depends upon interaction between soil
chemistry of salt composition and osmotic regimes of salt and water (Velagaleti et al
1990 Zahran 1991 Zahran and Sprent 1986) Salinity reduces plant growth directly
through ion and osmotic effects and indirectly by inhibiting Legume-Rhizobium
association (El-Shinnawi et al 1989) Studies demonstrated a more sensitive response of
rhizobial N-fixing mechanism than growth of plant to abiotic stresses including salinity
(Mhadhbi et al 2004) In nodules metabolic disturbance initiated with the production of
ROS leading to tissues injury and loss of nodule function (Becana et al 2000) In general
it slow down the nitrogenase activity and decrease nodule protein and leghemoglobin
content which decreased becteroid development (Mhadhbi et al 2008) In consequence
plant suffer directly by salt induced ion toxicity low water uptake and photosynthetic
damage and indirectly through weak association of symbionts due to high energy demand
for nodule function (Pimratch et al 2008) In our study the isolated rhizobial strain from
nodules of C cajan was found to be tolerant to salinity even up to 2 (ECw= 306 dSm-1)
NaCl (Figure 110 and 111) Some of the other species of Rhizobium such as Brady
Rhizobium have been shown salt tolerant even at higher concentration than their
leguminous hosts (Zahran 1999) For instance a number of rhizobial species can tolerate
up to 06 NaCl (Yelton et al 1983) while Rhizobium meliloti can tolerate 175 to
40 NaCl and R leguminosarum can tolerate can tolerate upto 2 NaCl (Abdel-Wahab
and Zahran 1979 Sauvage et al 1983 Breedveld et al 1991 Helemish 1991
Mohammad et al 1991 Embalomatis et al 1994 Mhadhbi et al 2011) Rhizobia
isolated from soybean and chickpea can tolerate up to 2 NaCl with a difference of fast-
growing and slow growing strains (El-Sheikh and Wood 1990 Ghittoni and Bueno 1996)
Similarly Rhizobium from Vigna unguiculata can survive up to up to 55 NaCl
(Mpepereki et al 1997)
56
Present study shows an increase in vegetative growth in terms of plant height and
fresh and dry weight of shoot with increasing time under non-saline and saline conditions
but the increase was rapid at early period of growth (Figure 112) All the vegetative
growth parameters determined were reduced under salinity stress compared to non-saline
control Measurements of shoot moisture succulence specific shoot length and RGR
(Figure 113) indicate that Z mauritiana adjusted in its water relation over coming
negative water and osmotic potential with increase in salinity levels increased There is
evidence that water and osmotic potentials of salt tolerant plants become more negative in
higher salinities (Khan et al 2000) These altered water relations and other physiological
mechanisms help plants to get by adverse abiotic stress like that of drought and salinity
(Harb et al 2010) However the results clearly showed that salinity had an inhibitory
effect on growth but the decline was less at early sixty days and more during later 60-120
days in compression to controls Growth inhibition in shoot has been observed in number
of plants including different species of halophytes (Keiffer and Ungar 1997) chickpea
(Cicer arietinum Kaya et al 2008) and different wheat cultivars (Triticum aestivum
Moud and Maghsoudo 2008)
Salinity also caused reduction in the number of branches and the number of flowers
in Z mauritiana however reduction in the number of flowers is non-significant in ECe=
72 dSm-1 salinity treatment in comparison with non-saline control (Figure 114) The main
reason for this reduction could be attributed to suppression of growth under salinity stress
during the early developmental stages (shooting stage) of the plants These results are
similar to those reported by Ahmad et al (1991) and Khan et al (1998) As affirmed by
Munns and Tester (2008) suppression of plant growth under saline conditions may either
be due to osmotic effect of saline solution which decreases the availability of water for
plants or the ionic effect due to the toxicity of sodium chloride High salt concentration in
rooting medium also reduced the uptake of soil nutrients a phenomenon which affected
the plant growth thus resulting in less number of branches per plant Various abiotic
stresses such as temperature drought salinity light and heavy metals altered plant
metabolism which ultimately affects plant growth and productivity Amongst these
salinity stress is a major problem in arid and semiarid regions of the world (Kumar et al
2010) Salinity has an adverse effect on several plant processes including seed
germination seedling establishment flowering and fruit formation and ripening (Sairam
and Tyagi 2004) Salinity stress also imposes additional energy requirements on plant
57
cells and less carbon is available for growth and flower primordial initiation (Cheesman
1988) The lesser decrease in number of flowers at lower salinity (ECe= 72 dSm-1) has
been attributed to the fact that the cells of apex are un-vacuolated and the incoming salts
accumulated in the cytoplasm Munns (2002) further suggested a well-controlled phloem
transport of toxic ions from these cells prevented any change in reproductive development
Our findings showed an increase in total chlorophyll contents particularly
chlorophyll b contents were enhanced more than chlorophyll a contents under salinity
stress (Figure 115) In general the total chlorophyll contents decreased under high salinity
stress and this may be due to accumulation of toxic ions in photosynthetic tissues and
functional disorder of stomatal opening and closing (Khan et al 2009) The increase in
total chlorophylls appearing at salinity levels is considered as an important indicator of
salinity tolerance in plants (Katsuhara et al 1990 Demiroglu et al 2001) In another
study on Z mauritiana (cv Banara sikarka) the chlorophyll contents has shown decrease
with increasing salinity and sodicity but the seedlings treated with low salinity (ECe of 5
mmhoscm-1) shows slightly higher values than controls (Pandey et al 1991) Our study
also suggests that increase in total chlorophylls adapted this plant increased its tolerance
to salt stress
Slight decrease in protein has been shown under salinity treatments compared to
controls (Figure 16) Proteins play diverse roles in plants including involvement in
metabolic pathways as enzyme catalyst source of reserve energy and regulation of osmotic
potential under salt stress (Pessarakli and Huber 1991 Mansour 2000) Salts may
accumulate in cell cytoplasm and alter their viscosity depending on the response of plant
to salinity stress (Hasegawa et al 2000 Paravaiz and Satyawati 2008) The decrease in
protein contents under increasing salinity has also been documented in several plants
including Lentil lines (Ashraf and Waheed 1993) sorghum (Ali et al 2013a) and sugar
beet (Jamil et al 2014)
Soluble sugars were also decreased with increasing salinity treatments in our study
(Figure 16) Decrease in soluble sugars due to salinity has also been reported in Viciafaba
(Gadallah 1999) some rice genotypes (Alamgir and Ali 1999) Bruguiera parviflora
(Parida et al 2002) and Lentil (Sidari et al 2008) However the accumulation of soluble
sugars under salinity stress is considered as strategy to tolerate stress condition due to their
58
involvement in osmoprotection osmotic adjustment and carbon storage (Parida et al
2002 Parvaiz and Satyawati 2008)
From these experiments it is evident that C cajan is a salt sensitive plant at every
level of its life cycle starting from germination to growth phases Germination capacity
and salt tolerance ability of this species can be enhanced by water presoaking treatment
Growth reduction with increasing salinity could be attributed to physiological and
biochemical disturbances which ultimately affect vegetative and plant reproductive
growth Its roots are well associated with nitrogen fixing rhizobia and these
microorganisms were salt tolerant in in-vitro cultures Another fruit baring species of
marginal lands Z mauritiana showed growth improvement in lower salinity and its growth
was not much affected in high saline mediums owing to its controlled biochemical
responses
59
2 Chapter 2
Intercropping of Z mauritiana with C cajan
21 Introduction
Increasing soil salinity fresh water scarcity and agricultural malpractice creating shortage
of food crops for human and animal consumption (Bhandari et al 2014) and making
prices high Traditional agriculture which has been practiced since centuries using multi
species at a time in a given space could be a potential solution to narrow down the growing
edges of this supply demand scenario Plant species with innate resilience to abiotic
stresses like salinity and drought could be considered suitable to serve this purpose
especially for arid regions where marginal lands can be utilized to generate economy
Presence of such type of local systems in the region highlight their potential advantage in
crop production income generation as well as sustainability (Somashekar et al 2015)
For instance reports are available on successful intercropping of multipurpose trees
shrubs and grasses like millets pulses and some oil seed and fodder crops Green part of
these species usually mixed and used for cattle feed especially during the lean period The
utilization of the inter-row spaces of fruit trees like Ziziphus mauritiana for growing edible
legumes can generate further income by similar input (Dayal et al 2015) As an option
to this Cajanus cajan could serve as better intercropped as it provides protein rich food
nutritious fodder and wood for fuel which helped to uplift the socio-economic condition
of poor farmers Integrated agricultural practices improve the productivity of each crop by
keeping cost of production under sustainable limits (Arabhanvi and Pujar 2015)
Keeping in mind the above mentioned scenario in present study the possibility to
increase production of a non-conventional salt tolerant fruit tree (Z mauritiana) by
intercropping with a leguminous plant (C cajan) was investigated to produce edible fruits
and fodder simultaneously from salt effected waste lands
60
22 Experiment No 7
Physiological investigations on Growth of Ziziphus mauritiana and
Cajanus cajan intercropped in drum pot (Lysimeter) culture being
irrigated with water of sea salt concentration at two irrigation intervals
221 Materials and Methods
2211 Growth and Development
Experiment was designed to investigate the effect of intercropping on growth and
development of Z mauritiana (a fruit tree) and C cajan (a leguminous fodder) in drum
pot culture irrigated with water of 03 sea salt concentrations at two irrigation intervals
2212 Drum pot culture
Drum pot culture as recommended by Boyko (1966) and modified by Ahmed and
Abdullah (1982) was used for the present investigation as described in chapter 1
2213 Experimental Design
Three sets of 18 plastic drums (lysimeter) were used in this experiment One plant of Z
mauritiana were grown in each lysimeter Three replicates were kept for each treatment
comprising of 06 drums in each set which was further divided in two sub-sets First sub-
set was irrigated at every 4th and second subset at every 8th day
Set ldquoArdquo =Ziziphus mauritiana (Sole crop)
Set ldquoBrdquo = Cajanus cajan (Sole crop)
Set ldquoCrdquo = Ziziphus mauritiana + Cajanus cajan (intercropped)
The effect of salinity on sole crops of C cajan and Z mauritiana on salinity created
by various dilutions of sea salt has been investigated in chapter 1 Concentration of 03
sea salt considered equal level to its 50 reduction has been selected in present
experiment In addition irrigation was given in sub-sets in two intervals to investigate to
have some idea of its water conservation
61
2214 Irrigation Intervals
Sub-set 1 Irrigation was given every 4th day
Sub-set 2 Irrigation was given every 8th day
In set lsquoArsquo and lsquoCrsquo six month old saplings of Ziziphus mauritiana (vern grafted
ber) plants of nearly equal height and good health were transplanted in drum pots Plants
were irrigated to start with non-saline tape water for about two weeks for purpose of
establishment All the older leaves fell down and new leaves immerged during
establishment period
In set lsquoBrsquo and lsquoCrsquo Ten healthy sterilized seeds of Cajanus cajan imbibed in distill
water were sown in each drum pot and irrigated to start with tap water and after
establishment of seedlings only six seedlings of equal size with eqal distance (about one
feet) between C cajan and that of Z mauritiana were kept for further study The sowing
time of cajanus cajan seeds in both sets (B and C) was the same In drum pot lsquoCrsquo it was
sown when sapling of Z mauritiana have undergone two weeks of their establishment
period in tap water
When seedlings of C cajan reached at two leaves stage irrigation in all the sets
(ABC ) was started with gradual increase sea salt concentration till it reached to the
salinity level of treatment (03) in which they were kept up to end of experiment Each
drum was irrigated with enough water sea salt solution which retains 15 liters in soil at
field capacity Rest of water drain down with leaching of accumulated salt in root
rhizosphere
Vegetative growth of Z mauritiana plant was noted monthly in terms of height
volume of canopy while in C cajan height and number of branches was noted Shoot
length root length number of leaves fresh and dry weight of leaf stem and root leaf
weight ratio root weight ratio stem weight ratio specific shoot and root length plant
moisture leaves succulence and relative growth rate was observed and calculated at final
harvest in both the plant species growing individually (sole) or as intercropping at two
irrigation intervals
Investigations were undertaken on nitrate content relative water content and
electrolyte leakage at grand period of growth Amount of photosynthetic pigments soluble
62
carbohydrates proline content soluble phenols and Protein contents were also investigated
in fully expended leaves
Activity of catalase (CAT) ascorbate peroxidase (APX) guaiacol peroxidase
(GPX) superoxide dismutase (SOD) (Anti-oxidant enzymes) and nitrate reductase (NR)
activity was also observed in on both the Z mauritiana and C cajan leaves growing as
sole and as intercropped at two different irrigation intervals
The procedures of above mentioned analysis as follows
Leaf succulence (dry weight basis) Specific shoot length (SSL) and relative
growth rate (RGR) were measured according to the equations given in chapter 1
2215 Estimation of Nitrate content
NO3 was estimated through Cataldo et al (1975) 01g fresh leaf samples were boiled in
50 mL distilled water for 10 min 01mL of sample were added to mixed in 04 mL 50
salicylic acid (wv dissolved in 96 H2SO4 ) and allowed to stand for 20 min at room
temperature 95 mL of 2N NaOH was slowly mixed at last The samples were permissible
to cool NO3 concentration was observed at 410 nm and was calculated according to the
standard curve expressed in mg g-1 fresh weight
2216 Relative Water content (RWC)
Young and fully expended leaf was excise from each plant removing dust particles
preceding to Relative water content (RWC) Fresh weights (FW) were taken to all leaf
samples and were immersed in distilled water at 4 degC for 10 hours The soaked leaf samples
were taken out and surfeit water was removed by tissue paper Weighted again these leaf
samples for turgid weight (TW) and were oven dried at 70 degC Dry weight (DW) was
recorded after 24 hrs The RWC of leaf was calculated by the following formula
RWC () = [FW ndash DW] [TW ndash DW] x 100
2217 Electrolyte leakage percentage (EL)
EL was measured according to Sullivon and Ross (1979) Young and fully expended
leaves removing dust particles were taken 20 disc of 6mm diameter were made through
63
porer and were placed in the test tube containing 10ml de-ionized water First electrical
conductivity (EC lsquoarsquo) was record after shaken the tubes These test tubes now were placed
at 45-50oC warmed water bath for 30 min and observed second Electrical conductivity (EC
lsquobrsquo) Finally tubes were placed at 100oC water bath for ten min and obtained third and final
Electrical conductivity (EC lsquocrsquo) The electrolyte leakage was calculated in percentage by
using following formula
EL () = (EC b ndash EC a) EC b x 100
2218 Photosynthetic pigments
Photosynthetic pigments including chlorophyll a chlorophyll b total chlorophyll
chlorophyll ab ratio and carotinoids were estimated according to the procedure given in
chapter 1
2219 Total soluble sugars
Dry leaf samples (01g) were milled in 5mL of 80 ethanol and were centrifuged at 4000
g for 10 minutes and were estimated according to the procedure described in chapter 1
22110 Proline content
The proline contents were determined through Bates et al (1973) Each dried leaf powder
sample (01 g) was grinded and homogenized in 5 ml of 3 (wv) sulphosalicylic acid and
were centrifuged at 5000 g for 20 minutes 2ml supernatant was boiled by adding 2 ml
glacial acetic acid and 2 ml ninhydrin reagent (prepared by dissolving 125 g ninhydrin in
30 ml of glacial acetic acid and 20 ml 6 M phosphoric acid) in caped test tube The tubs
were kept in boiling water bath (100oC) for 1 hour After cooling 4 ml of toluene was
added to each tube and vortex Two layers were appeared the chromophore layer of
toluene was removed and their absorbance was recorded at 590nm against reference blank
of pure toluene The proline concentrations in leaves were determined from a standard
curve prepared from extra pure proline of (Sigma Aldrich) and were calculated from the
equation and were expressed in mgg-1 of leaf dry weight
Proline (microgmL-1) = -074092 + 1660767 (OD) plusmn054031
64
22111 Soluble phenols
The dried leaf powder (01g) was milled in 3ml of 80 methanol and was centrifuged at
10000g for 15 min (Abideen et al 2015) Final volume (5ml) were adjusted by adding
80 methanol Soluble phenols were determined by using Singleton and Rossi (1965) ie
5 ml of Folin-Ciocalteu reagent (19 ratio in distilled water) and 4 ml of 75 Na2CO3
were added to 01 ml supernatant The absorbance was recorded at 765 nm after incubation
of 30 minutes at room temperature The soluble phenols concentration in leaf tissues was
determined from a standard curved prepared from Gallic acid
22112 Total soluble proteins
The protein contents were measured according to Bradford Assay reagent method against
Bovine Serum Albumin as standards (Bradford 1976) Procedure was followed as given
in chapter 1
22113 Enzymes Assay
Enzyme extract prepared as given below was used for study of enzymes mentioned in text
The juvenile and expended leaf excised was frozen in liquid nitrogen and were stored at -
20 degC These leaf samples (100mg) was firmed in liquid nitrogen and were mills in 3 ml
of ice chilled potassium phosphate buffer (pH = 7 01 M) with 1mM EDTA and 1 PVP
(wv) The homogenate was filtered through a four layers of cheesecloth and were
centrifuged at 21000 g using refrigeration centrifuge (Micro 17 TR Hanil Science
Industrial Co Ltd South Korea) at 4 degC for 20 min The supernatant was separated and
stored at -20 degC and used for investigation on following enzymes
i Superoxide dismutase (SOD)
SOD (EC 11511) antioxidant enzymeactivity was measured through Beauchamp and
Fridovich (1971) derived on the inhibition of nitroblue tetrazolium (NBT) reduction by
produced O2minus using riboflavin photo-reduction 50 mM of pH 78 phosphate buffer (with
01mM EDTA 13 mM methionine) 75 microM nitroblue tetrazolium (NBT) 2 microM riboflavin
and 100 microl of enzyme extract was added to 3ml reaction mixture Riboflavin was added at
the last before the reaction was initiated under fluorescent lamps for 10 min Exposed and
un-exposed to florescence lamp without enzyme extract were used to serve as calibration
65
standards Activity was measured at 560nm Unit of SOD activity was defined as the
amount of enzyme required for 50 inhibition of NBT conversion
ii Catalase (CAT)
CAT (EC 11116) antioxidant enzyme activity was precise according to Aebi (1984)
derived on H2O2 reduction at 240nm for 30 s (ε = 36 M-1 cm-1)100mM potassium
phosphate buffer (pH=7) with 30mM H2O2 and 50 microl of diluted enzyme extract (adding in
last) was added to 3ml reaction mixture The decrease in absorbance due to H2O2 reduction
was measured at 240 nm and expressed in micromol of H2O2 reduced m-1g-1 fresh weight at 25
degC
iii Ascorbate peroxidase (APX)
Nakano and Asada (1981) method was used for APX (EC 111111) antioxidant
enzymeactivity by measuring the decrease in ascorbate oxidation by H2O2 The reaction
mixture (3ml) contained potassium phosphate buffer (50mM pH=7) 01mM H2O2 050
mM Ascorbate and 100 microl of enzyme extract and were observed 290 nm for 1 min 25 degC
(extinction coefficient 28 mM-1cm-1)
iv Guaiacol peroxidase (GPX)
GPX (EC 11117) antioxidant enzymeactivity was estimated through Anderson et al
(1995) 3ml of 50 mM potassium phosphate buffer (pH 7) guaiacol 75 mM H2O2 10 mM
reaction mixture with 20 microl of enzyme extract adding at last Increase in absorbance was
observed due to the formation of tetra-guaiacol at 470 nm for 2 min (extinction coefficient
266 mM-1cm-1)
v Nitrate reductase (NR)
The NR activity in leaves was observed through Long and Oaks 1990 Fresh leaf samples
(01g) were placed in 5ml of 100mM potassium phosphate pH 75 (added to 10
Isopropanol and 25mM KNO3) Tubes were vacuumed for 10 min to remove air from the
mixture and were placed in water bath shaker at 33oC for 60 min in dark The tubes were
placed in hot water (100oC) for 5 min 15 mL from the reaction mixture were added in 05
mL 20 sulphanilamide (wv dissolve in 5N HCl) and 025 mL 008 N-1-Napthylene-
66
diamine dihydrochloride Final volume up to 60 ml was made by adding distilled water
Color developed over the next 20 min Absorbance was measured at 540 nm using
spectrophotometer
67
222 Observations and Results
Sole and intercropped Ziziphus mauritiana
2221 Vegetative growth
Growth of Z mauritiana in terms of shoot root and plant length and number of leaves in
two different cropping system (sole and intercrop with C cajan) in two different irrigation
intervals has been presented in Figure 21 Appendix-XII A significant increase (plt0001)
in plant length was observed in 8th day irrigation in both the cropping systems in Z
mauritiana At 4th day of irrigation interval a non-significant increase in length was
observed in intercropped plants compared to sole crop Similarly at 8th day of irrigation
plants attain almost same heights in both the cropping systems
A significant increase (plt001) in root length was observed in sole Z mauritiana
at 8th day of irrigation compared to other treatments Smallest root length revealed in plants
that were irrigated at 4th day under sole crop system
The shoot length was significantly increase (plt0001) in plants which were
irrigated at 8th day under intercropped system However shoot length remains unaffected
when comparing the different cropping system at both the irrigation intervals
A significant increase (plt0001) in number of leaves was observed in intercropped
Z mauritiana plants compared to plants cultivated according to sole system However
more increase was observed in 4th day irrigated intercropped plant as compared to 8th day
The difference in number of leaves in sole crop at both irrigating intervals remains same
i Fresh weight
Figure 22 Appendix-XII showed fresh and dry weight of stem root and leaf of Z
mauritiana plant in two different cropping system (sole and intercrop with C cajan) in
two different irrigation intervals A significant increase (plt0001) in fresh weights of leaf
stem and root was observed in intercropping (with C cajan) 4th and 8th day of irrigation
interval compared to individual cropping of Z mauritiana In 4th day of irrigation the
increment was more pronounced in fresh weights of root (7848) leaves (4130) and
stem (4047) respectively with comparison to the crop growing alone Similarly
intercropping in 8th day of irrigation showed better growth of leaves (28) stem (12)
68
and root (31) against sole crop Whereas decrease in leaves 33 (plt005) stem 70
(plt0001) and root 60 (plt0001) fresh weights were observed in 8th day of irrigation
compared to 4th day intercropping However the difference was non-significant between
two sole crops irrigated at 4th and 8th day interval
ii Dry weight
Intercropping with comparison to the sole crop showed significant (plt0001) increase in
dry weights of leaves root and stem of Z mauritiana at 4th and 8th day of irrigation (Figure
22 Appendix-XII) At 4th day of irrigation intercropping showed an increment in dry
weights of Leaves (4366) stem (4109) and root (754) compared to the sole crop
Similar increase was observed in leaves (plt0001) stem (plt0001) and root (plt0001)
weights after 8th day of irrigation However intercropping at 8th day irrigation showed an
increment in root (19) stem (11) whereas a slight decrease (1) in leaves dry weight
When comparing irrigation time an increase in stem dry weight at 4th day whereas decline
in leaves dry weight was observed Root dry weights were more or less similar at both
irrigation intervals
iii Leaf weight ratio (LWR) root weight ratio (RWR) stem weight
ratio (SWR)
Leaf weight ratio (LWR) root weight ratio (RWR) stem weight ratio (SWR) of Z
mauritiana plant grown in two different cropping system (sole and intercrop with C cajan)
in two different irrigation intervals has been presented in Figure 23 Appendix-XII An
increased in LWR and SWR was recorded at 8th day of irrigation compared to 4th day of
irrigation in both cropping systems whereas decrease in RWR was observed LWR and
SWR remained un-change in sole and inter crop system However RWR increased in
intercrop system compared to sole crop system
iv Specific shoot length (SSL) specific root length (SRL)
Specific shoot length (SSL) specific root length (SRL) of Z mauritiana plant grown in
two different cropping system (sole and intercrop with C cajan) in two different irrigation
intervals has been presented in Figure 23 Appendix-XII SSL was observed higher in 8th
day of irrigation compared to 4th day in both the cropping systems However the increase
69
in SSL was lesser in sole crop compared to intercropping Similarly SRL was recorded
lesser in 4th day of irrigation compared to 8th day of irrigation in both cropping systems
Intercropped plants showed decline in SRL compared to sole crop plants Greatest SRL
revealed in plants that were irrigated after 8th day and planted according to sole crop
system
v Plant moisture
The moisture content of Z mauritiana plant grown in two different cropping system (sole
and intercrop with C cajan) in two different irrigation intervals has been presented in
Figure 23 Appendix-XII The moisture content of plants was significantly decreased
(plt005) in sole crop while increased (plt005) in intercropping at 8th day of irrigation
compared to 4th day At 4th day moisture remained same in both cropping system
However significant increase in moisture contents was observed in inter-crop system
compared to sole crop system after 8th day of irrigation
vi Plant Succulence
Succulence of Z mauritiana plant grown in two different cropping system (sole and
intercrop with C cajan) in two different irrigation intervals has been presented in Figure
23 Appendix-XII Plant succulence in 8th day was significantly reduced in sole crop
whereas increased in intercropping system In 4th day irrigated plants decrease in
succulence was noticed compared to plants that were irrigated at 8th day under sole crop
system However significant increase (plt0001) was observed in intercropped plants
irrigated at 4th day compared to 8th day
vii Relative growth rate (RGR)
Relative growth rate (RGR) of Z mauritiana plant grown in two different cropping system
(sole and intercrop with C cajan) in two different irrigation intervals has been presented
in Figure 23 Appendix-XII Relative growth rate remains unchanged at both irrigation
times under sole crop system However decline in 8th day was observed compared to 4th
day of irrigation under intercrop system Greatest RGR was recorded in plants that were
irrigated at 4th day under intercrop system
70
2222 Photosynthetic pigments
Photosynthetic pigments including Chlorophyll a chlorophyll b total chlorophyll
Chlorophyll ab ratio and carotinoids of Z mauritiana plant grown in two different
cropping system (sole and intercrop with C cajan) in two different irrigation intervals has
been presented in Figure 24 Appendix-XII
i Chlorophyll contents
A significant increase (plt0001) in chlorophyll a b and total chlorophyll was observed in
plants growing as sole crop compared to intercropped system at both the irrigation
intervals Higher chlorophyll contents were also recorded in plants that were irrigated at
8th day compared to 4th day of irrigation The chlorophyll ab ratio increased in 4th day
while decline in 8th day in intercropped system compared to sole crop However overall
results showed non-significant changes
ii Carotinoids
A significant increase (p lt 0001) in leaf carotinoids was observed in sole crop compare
to intercropped system at both irrigation times in Z mauritiana Least carotene content
was estimated in plants that were irrigated at 4th day under intercrop system
2223 Electrolyte leakage percentage (EL)
Electrolyte leakage percentage (EL) of Z mauritiana plant grown in two different
cropping system (sole and intercrop with C cajan) in two different irrigation intervals has
been presented in Figure 25 Appendix-XII A non-significant result was observed in
electrolyte leakage in plant growing at varying cropping system and irrigating intervals
2224 Phenols
Total phenolic contents in leaves of Z mauritiana plant grown in two different cropping
system (sole and intercrop with C cajan) in two different irrigation intervals has been
presented in Figure II25 Appendix-XII A significant increase (plt001) in total phenolic
contents was observed in intercropped growing at both irrigation interval compared to sole
crop However the increase was more pronounced at 8th day of irrigation Maximum
phenolic contents were measured in plants irrigated at 8th day under intercropped plants
71
2225 Proline
Total proline contents in leaves of Z mauritiana plant grown in two different cropping
system (sole and intercrop with C cajan) in two different irrigation intervals has been
presented in Figure 25 Appendix-XII A significant decreased (plt0001) was observed
in Z mauritiana cultivated according to intercropped system in both irrigation intervals
Maximum decrease was observed in intercropped plants irrigated at 8th day whereas
highest phenolic contents were observed in plants irrigated at 4th day under sole crop
system
2226 Protein and sugars
Protein and sugar contents in leaves of Z mauritiana plant grown in two different cropping
system (sole and intercrop with C cajan) in two different irrigation intervals has been
presented in Figure 26 Appendix-XII A nonsignificant difference in total protein and
sugar contents in Z mauritiana plants was observed in two different (4th and 8th day)
irrigation intervals However the interaction with time and irrigation interval also showed
nonsignificant result
2227 Enzyme essays
Antioxidant enzymes like Catalase (CAT) Ascorbate peroxidase (APX) Guaiacol
peroxidase (GPX) Superoxide dismutase (SOD) and Nitrate reductase activity in leaf of
Z mauritiana plant grown in two different cropping system (sole and intercrop with C
cajan) in two different irrigation intervals has been presented in Figure 27 and 28
Appendix-XII
i Catalase (CAT)
A significant decreased (plt0001) in catalase activities was observed in Z mauritiana
leaves in intercropped system in both time interval with compare to sole crop at 4th day
irrigated plant However maximum decline was in sole plants irrigated at 8th day interval
However their interaction with time was nonsignificant
72
ii Ascorbate peroxidase (APX)
A significant increase (plt0001) in APX activity was observed in 8th day irrigation in both
sole and intercropped plants with compare to sole and intercropped at 4th day irrigation
interval More increase (plt0001) was observed in intercropped Z mauritiana at 8th day
Whereas nonsignificant decrease was observed in two different cropping system in 4th day
irrigation interval However interaction between time and the treatments shows significant
values
iii Guaiacol peroxidase (GPX)
A significant (plt0001) increase in GPX was observed in 8th day intercropped Z
mauritiana plant with compare to irrigation intervals as well as cropping system However
at 4th day both cropping system showed nonsignificant difference Whereas more decline
was observed in 8th day sole crop The ANOVA reflects significant (plt005) interaction
between time and the cropped system
iv Superoxide dismutase (SOD)
A nonsignificant increase in SOD was observed in intercropped at 8th day irrigation
interval Whereas there was nonsignificant differences in 4th day intercropped and at both
time intervals of sole crop However interaction between time interval and the two
cropping system shows nonsignificant result
v Nitrate and Nitrate reductase
A significant increase (plt0001) in nitrate content and activity of nitrate reductase was
observed in intercropped plants of both irrigation intervals Increase in activity was
observed (plt0001) in intercropped Z mauritiana at 4th day
73
Sole and intercropped Cajanus cajan
2228 Vegetative growth
Growth of C cajan in terms of shoot root and plant length and number of leaves was
observed in two different cropping system (sole and intercrop with Z mauritiana) in two
different irrigation intervals has been presented in Figure 21 Appendix-XIII XIV A
significant increase (plt001) in plant length was observed in intercropped C cajan
compared to sole crop at both irrigation interval Whereas sole crop at 8th day interval
showed better results as compare to sole of 4th day Similarly root length remains
unaffected and showed non-significant change in both cropping systems and even at two
different irrigation intervals While shoot length was significantly (Plt001) decreased in
sole crop compared to intercropped at 4th day irrigation Whereas non-significant
difference be observed in rest of cropping systems growing at different irrigation interval
A significant increase (plt001) in leaves number was observed in intercropped
plants compared to sole crop at 4th and 8th day irrigation interval However most
significant decrease (plt0001) was observed in sole crop at 4th day
i Fresh weight
Figure 22 Appendix-XIV showed fresh and dry weight of stem root and leaf of C cajan
plant in two different cropping system (sole and intercrop with C cajan) in two different
irrigation intervals A significant increase (plt001) in fresh weight of leaf was observed in
intercropping (with Z mauritiana) at 4th and 8th day of irrigation interval compared to
individual cropping of C cajan The increase in intercropped system compared to sole
crop was more pronounced at 4th day (42) of irrigation than the 8th day (1701) Plants
showed higher leaves fresh weights in 8th day of irrigation compared to 4th day Similarly
the interaction between cropping system and the irrigation interval was significant
(Plt005)
An insignificant difference was observed in stem at 4th (15) and 8th (12) days
fresh weights in both intercropping system at two different irrigation intervals The
interaction between cropping system and the irrigation interval also showed non-
significant result
74
A non-significant difference in root fresh weight was observed in two different
cropping systems (sole and intercropped) in 4th and 8th day of irrigation intervals However
fresh weight of crop at 8th day irrigation interval was significantly increase (plt0001) over
4th day irrigation interval Similar pattern was observed in 4th day irrigated sole and
intercropped C cajan
ii Dry weight
A significant increase in leaves (42) stem (24) and root (18) dry weights were
observed in 4th day irrigation under intercropped system compared to sole However in 8th
day of irrigation this increase of dry weights was not much prominent Under sole crop
system dry weights of leaves stem and root was increased markedly in 8th day compared
to 4th day However in intercrop system the difference in dry weights was insignificant
between 8th and 4th day of irrigation
iii Leaf weight ratio (LWR) root weight ratio (RWR) stem weight
ratio (SWR)
Leaf weight ratio (LWR) root weight ratio (RWR) stem weight ratio (SWR) of C cajan
grown in two different cropping system (sole and intercrop with Z mauritiana) in two
different irrigation intervals has been presented in Figure 23 Appendix-XIV A
significant increase (plt0001) in LWR was observed at 8th day of irrigation compared to
4th day intercropped Similar pattern was noticed in RWR however SWR showed
insignificant difference between 4th and 8th day of irrigation A slight increase in LWR was
noticed in intercropped plants compared to sole Whereas RWR declined in intercrop
compared to sole and SWR remains un-changed
iv Specific shoot (SSL) root length (SRL)
Specific shoot length (SSL) specific root length (SRL) of C cajan grown in two different
cropping system (sole and intercrop with Z mauritiana) in two different irrigation
intervals has been presented in Figure 23 Appendix-XIV SSL and SRL were observed
to increase in sole crop compared to intercrop at 4th day of irrigation However increase
SSL and SRL was recorded in intercropped compared to sole at 8th day of irrigation A
general decline in SSL and SRL was noticed in 8th day of irrigation compared to 4th day
75
v Plant moisture
The moisture content of C cajan plant grown in two different cropping system (sole and
intercrop with Z mauritiana) in two different irrigation intervals has been presented in
Figure 23 Appendix-XIV The moisture content of plants was decreased significantly
(plt005) at 8th day irrigation interval compared to 4th day in sole crop Whereas non-
significant increase was observe in intercrop plants at 8th day of water irrigation
vi Plant succulence
Succulence of C cajan plant grown in two different cropping system (sole and intercrop
with Z mauritiana) in two different irrigation intervals has been presented in Figure 23
Appendix-XIV A significant increase (plt001) was observed in intercropped plants of C
cajan compared to sole crop at both irrigation interval However succulence increased in
sole crop and decreased in intercrop plants at 8th day of irrigation compared to 4th day
vii Relative growth rate (RGR)
Relative growth rate (RGR) of C cajan plant grown in two different cropping system (sole
and intercrop with Z mauritiana) in two different irrigation intervals has been presented
in Figure 23 Appendix-XIV A significant increase in RGR was observed in 8th day
compared to 4th day in both the cropping systems Highest increase was observed in
intercropped at 8th day irrigation At 4th day irrigation intervals intercropped plants
showed better RGR compared to Sole crop
2229 Photosynthetic pigments
Photosynthetic pigments including Chlorophyll a chlorophyll b total chlorophyll
Chlorophyll ab ratio and carotinoids of C cajan plant grown in two different cropping
system (sole and intercrop with Z mauritiana) in two different irrigation intervals has been
presented in Figure 24 Appendix-XIV
i Chlorophyll contents
A significant increase (plt005) in Chlorophyll a b and total chlorophyll was observed in
intercrop plants at 8th day irrigation interval Whereas at 4th day irrigation interval Sole
76
plants showed better results as compare to intercrop plants Plants at 8th day significantly
increase chlorophyll a b and total chlorophyll compared to 4th day of irrigation
Interactions between cropping systems and irrigation intervals were found significant
(chlorophyll a (plt001) chlorophyll b (plt001) and total chlorophyll (plt0001)
respectively) However the ratio of chlorophyll ab showed non-significant values in
cropping irrigation interval and their interaction
ii Carotenoids
A significant increase (plt001) in carotinoids was observed in intercropped C cajan at 8th
day of irrigation Whereas non-significant increase was observed in sole crop at 4th day
irrigation interval with compare to intercrop However the irrigation intervals showed
significant (plt0001) difference Whereas interaction of cropping system with irrigation
time also showed significant correlation (plt0001)
22210 Electrolyte leakage percentage (EL)
Electrolyte leakage percentage (EL) of C cajan plant grown in two different cropping
system (sole and intercrop with Z mauritiana) in two different irrigation intervals has been
presented in Figure 25 Appendix-XIV A non-significant increase in EL percentage was
observed in sole crop compared to intercrop plants growing at 4th and 8th day of irrigation
No significant change was noticed between the irrigation times to C cajan The interaction
between cropping system (sole and intercropped) and irrigation interval (4th and 8th day)
also showed non-significant
22211 Phenols
Total phenolic contents in leaves of C cajan plant grown in two different cropping system
(sole and intercrop with Z mauritiana) in two different irrigation intervals has been
presented in Figure 25 Appendix-XIV A nonsignificant result was observed in total
phenolic contents of C cajan growing as sole and intercropped system at two different
irrigation intervals However the interaction between irrigation intervals with crop system
showed significant (p lt 005) results
77
22212 Proline
Total proline contents in leaves of C cajan plant grown in two different cropping system
(sole and intercrop with Z mauritiana) in two different irrigation intervals has been
presented in Figure 25 Appendix-XIV Proline contents in leaves of C cajan showed
nonsignificant increase at 4th day of irrigation interval in both sole and intercropped
system Whereas the interaction between irrigation intervals showed significant (Plt001)
results
22213 Protein and Sugars
Protein and sugar contents in leaves of C cajan plant grown in two different cropping
system (sole and intercrop with Z mauritiana) in two different irrigation intervals has been
presented in Figure 26 Appendix-XIV A less significant difference (plt005) was
observed in two different (4th and 8th day) irrigation intervals However there was
nonsignificant difference in two cropped system More decrease was observed at 4th day
intercropped plants Whereas nonsignificant increase in 8th day intercropped and 4th day
sole plants were observed However interaction between crop and time of irrigation
showed significant results (plt0001)
22214 Enzyme assay
Antioxidant enzymes like Catalase (CAT) Ascorbate peroxidase (APX) Guaiacol
peroxidase (GPX) Superoxide dismutase (SOD) and Nitrate reductase activity in leaf of
C Cajan plant grown in two different cropping system (sole and intercrop with Z
mauritiana) in two different irrigation intervals has been presented in Figure II27
Appendix-XIV
i Catalase (CAT)
A significant increase (plt001) in catalase activity was observed in intercropped C cajan
at 8th day of irrigation with compare to other irrigation time and cropped system Whereas
increase was observed in sole crop at 4th day irrigation interval with compare to 8th day
However the irrigation intervals and the interaction between cropping system with
irrigation interval also showed nonsignificant correlation
78
ii Ascorbate peroxidase (APX)
A non-significant increase in APX was observed in intercropped plant in 4th and 8th day
irrigation interval with compare to sole crops Sole crop at 8th day showed maximum
decline However the difference between cropping system and their interaction with
irrigation interval also showed nonsignificant results
iii Guaiacol peroxidase (GPX)
A significant increase (plt005) in GPX activity was observed in 8th day sole crop
However there was nonsignificant difference among intercropped at two time interval and
sole crop at 4th day irrigation Whereas interaction with time to irrigation interval also
showed less significant results
iv Superoxide dismutase (SOD)
A significant decrease (plt0001) in SOD activity was observed in intercropped at 8th day
irrigation interval with compare to 4th day Maximum decrease was observed in 8th day
intercropped Whereas sole crop at 8th day also showed better result to 4th day sole crop
However ANOVA showed significant correlation among crop system at two time interval
and 4th day irrigation
v Nitrate and Nitrate reductase
Nitrate content and activity of nitrate reductase was nonsignificant in both cropping
system using both irrigation intervals However nonsignificant increase was observed in
nitrate content and activity of nitrate reductase in intercropped Z mauritiana at 8th day
79
Sole IntercropSole Intercrop
No o
f le
aves
0
20
40
60
Len
gth
(cm
)
0
40
80
120
160
200
2404
th day
Cajanus cajan
a
RootShoot
ab
a
a
b
a
a
8th
day
Figure 21 Vegetative parameters of Z mauritiana and C cajan at grand period of growth under sole and
intercropping system at 4th and 8th day irrigation intervals (Bars represent means plusmn standard error
of each treatment and significance among the treatments was recorded at p lt 005)
Sole IntercropSole Intercrop
No of
leav
es
0
200
400
600
Len
gth
(cm
)
0
40
80
120
160
200
240
Ziziphus mauritiana
RootShoot
4th
day 8th
days
b b
a a
a
b
cc
80
Sole Intercrop
Dry
wei
ght
(g)
50
100
150
200
250
300
Fre
sh w
eight
(g)
100
200
300
400
500
Sole Intercrop
4th
day 8th
day
a
b
c
a
b b aa
b
b
c c
a
bc
a
c
ba
b
c
a
b
c
Leaf Stem Root
Ziziphus mauritiana
Sole Intercrop
Dry
wei
ght
(g)
2
4
6
8
10
12
Fre
ah w
eight
(g)
5
10
15
20
25
30
35
40
Sole Intercrop
4th
day 8th
day
aa
b
a
a
b
a
b
c
a
b
c
a
c
b
a a
b
a
b
c
a
b
c
Leaf Stem Root
Cajanus cajan
Figure 22 Fresh and dry weight of Z mauritiana and C cajan plants under sole and intercropping system
at 4th and 8th day irrigation intervals (Bars represent means plusmn standard error of each treatment
and significance among the treatments was recorded at p lt 005)
81
Figure 23 Leaf weight ratio (LWR) root weight ratio(RWR) shoot weight ratio(SWR)specific shoot
length (SSL) specific root length (SRL) plant moisture Succulence and relative growth rate (RGR) of
Zmauritiana and C cajan grow plants under sole and intercropping system at 4th and 8th
day irrigation
intervals (Bars represent means plusmn standard error of each treatment and significance among the treatments
was recorded at p lt 005)
Sole Intercrop
Mo
istu
re (
)
0
20
40
60
80
SS
L (
cm g
-1)
01
02
03
04
05
06
RW
R (
g g
-1 D
W)
005
010
015
020
LW
R (
g g
-1 D
W)
01
02
03
04
05
06
07
Sole Intercrop
Su
ccu
lan
ce
(g H
2O
g-1
DW
)00
05
10
15
20
25
RG
R
(g g
-1 d
ay-1
)
001
002
003
004
005
SR
L (
cm g
-1)
05
10
15
20
25
SW
R (
g g
-1 D
W)
02
04
06
08
10
Ziziphus mauritiana
a a
bb
b
a
bb
a
b
aa
a aa
b
a
bb
c
b
a
bb
b
aa a
ba
bc
4th day
8th day
82
(Figure 23 continuedhellip)
Sole Intercrop
Mo
istu
re (
)
0
20
40
60
80
SS
L (
cm g
-1)
2
4
6
8
10
12
RW
R (
g g
-1 D
W)
002
004
006
008
010
012
014
LW
R (
g g
-1 D
W)
01
02
03
04
05
06
07
08
Sole Intercrop
Su
ccu
lan
ce
(g H
2O
g-1
DW
)
00
05
10
15
20
25
RG
R
(g g
-1 d
ay-1
)
001
002
003
004
005
SR
L (
cm g
-1)
5
10
15
20
25
SW
R (
g g
-1 D
W)
02
04
06
08
10
Cajanus cajan
a aab
a aaa
a
bba
a
b b
c
a aab
a
bbb
abbb
aa
bc
8th day
4th day
83
Sole Intercrop
Car
oti
noid
s (m
g g
-1 F
W)
00
01
02
03
04
05
Ch
loro
phyll
(m
g g
-1 F
W)
00
03
06
09
12
15
Sole Intercrop
4th
day 8th
day
Ch
loro
phyll
ab
rat
io
00
05
10
15
20
25Chl ab
Ziziphus mauritiana
a a
bb
a
b
a
b
a ab
b
Chl aChl b
Figure 24 Leaf pigments of Zmauritiana and C cajan grow plants under sole and intercropping system at
4th and 8th
day irrigation intervals (Bars represent means plusmn standard error of each treatment and
significance among the treatments was recorded at p lt 005)
Sole Intercrop
Car
oti
noid
s (m
g g
-1 F
W)
00
01
02
03
04
05
Ch
loro
phyll
(m
g g
-1 F
W)
00
03
06
09
12
15
18
Sole Intercrop
4th
day 8th
day
ab r
atio
00
05
10
15ab
ab
Cajanus cajan
bb b
a
a
b
cc
bb b
a
84
Ele
ctro
lyte
lea
kag
e(
)
0
5
10
15
4th
day 8th
dayP
hen
ols
(m
g g
-1)
0
5
10
15
20
25
30
Sole Intercrop
Pro
line
( g g
-1)
0
10
20
30
40
Sole Intercrop
Ziziphus mauritiana
a a a
a
b b ba
a
b
c
d
Figure 25 Electrolyte leakage phenols and prolein of Z mauritiana and C cajan at grand period of growth
plants under sole and intercropping system at 4th and 8
th day irrigation intervals (Bars represent
means plusmn standard error of each treatment and significance among the treatments was recorded at
p lt 005)
85
(Figure 25 continuedhellip)
E
lect
roly
te l
eakag
e(
)
0
20
40
60
80
4th
day 8th
day
Phen
ols
(m
g g
-1)
0
2
4
6
8
10
12
Sole Intercrop
Pro
line
( g g
-1)
000
003
006
009
012
015
018
Sole Intercrop
Cajanus cajan
a aa
a
a a aa
aa a
a
86
Sole Intercrop
Sugar
s (m
g g
-1)
0
20
40
60
Sole Intercrop
Pro
tein
(m
g g
-1)
00
02
04
06
4th
day 8th
day
Ziziphus mauritiana
a aa a
a
a a a
Sole Intercrop
Sugar
s (m
g g
-1)
0
10
20
30
Sole Intercrop
Pro
tein
(m
g g
-1)
00
02
04
06
08
10
4th
day 8th
dayCajanus cajan
ab
a
c
a
b
cc
Figure 26 Total protein and sugars in leaves of Z mauritiana and C cajan plants under sole and
intercropping system at 4th and 8th
day irrigation intervals (Bars represent means plusmn standard
error of each treatment and significance among the treatments was recorded at p lt 005)
87
Sole Intercrop
SO
D (
Unit
s m
g-1
)
0
2
4
6
8
10
12
14
Sole Intercrop
Cat
alas
e (U
nit
s m
g-1
)
0
5
10
15
20
25
AP
X (
Unit
s m
g-1
)
0
20
40
60
80
GP
X (
Unit
s m
g-1
)
00
01
02
03
04
05
4th
day 8th
day
Ziziphus mauritiana
a
bc
c
a
b
cc
a
c
b
b
b bb
a
Figure 27 Enzymes activities in leaves of Z mauritiana and C cajan plants under sole and intercropping
system at 4th and 8th
day irrigation intervals (Bars represent means plusmn standard error of each
treatment and significance among the treatments was recorded at p lt 005)
88
(Figure 27 continuedhellip)
Sole Intercrop
SO
D (
Unit
s m
g-1
)
0
1
2
3
4
5
Sole Intercrop
Cat
alas
e (U
nit
s m
g-1
)
0
2
4
6
8
4th
day 8th
dayG
PX
(U
nit
s m
g-1
)
00
05
10
15
20
25
Cajanus cajan
aA
PX
(U
nit
s m
g-1
)
0
20
40
60
80
100
bb
b
aaa
b
a
bbb
a
c
a
b
89
Sole Intercrop
NO
3 (
mM
ol
g-1
)
00
02
04
06
08
10
12
14
8th
day
Sole Intercrop
Nit
rate
Red
uct
ase
(mM
ol
g-1
)
0
1
2
3
4
4th
day
Nitrate reductaseNO
3
Ziziphus mauritiana
a
b
c
cb
b
b
a
Sole Intercrop
NO
3 (
mM
ol
g-1
)
00
02
04
06
08
10
12
8th
day
Sole Intercrop
Nit
rate
Red
uct
ase
(mM
ol
g-1
)
0
2
4
6
8
10
12
4th
dayCajanas cajan
a
bb
b
aa
aa
Nitrate reductase NO3
Figure 28 Nitrate reductase activity and nitrate concentration in leaves of Z mauritiana and C cajan plants
under sole and intercropping system at 4th and 8th
dayirrigation intervals (Values represent means
plusmn standard error of each treatment and significance among the treatments was recorded at p lt
005)
90
23 Experiment No 8
Investigations of intercropping Ziziphus mauritiana with Cajanus cajan
on marginal land under field conditions
231 Materials and Methods
2311 Selection of plants
Ziziphus mautitiana and Cajanus cajan were selected for this study as described in chapter
1
2312 Experimental field
Field of Fiesta Water Park was selected to investigate intercropping of Z mauritiana with
Ccajan It is situated about 50 km from University of Karachi at super highway toward
HyderabadThe area of study has subtropical desert climate with average annual rain fall
is ~20 cmmost of which is received during the monsoon or summer seasonSince summer
temperature (April to October) are approx 30-35 degC and the winter months (November to
March) are ~20 degC Wind velocity is generally high all the year Topography of the area
was uneven with clay- loam soil having gravels Xerophytic plants are pre-dominantly
present in the area including Prosopis spp Acacia spp Euphorbia spp Caparus
deciduas etc
2313 Soil analysis
Before conducting experiment soil of Fiesta Water Park field was randomly sampled at
three locationsatone feet of depthusing soil augerThese soil samples were analyzed in
Biosaline Research Laboratory Department of Botany University of Karachi to
determine its physical and chemical properties
i Bulk density
Bulk density was determinedin accordance with Blake and Hartge (1986) by using the
following formula
Bulk density = Oven dried soil (g) volume of soil (cm3)
91
ii Soil porosity
Soil porosity was calculated in accordance with Brady and Weil (1996) by using the
following formula
Soil porosity = 1- (bulk density Particle density) times 100
Where particle density = 265 gcm3
iii Soil texture and particle size
Soil particle size was determined by Bouyoucos hydrometric method in accordance with
Gee and Or (1986)On the basis of clay silt and sand percentages soil texture was
determined by using soil texture triangle presented in Figure 31
iv Water holding capacity
Water holding capacity in percentages was calculatedaccording to George et al (2013)
v pH and Electrical conductivity of soil (ECe)
Soil saturated paste was made with de-ionized water and leave for 24 hours Soil solution
was extracted through Buckner funnel and suction pump (Rocker 300) pH of soil
solution was taken on Adwa AD1000 pHMV meter and ECe was taken on electrical
conductivity meter (4510 Jenway)
2314 Experimental design
Six months old grafted Ziziphus mauritiana saplings were carefully transported in field of
Fiesta Water Park
Three equal size plots of 100times10 sq ft were prepared for this experiment
Plot ldquoArdquo = Ziziphus mauritiana (Sole crop)
Plot ldquoBrdquo = Cajanus cajan (Sole crop)
Plot ldquoCrdquo = Ziziphus mauritiana + Cajanus cajan (intercropped)
In plot lsquoArsquo and lsquoCrsquo pits of two cubic feet depth were prepared in two parallel rows
at a distance of 10 feet (Yaragattikar amp Itnal 2003)so that the distance of pits within the
row and the distance of pits between the rows were same Each row bears nine pits
Eighteen healthy saplings of nearly equal height and vigor of Z mauritiana were
92
transplanted in the pits and were fertilized with cow-dong manure Plants were irrigated
with underground (pumped) water initially on alternate day for two weeks older leaves
fall down completely and new leaves appeared in this establishment period Later the
irrigation interval was kept fortnightly Electrical conductivity of irrigated water (ECiw)
was 24 plusmn 05 dSm-1
After establishment of Z mauritiana water soaked seeds of intercropping plant (C
cajan) were sown in plot lsquoCrsquo Three vertical lines (strips design) of equal distance were
made between the rows of Z mauritiana The distance between the line was one feet
Eleven C cajan were maintained in each line at a distance of one feet which constitute a
total of 33 C cajan in 3 lines There were 264 plants of C cajan arranged in strip pattern
as intercrop for eighteen Z mauritiana A sole crop of C cajan in plot lsquoBrsquo was arranged
with the same manner to serve as control Similarly plot lsquoArsquo was served as control of Z
mauritianaThe experiment was observed up to reproductive yield of each plant
Field diagram Theoritical model of intercropping system used in this study showing sole crop in Plot lsquoArsquo
(Z Mauritiana) and Plot lsquoBrsquo (C cajan) while Plot lsquoCrsquo represents intercropping of both
species at marginal land
Six Z mauritiana plants were randomly selected from their two rows of block lsquoCrsquo
which were facing two rows of C cajan on either sides Similarly ten plants of C cajan
facing Z mauritiana were randomly selected for further study At the same manner six Z
mauritiana from block lsquoArsquo and ten C cajan from block lsquoBrsquo grown as sole crop were
selected as control for further study
93
2315 Vegetative and reproductive growth
Vegetative growth of Z mauritiana plant was noted in terms of height volume of canopy
while height and number of branches in Ccajan bimonthly after establishment Fresh and
dry weightsof leaves stem and root were observed at final harvest in both plant species
growing as sole or intercropping
Reproductive growth of Z mauritiana such as number length and diameter fruit
weight per ten plant and average fruit yield was measured at termination of the experiment
Whereas reproductive growth in C cajan was monitored in terms of number of pods
number of seeds weight of pods and weight of seed
2316 Analyses on some biochemical parameters
Following biochemical analysis was conducted in Fully expended leavesof Z mauritiana
and C cajan growing as sole and as intercropped at grand period of growth Additionally
fruits of Z mauritiana were also analyzed for their protein soluble and insoluble sugars
and total phenolic contents
i Photosynthetic pigments
Photosynthetic pigments including chlorophyll a chlorophyll b and total chlorophyll were
estimated in leaves of Z mauritiana and C cajan according to procedure described in
chapter 1
ii Protein in leaves
Protein contents were estimated in leaves of Z mauritiana and C cajan according to
procedure described in chapter 1
iii Total soluble sugars in leaves
Total soluble sugars were estimated in leaves of Z mauritiana and C cajanaccording to
procedure described in chapter 1
94
iv Phenolic contents in leaves
Phenolic content were estimated in leaves of Z mauritiana and C cajan according to
procedure described in chapter 1
2317 Fruit analysis
i Protein in fruit
Protein content in fruit of Z mauritiana was estimated according to procedure described
in chapter 1
ii Total soluble sugars in fruits
Total soluble sugars in ripe fruits of Z mauritiana were estimated according to procedure
described in chapter 1
iii Phenolic contents in fruits
Phenolic contents in fruits of Z mauritiana were estimated according to procedure
described in chapter 1
2318 Nitrogen estimation
Nitrogen was also estimated in root zone soil as well as in fully expended leaves of Z
mauritiana and C cajan plants
Total nitrogen in leaves and soil was estimated through AOAC method 95504
(2005) One g of dried powdered sample in round bottle flask was digested in presence of
20 mL H2SO4 15 mL K2SO4 and 07g CuSO4 at 400oC heating mental After digestion 80
ml distilled water was added in digest Then distillation was done at 100oC by adding 100
mL of 45 NaOH (drop wise) in digested solution Steam was collected in 35 mL of 01M
HCl in a flask Three samples of 10 mL each steam collected solution were taken and 2-3
drops of methyl orange was added as indicator Titration was made with 01M NaOH
Changeappearance of color indicates the completion of reactionPercent nitrogen was
calculated through following equation
N = (mL of acid times molarity) ndash (mL of base times molarity) times 14007
95
2319 Land equivalent ratio and Land equivalent coefficient
The LER defined the total land area needed for sole crop system to give yield obtained
mixed crop It is mainly used to evaluate the performance of intercropping (Willey 1979)
Land equivalent ratio (LER) of two crops was estimated according to (Willey 1979) by
using formula
Whereas partial LER of Z mauritiana calculated according to
Similarly Partial LER of Ccajan were calculated as
Land equivalent coefficient (LEC) an assess of dealings the effectiveness of relationship
of two crops (Alhassan et al 2012) was calculated by using (Adetiloye et al 1983)
equation as
Yield was calculated in gram fresh weight LER and LEC of height and total chlorophyll
were also calculated by using above formula by substituting their values with yield (fruits
of Z mauritiana and seeds of C cajan) to height fruits and chlorophyll respectively
23110 Statistical analysis
Data were analyzed by using (ANOVA) and the significant differences between treatment
means wereexamined by least significant difference (Zar 2010) All statistical analysis
was performed using SPSS for windows version 14 and graphs were plotted using Sigma
plot 2000
LER= Yield of Z mauritiana + Yield of C cajan (in intercropped) + Yield of C cajan + Yield of Z mauritiana (in intercropped)
Yield of Z mauritiana (sole) Yield of C cajan (sole)
Partial LER = Yield of Z mauritiana + Yield of C cajan (in intercropped)
Yield of Z mauritiana (sole)
Partial LER = Yield of C cajan + Yield of Z mauritiana (in intercropped)
Yield of C cajan (sole)
LEC = Partial LER of Z mauritiana times Partial LER of C cajan
96
232 Observations and Results
2321 Vegetative parameters
Vegetative growth parameters of Z mauritiana include plant height volume of canopy
grown individually as well as intercropped with C cajan is presented in Figure 29
Appendix-XV A significant increase in height and canopy volume of Z mauritiana with
time (p lt 0001) and cropping system (p lt 005) was observed However the interaction
between time and cropping system showed non-significant results In general the
intercropped plants were showed higher values in all vegetative parameters than sole crop
and this increase was more pronounced after 60 days
Figure 29 Appendix-XVII showed the vegetative growth parameters of C cajan
including height and number of branches Height of C cajan was significantly increased
(plt0001) with increasing time in plants growing sole and as intercropped with Z
mauritiana The interaction with time to crop height also showed significant (plt0001)
results in both cropping systems However slight decline in height of intercropped C
cajan was noticed at 120 days compared to sole crop Number of branches was significant
increased (plt0001) in both crops with increasing time The interaction of time with
branches also showed significant (plt0001) results in both cropping systems However
number of branches was slightly increased in intercropped plants at 120 days compared to
sole crop
2322 Reproductive parameters
i Fruit number and weight (fresh and dry)
Reproductive parameters of Z mauritiana and C cajan at grand period of growth under
sole and intercropping system has been presented in Figure 210 Appendix-XVI XVIII
Individual and interactive effect of time (p lt0001) and treatment (plt001) on number and
fresh weight of fruits of Z mauritiana was showed significant results Similarly plants
grown with C cajan showed significant increase (p lt0001) in fresh weight of fruits (p
lt005) whereas fruit dry weight and circumference was non-significant in comparison to
sole crop
97
In C cajan flowers were appeared only at blooming phase (during 60 days of treatment)
and no difference in number of flowers was observed in both cropping systems (sole and
with Z mauritiana (Figure 210 XVII)
Leguminous pods were initiated soon after flowering period (during 60 days) and
last till end of the experiment (120 days) A significant increase (plt0001) in pod numbers
was observed with increasing time in both sole and intercropped system But non-
significant differences in number of pods of both cropping system and their interaction
with time were observed
Similarly number and weight of C cajan seeds were showed non-significant difference
in both cropping systems
2323 Study on some biochemical parameters
i Photosynthetic pigments
Leaf pigments of Zmauritiana and C cajan grow plants under sole and intercropping has
been presented in Figure 211 Appendix-XVI XVIII In Z muritiana leaves A significant
increase (plt005) in chlorophyll a chlorophyll b total chlorophyll and carotinoids was
observed when grown as intercrop whereas the effect on chlorophyll ab ratio was non-
significant as that of sole one
In C cajan a slight decrease (plt005) in chlorophyll lsquobrsquo and total chlorophyll
(plt001) was observed in intercropped plants compare to sole one Whereas chlorophyll
lsquoarsquo chlorophyll ab ratio and carotinoids showed nonsignificant difference between sole
and intercropped C cajan
ii Total proteins sugar phenols
Sugars protein and phenols in leaves of Z mauritianaand C cajan at grand period of
growth under sole and intercropping system is presented in Figure 212 Appendix-XVI
XVIII Total proteins and soluble and insoluble sugar content of Z mauritiana leaves was
unaffected throughout the experiment However an increase in total phenolic content
(plt001) was observed in intercropped Z mauritiana plants than grown individually
98
In C cajan total soluble sugars protein and phenols in leaves showed non-
significant differences between sole to intercropped plants
Sugars protein and phenols in fruits of Z mauritiana grown under sole and
intercropping system is presented in Figure 213 Appendix-XVI A non-significant
increase was observed in phenolic as well as in soluble insoluble and total sugar contents
in fruits of Z mauritiana plants grown with C cajan (intercrop) as compare to the fruits
of sole crop
2324 Nitrogen Contents
Nitrogen in leaves and in soil of Z mauritiana and C cajan growing under sole and
intercrop system is presented in Figure 214 Appendix-XVI XVIII ANOVA showed a
non significant effect on nitrogen content of leaf as well as root zone soil of Z mauritiana
and C cajan grown individually or as intercropping system
2225 Land equivalent ratio (LER) and land equivalent coefficient
(LEC)
Land equivalent ratio (LER) Land equivalent coefficient (LEC) of height chlorophyll and
yield of of Z 98auritiana and C cajan growing as sole and intercropping system in has
been presented in Table 22 The LER using height of both species was nearly 2 in which
PLER of Z mutitania was 48 and PLER of C cajan was 519 Whereas the calculated
values of the land equivalent coefficient (LEC) of Z mauritiana and C cajan remained
9994
The LER using yield of both species was above 2 in which PLER of Z mauritiana
was 46 Whereas PLER of C cajan was 543 However the calculated values of LEC
of both species were 100
The LER using total chlorophylls of both species were more than 25 in which
PLER of Z mauritiana was 344 and as that of PLER of C cajan was 655 Whereas
the calculated values of LEC was 999 of both the species
99
Table 21 Soil analysis data of Fiesta Water Park experimental field
Serial number Parameters Values
1 ECe (dSm-1) 4266plusmn0536
2 pH 8666plusmn0136
3 Bulk density (gcm3) 123plusmn0035
4 Porosity () 53666plusmn1333
5 Water holding capacity () 398plusmn2811
6 Soil texture Clay loam
7 Sand () 385plusmn426
8 Silt () 3096plusmn415
9 Clay () 305plusmn1
Ece is the electrical conductivity of saturated paste of soil sample
Figure 29 Soil texture triangle (Source USDA soil classification)
100
Ziziphus mauritiana
Days
0 60 120
Volu
me
(m3)
0
10
20
30
Days
0 60 120
Hei
ght
(cm
)
0
50
100
150
200
250
Sole Intercrop
a
a
bb
c c
aa
bb
c c
Cajanus cajan
Days
0 60 120
Bra
nch
es (
)
0
10
20
30
Days
0 60 120
Hei
ght
(cm
)
0
50
100
150
200
250
300
Sole Intercrop
aa
bb
c c
aa
bb
c c
Figure 210 Vegetative growth of Z mauritiana and C cajan growing under sole and intercropping
system (Bars represent means plusmn standard error of each treatment and significance among the
treatments was recorded at p lt 005)
101
Ziziphus mauritiana
Fresh Dry
Fru
it w
eig
ht
(g)
0
50
100
150
200
Days
0 60 120 180
Nu
mb
er o
f F
ruit
s
0
100
200
300
Sole Intercrop
a
b
a
b
c
c
dd
Cajanus cajan
0 60 120
Num
ber
of
Pods
0
50
100
150
200
Days
0 60 120
Num
ber
of
Flo
wer
s
0
50
100
150
Sole Intercrop
Days
aa
bb
c c
Sole Intercrop
Num
ber
of
See
ds
0
100
200
300
400
500
See
d W
eight
(g)
0
10
20
30
40
50
60Number of seedsSeed weight
Figure 211 Reproductive growth of Z mauritiana and C cajan growing under sole and intercropping
system (Bars represent means plusmn standard error of each treatment and significance among the
treatments was recorded at p lt 005)
102
Ziziphus mauritiana
Cajanus cajan
Figure 212 Leaf pigments of Zmauritiana and C cajan growing under sole and intercropping (Bars
represent means plusmn standard error of each treatment and significance among the treatments was
recorded at p lt 005)
Sole Intercrop
Car
ote
noid
s (m
g g
-1)
00
01
02
03C
hlo
rophyl
l (m
g g
-1)
00
02
04
06
08
ab r
atio
00
05
10
15
20
25
ab
ab
Sole Intercrop
Car
ote
no
ids
(mg
g-1
)
00
01
02
03
Ch
loro
ph
yll
(m
g g
-1)
00
02
04
06
08
10
ab
rat
io
0
1
2
3
4ab
ab
103
Ziziphus mauritiana
Sole Intercrop
Lea
f P
hen
ols
(m
g g
-1)
0
2
4
6
8
10
12
Lea
f P
rote
ins
(mg
g-1
)
0
2
4
6
8
Lea
f S
ug
ars
(mg
g-1
)
0
5
10
15
20
25
30
35SoluableInsoluable
Figure 213 Sugars protein and phenols in leaves of Z mauritiana and C cajan at grand period of growth under
sole and intercropping system (Bars represent means plusmn standard error of each treatment and
significance among the treatments was recorded at p lt 005)
104
(Figure 212 continuedhellip)
Cajanus cajan
Sole Intercrop
Lea
f P
hen
ols
(m
g g
-1)
0
2
4
6
8
Lea
f P
rote
ins
(mg g
-1)
00
05
10
15
20
Lea
f S
ugar
s (m
g g
-1)
0
2
4
6
8
105
Ziziphus mauritiana
Sole Intercrop
Fru
it P
hen
ols
(m
g g
-1)
0
2
4
6
8
10
12
14
Fru
it P
rote
ins
(mg g
-1)
00
02
04
06
08
10
Fru
it S
ugar
s (m
g g
-1)
0
5
10
15
20
25
30
35 SoluableInsoluable
Figure 214 Sugars protein and phenols in fruits of Z mauritiana grown under sole and intercropping
system (Bars represent means plusmn standard error of each treatment and significance among the
treatments was recorded at p lt 005)
106
Z mauritiana
Sole Intercrop
Nit
rogen
(
)
0
1
2
3
4
5
6
7 LeafSoil
Cajanus cajan
Sole Intercrop
Nit
rogen
(
)
0
1
2
3
4
5
6
7 LeafSoil
Figure 215 Nitrogen in leaves and in soil of Z mauritiana and C cajan growing under sole and intercrop
system (Bars represent means plusmn standard error of each treatment and significance among the
treatments was recorded at p lt 005)
107
Table 22 Land equivalent ratio (LER) and Land equivalent coefficient (LEC) with reference to height chlorophyll and yield of of Z mauritiana and C cajan growing
under sole and intercropping system
Plant species Parameters Formulated with
reference to Height
Formulated with
reference to Total
Chlorophyll
Formulated with reference to Yield
(fresh weight of Z mauritiana fruit
and seed of C cajan)
Z mauritiana Partial LER 1027 1666 1159
C cajan Partial LER 0950 0877 0993
Intercropped
Total LER 1977 2543 2152
Z mauritiana amp C cajan
(Sole and intercropped) LEC 0975 1461 1151
107
108
24 Discussion
Intercropping is a common practice used to obtain better yield on a limited area through
efficient utilization of given resources which may not be achieved by growing each crop
independently (Mucheru-Muna et al 2010) In this system selection of appropriate crops
planting rates and their spatial arrangement can reduce competition for light water and
nutrients (Olowe and Adeyemo 2009) In general increased growth (biomass height
volume circumference biomass succulence SSL SRL SSR LWR SWR RWR and
RGR) of each species is a good indicator of successful intercropping The SRL and SSL
measure the ratio between the lengths of root or shoot per unit dry weight of respective
tissues (Wright and Westoby 1999) The weight ratio of leaf stem and root to total plant
weight (LWR SWR and RWR) describes the allocation of biomass towards each organ to
maximize overall relative growth rate (RGR) which explains how plant responds to certain
type of condition (Reynolds and Antonio 1996) In this study height and canopy volume
of Z mauritiana and height and branches of C cajan were increased when grown together
in comparison to sole crop in field experiment (Figure 29) Whereas in drum pot culture
biomass generally the length of plant canopy volume number of leaves RGR LWR
SWR RWR SSL and SRL were either higher or unaffected in both species growing in
intercropping at 4th and 8th days intervals (Figure 21-23) Similar beneficial effects on
growth of other intercrops have also been reported under different conditions (Yamoah
1986 Atta-Krah 1990 Kass et al 1992 Singh et al 1997) Dhyani and Tripathi (1998)
observed increased height stem diameter crown width and timber volume of three
intercropped species than sole crop Bhat et al (2013) also revealed significant
improvement in annual extension height and spread in apple plants intercropped with
leguminous plants
The increased growth of both intercropped plants of this study was well reflected
by their biochemical parameters Leaf pigments like chlorophyll a chlorophyll b and total
chlorophyll were either higher or remained unaffected (Figure 211) in both intercropped
plants than sole crops of field experiments Whereas in drum pot culture chlorophyll
content (Figure 24) was higher only in intercropped C cajan (specially in 8th days) Bhatt
et al(2008) and Massimo and Mucciarelli (2003) also reported the increased accumulation
of chlorophyll a b and total chlorophylls in leaves of soybean and peppermint when
109
grown with their respective intercrops Our results are also in agreement with Liu et al
(2014) and Otusanya et al (2008) reported similar results in Lycopersican esculentum and
later in Capsicum annum as well Some other reports are also available which shows non-
significant effect on leaf pigments in both cropping systems (Shi-dan 2012 Luiz-Neto-
Neto et al 2014)The synthesis and activity of chlorophyll depends on severity and type
of applied stress it generally increase in low saline mediums (Locy et al 1996) or
remained unaffected however sometimes stimulated (Kurban et al 1999 Parida et al
2004 Rajesh et al 1998)
Proteins and carbohydrates (sugars) perform vast array of functions which are
necessary for plant growth and reproduction (Copeland and McDonald 2012) Variation
in their contents helps to predict plant health which is usually decreased with applied stress
(Arbona et al 2013) Both are also the compulsory factors of animals diet since they
cannot manufacture sugars and some of the components of proteins which must be
obtained from food (Bailey 2012) In our experiment protein content was either remained
unchanged or increased which indicated a good coordination of both intercrops in field
and drum pot experiments (Figure 26 and 212) Liu et al (2014) also found that protein
and sugars were not affected in tomatogarlic intercrops In another experiment similar
results were found when corn was grown with and without intercropping (Borghi et al
2013)
Reactive oxygen species (ROS) are produced as a spinoff of regular metabolism
however under stress the overproduction of ROS may lead to oxidative damage (Baxter et
al 2014) In low concentrations ROS worked as messengers to regulate several plant
processes and also helps to improve tolerance to various biotic and abiotic stresses (Miller
et al 2009 Nishimura and Dangl 2010 Suzuki et al 2011) but when the concentration
goes beyond the critical limit ROS would become self-threatening at every level of
organization (Foreman et al 2003) To maintain a proper workable redox state an
efficient scavenging system of enzymatic (SOD CAT GPX and APX) andor non-
enzymatic (polyphenols sugars glutathione and ascorbic acid) antioxidants is required
which would be of critical importance when plant undergoes stress (Sharma et al 2012)
Among these enzymes SOD is a first line of defense which converts dangerous superoxide
radicals into less toxic product (H2O2) In further CAT APX and GPX worked in
association to get rid off from the excessive load of other oxygen radicals or ions (H2O2
110
OH- ROO etc) In this study antioxidant enzymes (SOD CAT GPX and APX) were
found to work in harmony which was not affected during 4th day treatment in both species
in comparison to sole crop (Fig 27) showing strong antioxidant defense which was not
compromised by cropping system When comparing in 8th day treatment a significant
general increase in all enzyme activities were observed in both species except for SOD
and GPX of C cajan (Fig 27) These results displayed relatively better performance and
tight control over the excessive generation of ROS which would be predicted in this case
due to less availability of water than in 4th day treatment (Karatas et al 2014 Doupis et
al 2013) Similarly by coping oxidative burst and maintaining cellular redox equilibrium
plants were able to improve growth performance especially in Z mauritiana (Fig 21)
Water deficit affect stomatal conductance which could bring about changes in
photosynthetic performance hence overproduction of ROS is usually found among
different crops (Moriana et al 2002 Miller et al 2010) As a response tolerant plants
overcome this situation by increased activity of antioxidant enzymes which was evident in
Wheat Rice olive etc (Zhang and Kirkham 1994 Sharma and Dubey 2005 Guo et al
2006 Sofo et al 2005)
Phenolic compounds despite their role in physiological plant processes are
involved in adsorbing and neutralizing reactive oxygen species (ROS Ashraf and Harris
2004) The overproduction of ROS may cause several plant disorders Plants produce
secondary compounds like polyphenols to maintain balance between ROS generation and
detoxification (Posmyk et al 2009) Increased synthesis and accumulation of phenolic
compounds is reported to safeguard cellular structures and molecules especially under
biotic abiotic constraints (Ksouri et al 2007 Oueslati et al 2010) In this study
intercropped Z mauritiana of field and both species in drum pot culture showed higher
phenolic content than individual crop (Figure 25 and 212) which may be attributed to
adaptive mechanism for scavenging free radicals to prevent cellular damage (Rice-Evans
1996)
In terms of fruit yield we observed that Z mauritiana is suitable for intercropping
as suggested by Yang et al (1992) Number of flowers fruits and fruit fresh weight of
both species either increased considerably or no-affected in intercropped plants compared
to individual ones (Figure 210) Moreover fruit quality of Z mauritiana includes proteins
phenols and soluble extractable and total sugars were also higher in intercropped plants
111
(Figure 213) Results of this study are better than other experiments reported by
Sharma (2004) Kumar and Chaubey (2008) and Kumar et al (2013) who did not find
influence of other understory forage crops (like Aonla) on the yield of Z mauritiana
However in other case the yield of intercropped ber was some time higher (Liu 2002)
Singh et al 2013 found no adverse effects on the yield of pigeonpea when intercropped
with mungbean however it improved the grain yield of associated species
A leguminous plant C cajan is used in this experiment as secondary crop which
can supplement Z mauritiana by improving soil fertility Results of both experiments
showed that the nitrogen was higheror un-affected (Figure 214) in soils of intercropped
plants which supports our hypothesis that leguminous intercrop increase N supply This
can be achieved by acquisition of limited resources to manage rootrhizosphere
interactions which can improve resource-use efficiency (Zhang et al 2010
Shen et al 2013 White et al 2013b Ehrmann and Ritz 2014 Li et al 2014) As a
consequence it impact on overall plant performance which starts from high photosynthetic
activity by increasing chlorophyll results in more availability of photoassimilate for
growth and reproductive allocation (Eghball and Power 1999) Use of C cajan in tree
intercropping proved beneficial for producing high yield crops and for the environment
(Gilbert 2012 Glover et al 2012)
Land equivalent ratio (LER) is commonly used to evaluate the effectiveness of
intercropping by using the resources of same environment compared with sole crop
(Vandermeer 1992 Rao et al 1990 1991 Cao et al 2012) It is the ratio of area for sole
crop to intercrop required to produce the equal amount of yield at the same management
level (Mead and Willey 1980 Dhima et al 2007) On the other hand land equivalent
coefficient (LEC) describe an association that concern with the strength of relationship It
is the proportion of biomassyield of one crop explained by the presence of the other crop
The LER 1 or more indicate a beneficial effect of both species on each other which increase
the yield of both crops as compare to single one (Zada et al 1988) In this experiment all
LER values were about 2 or more than 2 while LEC values were around 1 or more than
one in ZizyphusCajnus intercropping Both LER and LEC values were in descending
order of chlorophylls gt yield gt height (Table 22) However the partial LER was higher in
Zizyphus than Cajanus in all cases These results describe the superiority of intercropping
over sole cropping where LER values are even gt2 Some other studies reported LER from
112
09-14 (Bests 1976) 12-15 (Cunard 1976) and up to 2 (Andrews and Kassam 1976)
Similar results were reported in poplarsoybean system (Rivest et al 2010) black
locustMedicago sativa (Gruenewald et al 2007) wheatjujube (Zhang et al 2013)
Acacia salignasorghum (Droppelmann et al 2000 Raddad and Luukkanen 2007) The
high LER values in our system indicating a harmony in resource utilization in both species
which was also corroborated with their respective LEC values The greater LEC values (gt
025) suggesting an inbuilt tendency of studied crops to give yield advantage (Kheroar and
Patra 2013) Experiments based on traditional practices of growing legumes with cereals
demonstrated greater and continuous cash returns than individual-crops (Baker 1978) In
addition the same authors found further increase in cash returns by increasing the
proportion of cereal and incorporating maize with sorghum and millet In agreement with
our findings similar reports are also available from different intercropping systems
including sesamegreengram (Mandal and Pramanick 2014) maizeurdbean (Naveena et
al 2014) and pegionpeasorghum (Egbe and Bar-Anyam 2010)
After detailed investigations of both species using two different experiment designs
(drum pot and field) it is evident that intercropping had beneficial effects on growth
physiology biochemisty and yield of both species Furthermore by using this system
higher outcome interms of edible biomass and green fodder using marginal lands can be
obtained in a same time using same land and water resources which can help to eliminate
poverty and uplift socio-economic conditions
113
3 Chapter 3
Investigations on rang of salt tolerance in Carissa carandas
(varn karonda) for determining possibility of growing at waste
saline land
31 Introduction
Carissa carandas commonly known as Karonda or lsquoChrist thornrsquo belonging to family
Apocynaceae shows capability of growing under haloxeric conditions It is an important
plant which has established well at tropical and subtropical arid zone under high
temperatures It is large evergreen shrub and having short stem It has fork thorn and hence
used as hedges or fence around fields The leaves are oval or elliptic 25 to 75 cm long
dark green leathery and secrete white milk if detached The fruits are oblong broad- ovoid
or round 125- 25 cm long It has thin but tough epicarp Fruits are in clusters of 3-10
Young fruits are pinkish white and become red or dark purple on maturation
The plant is propagated through seed in August and September Budding and cutting
could also be undertaken Planting is started after first shower of monsoon Plants raised
from seeds are able to flower within two years Flowering starts in March and fruit ripen
from July to September (Kumar et al 2007) The fruit possess good amount of pectin and
acidity hence used in prickle jelly jam squash syrup and in chutney by the commercial
name lsquoNakal cherryrsquo (Mandal et al 1992) They are rich in vitamin C and good source
of Anthocyanin (Lindsey et al 2000) Its fruits also are one of the richest source of iron
(391 mg 100gm) (Tyagi et al 1999) Juice of its root is also used to treat various
microbial diseases such as diarrhea dysentery and skin disease (Taylor et al 1996)
Hence its range of salt and suitability for cultivation at waste saline land or with saline
water irrigation is being undertaken for commercial exploitation by preparing jams jellies
and prickles (Kumar 2014) Investigations on its growth and development at higher range
of salinities are being undertaken with an interest to cultivate it if profitable at highly saline
waste land
114
32 Experiment No 9
Investigation on the effect of higher range of salinities on growth of
Carissa carandas (varn karonda) created by irrigation of different
dilutions of sea salt
321 Materials and methods
3211 Drum Pot Culture
Drum pot culture as recommended by Boyko (1966) and modified by Ahmed and
Abdullah (1982) was used for the present investigation which was been already described
in Chapter 1 earlier
3212 Plant material
About six months old sapling of Carissa carandas (varn Karonda) having almost equal
height and volume poted in polythene bag in 3kg of soil fertilized with cow-dong manure
were purchased from the Noor nursery Gulshan-e-Iqbal Karachi Sindh and were
transported to the Biosaline research field department of Botany University of Karachi
3213 Experimental setup
Plants were transplanted in drum pot (Homemade lysimeter) filled with sandy loam mixed
with cow dung manure (91) Each drum pot was irrigated weekly during summer and
fortnightly during winter months with 20 liters tap water (Eciw= 0 6 dSm-1) or water of
sea salt concentrations of various ie 03 (Eciw = 42 dSm-1) 04 (Eciw =61 dSm-1)
06 (Eciw = 99 dSm-1) and 08 (Eciw = 129 dSm-1) The plants were established initially
by irrigation with tap water for two weeks and later salinity was gradually increased till
desired percentage is achieved for different treatments by dessolving of sea salt in
irrigation water Three replicates were maintained for each treatment Urea DAP and
KNO3 were the source of NPK provided in the ratio 312 50g granules Osmocot (Scotts-
Sierra Horticulture Products) and 50g Mericle-Gro (Scotts Miracle-Gro Products Inc)
were dissolved in irrigation water per drum after six months at six monthly intervals
Height and volume of canopy of these plants were recorded prior to the starting the
experiment and then after every six months interval
115
Since the vegetative growth performance in plants irrigated with 03 sea salt (Eciw = 42
dSm-1) was found comparatively better than control and only 26 decrease was noticed
in volume of canopy at plant irrigated with 04 sea salt (Eciw = 61 dSm-1) (Table III41)
the onward investigations were focused at higher salinity levels and plants were irrigated
with 06 (Eciw = 99 dSm-1) and 08 (Eciw = 129 dSm-1) sea salt in rest of experiment
3214 Vegetative parameters
Vegetative growth on the basis of plant height and volume were recorded while
reproductive growth was observed on the basis of number of flowers and number and
weight of fruits per plant Length and diameter of fruit were also recorded in ten randomly
selected fruits
3215 Analysis on some biochemical parameters
Following biochemical analysis of leaves was performed at grand period of growth (onset
of flowers)
i Photosynthetic pigments
Fresh fully expended leaves (01g) was crushed in 80 chilled acetone Further procedure
was followed described in chapter 1
ii Soluble sugars
Dry leaf samples (01g) were milled in 5mL of 80 ethanol and were centrifuged at 4000
g for 10 minutes Same procedure was followed as described in chapter 1
iii Protein content
The protein contents were measured according to Bradford Assay reagent method against
Bovine Serum Albumin which was taken for standard (Bradford 1976) as described in
chapter 1
iv Soluble phenols
The dried leaf powder (01g) was milled in 3ml of 80 methanol and was centrifuged at
10000g for 15 min Further procedure has been described in chapter 2
116
3216 Mineral Analysis
Estimation of Na+ and K+ were made according to Chapman and Pratt (1961) Oven dried
grinded Leaves (1g) furnace at 550ordmC for 6 hours and were digested in 5 ml of 2N HCl
Diluted and filtered solution was used to estimated Na+ and K+ in flame photometer
(Petracourt PFP I) The concentration of these ions was calculated against the following
standard curve equations
Na+ (ppm) = 0016135x1879824
K+ (ppm) = 0244346x1314603
117
322 Observations and Result
3221 Vegetative parameters
Vegetative growth in terms of height and volume of canopy of C carandas growing under
salinities created by irrigation of different dilutions of sea salt is presented in Table 32
Appendix-XIX A significant increase (plt0001) in plant height and volume of canopy
was observed with increasing time but the increase was rapid at early period of growth
However there was significant (plt0001) reduction under salinity stress The interaction
of time and salinity also showed significant (plt001) effect on plant parameters but the
increase in height and volume of canopy at Eciw= 42dSm-1of sea salt salinity was more
than control Plants irrigated with Eciw= 61 dSm-1 and Eciw= 99 dSm-1sea salt solution
showed decrease in height with respect to control but the difference between their
treatments was insignificantly higher decrease was observed in Eciw= 129 dSm-1 sea salt
irrigated plants
3222 Reproductive parameters
Reproductive growth in terms of flowers and fruits numbers flower shedding percentage
fresh and dry weight of ten fruit their length and diameter under salinities created by
irrigation of different dilutions of sea salt is presented in Table 33 Appendix-XX Number
of flowers and fruits significantly (plt0001) decreased with increasing salinity treatment
Difference in flower initiation seems non-significant at early growth period in controls and
salinity treatments However drastic decrease was observed in plants irrigated beyond
Eciw= 99 dSm-1 with increase in salinity
Flowers shedding percentage (Table 33 Appendix-XX) show an increase directly
proportional with increase in salinity however the difference in number of flowers
between the plants irrigated with Eciw= 99 dSm-1 and Eciw= 129 dSm-1 sea salt solution
is of little significance level (plt001)
Fresh and dry weight of average fruits (plt001) and their diameter (plt001) showed
decrease with increasing salinity whereas diameter and length of fruits showed non-
significant difference
118
3224 Study on some biochemical parameters
i Photosynthetic Pigments
Photosynthetic Pigments including Chlorophyll a chlorophyll b total chlorophyll
chlorophyll a b ratio and carotenoids of C carandas growing under salinities created by
irrigation of different dilutions of sea salt is presented in Figure 31 Appendix-XX The
chlorophyll contents of leaves significantly decreased (plt0001) over control with
increasing salinity however Chlorophyll rsquobrsquo at Eciw= 99 dSm-1salinity shows significant
increase (plt0001) over control Similarly Carotenoids at Eciw= 99 dSm-1 salinity show a
bit less significant increase (plt001) compare to control while at higher salinity (Eciw=
129 dSm-1) the decline is observed at all above mentioned parameters
iii Protein Sugars and phenols
Some biochemical parameters including Protein sugars and phenolic contents of C
carandas growing under salinities created by irrigation of different dilutions of sea salt is
presented in Figure 31 Appendix-XX Soluble proteins in leaves show non-significant
decrease at Eciw= 99 dSm-1salinity as compared with controls but a significant decrease
(plt005) was noted at Eciw= 129 dSm-1 salinity Sugars also showed non-significant
decrease at both the salinity whereas on contrary soluble phenols showed significant
increase (plt0001) with increasing salinity
3225 Mineral analysis
Mineral analysis including Na and K ions performed in leaves of C carandas growing
under salinities created by irrigation of different dilutions of sea salt is presented in Figure
32 Appendix-XX Sodium significantly increased (plt0001) all the way with increasing
salinity of growth medium Whereas significant decrease (plt0001) was observed in
Potassium with increasing salinity K+Na+ ratio show continuous increase with increasing
salinity
119
Table 31 Electrical conductivities of different sea salt concentration used for determining
their effect on growth of C carandas
Treatment
Sea salt ()
ECiw of irrigation water (dSm-1) ECe of soil saturated paste
(dSm-1)
Non-saline control 06 09
03 42 48
04 61 68
06 99 112
08 129 142
Whereas ECiw and ECe are the electrical conductivities of irrigation water and soil saturated past measured in deci semen per meter
120
Table 32Vegetative growth in terms of height and volume of canopy of C carandas growing under salinities created by irrigation of different dilutions of
sea salt
Treatment
Sea salt
(ECiw dSm-1)
Initial values prior to
starting saline water
irrigation
Growth at different salinities after 06 months
Height Volume Height Volume of canopy
cm m3 cm
increase
over initial
values
increase
decrease over
control
m3 increase over
initial values
increase
decrease
over control
Control 3734plusmn455 0029plusmn0001 8227plusmn4919 5363plusmn830 - 014plusmn0015 7952plusmn269 -
42 3674plusmn1415 0026plusmn0003 9930plusmn6142 6280plusmn205 +1710 019plusmn0017 8593plusmn098 +806
61 3752plusmn1243 0026plusmn0001 6490plusmn5799 4132plusmn485 -2305 012plusmn0010 7740plusmn117 -282
99 3819plusmn4499 0028plusmn0005 5793plusmn5821 3123plusmn1446 -4185 009plusmn0008 6759plusmn377 -1499
129 3676plusmn3114 0026plusmn0008 5250plusmn4849 2775plusmn1276 -4836 006plusmn0005 5690plusmn1110 -2844
LSD0 05
Salinity
Time Fisherrsquos least significant difference
91
172
002
0005
Values represent means plusmn standard error of each treatment and significance among the treatments was recorded at p lt 005
120
121
Table 33 Vegetative growth in terms of height and volume of canopy of C carandas growing under salinities
created by irrigation of different dilutions of sea salt
Treatment
Sea salt
(ECiw dSm-1)
Growth at different salinities after 12 months
Height Volume of canopy
cm
increase
over initial
values
increase
decrease over
control
m3
increase
over initial
values
increase
decrease over
control
Control 16214 plusmn633 7674plusmn307 - 077plusmn012 9689plusmn449 -
99 9736plusmn1048 6056plusmn561 -2109 034plusmn006 9367plusmn412 -333
129 6942plusmn565 4741plusmn480 -3822 022plusmn002 9064plusmn623 -645
Table 33 continuedhellip
Treatment
Sea salt
(ECiw= dSm-1)
Growth at different salinities after 18 months
Height Volume of canopy
Cm
increase
over initial
values
increase
decrease over
control
m3
increase
over initial
values
increase
decrease over
control
Control 1676plusmn1135 7776plusmn756 - 094plusmn011 9701plusmn578 -
99 10547plusmn842 6351plusmn666 -1833 045plusmn010 9445plusmn1024 -264
129 7581plusmn593 5154plusmn716 -3372 030plusmn003 9318plusmn580 -395
Table 33 continuedhellip
122
Table 33 continuedhellip
Treatment
Sea salt
(ECiw= dSm-1)
Growth at different salinities after 24 months
Height Volume of canopy
Cm
increase
over initial
values
increase
decrease over
control
m3
increase
over initial
values
increase
decrease over
control
Control 1911plusmn6
05 8055plusmn941 - 121plusmn015 9837plusmn522 -
99 1110plusmn5
31 6557plusmn543 -1859 053plusmn002 9509plusmn1032 -334
129 8754plusmn10
67 5990plusmn801 -2564 040plusmn008 9287plusmn745 -560
Table 33 continuedhellip
Treatment
Sea salt
(ECiw= dSm-1)
Growth at different salinities after 30 months
Height Volume of canopy
Cm
increase
over initial
values
increase
decrease over
control
m3
increase
over initial
values
increase
decrease over
control
Control 2052plusmn1126 8182plusmn676 - 146plusmn029 9873plusmn729 -
99 11700plusmn816 6743plusmn610 -1759 070plusmn011 9565plusmn850 -312
129 9628plusmn552 6189plusmn573 -2436 050plusmn004 9417plusmn1011 -462
LSD0 05 Salinity 77 007
Time 168 016
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and significance among the treatments was recorded at p lt 005
123
Table 34 Reproductive growth in terms of flowers and fruits numbers flower shedding percentage fresh and dry weight of ten fruit and their totals
perplant fruit length and diameter of C carandas growing under salinities created by irrigation of different dilutions of sea salt
Treatment
Sea salt
(ECiw= dSm-1)
Flower Fruits Flower
shedding
Weight of
Ten
fruit(fresh)
Weight of
Ten
fruit(dry)
Weight of
total fruitplant
(fresh)
Weight of
total fruitplant
(dry)
length
fruit
diameter
fruit
Numbers Numbers g g g g mm mm
Control 19467plusmn203 16600plusmn231 1468plusmn208 2282plusmn022 605plusmn009 37891plusmn891 10047plusmn283 1800plusmn003 1423plusmn006
99 12050plusmn202 7267plusmn491 3980plusmn307 1880plusmn035 530plusmn029 13695plusmn1174 3880plusmn469 1732plusmn037 1297plusmn011
129 12567plusmn549 6967plusmn203 4449plusmn082 1541plusmn023 435plusmn026 10742plusmn470 3041plusmn268 1711plusmn015 1233plusmn038
LSD0 05 Salinity 1514 1417 929 115 097 3785 1494 0971 097
Fisherrsquos least significant difference
Values represent means plusmn standard error of each treatment and significance among the treatments was recorded at p lt 005
123
124
Sea Salt (ECiw
= dSm-1
)
Cont 99 129
Car
ote
nio
ds
(mg
g-1
)
00
01
02
03
04
Ch
loro
ph
yll
(m
g g
-1)
00
01
02
03
04
05
06
ab
rat
io
00
05
10
15
20
25
30
35
ab
Chl a Chl b
a
a
a a
b
bcbc
a
b
c
a a
b
Figure 31 Chlorophyll a chlorophyll b total chlorophyll chlorophyll a b ratio carotenoids contents of C
carandas growing under salinities created by irrigation of different dilutions of sea salt (Bars
represent means plusmn standard error of each treatment and significance among the treatments was
recorded at p lt 005)
125
Sea Salt (ECiw
= dSm-1
)
Cont 99 129
Ph
eno
ls (
mg
g-1
)
0
5
10
15
20
Pro
tein
s (m
g g
-1)
0
1
2
3
4
Su
gar
s (m
g g
-1)
0
30
60
90
120
150Soluble Insoluble
a
a
a
a
a
a
b
b
b
c
ab
a
a
b
Figure 32 Total protein sugars and phenolic contents of C carandas growing under salinities created by
irrigation of different dilutions of sea salt (Bars represent means plusmn standard error of each treatment
and significance among the treatments was recorded at p lt 005)
126
Sea Salt (ECiw
= dSm-1
)
Cont 99 129
Ions
(mg
g-1
DW
)
0
20
40
60
80
100
120
KN
a ra
tio
00
01
02
03
04
05
06
07
Na K KNa
c
a
b
b
a
c
a
b
c
Figure 33 Mineral analysis including Na and K ions was done on leaves of C carandas growing under salinities
created by irrigation of different dilutions of sea salt (Bars represent means plusmn standard error of each
treatment and significance among the treatments was recorded at p lt 005)
127
33 Discussion
The volume and height of plants were increased per unit time under saline conditions This
increase was observed after six months in 03 sea salt (ECiw = 42 dSm-1) treated plants in
comparison to control (Table 32) Slight decrease was observed at 04 sea salt
(ECiw=61dSm-1) irrigation after which (Eciw= 99 dSm-1 and Eciw = 129 dSm-1sea salt) the
growth was significantly inhibited (Table 33) Noble and Rogers (1994) also noticed a general
decrease in growth of some of the glycophytes Humaira and Ahmad (2004) and Rivelli et al
(2004) also reported a proportional decrease in height of canola with increasing salinity
Cotton plants irrigated with saline water or those grown at saline soil are reported to increase
Na+ content in leaves accompanied by significant reduction in vegetative biomass (Meloni et
al 2001) Bayuelo-Jimenez et al (2003) observed salt induced growth inhibition of tomato
plant which was higher in shoot than root
Reproductive growth in terms of number of flowers number of fruits fruit length and
diameter were decreased and percent flower shedding increased with increasing salinity
(Table 34) These effects were higher at Eciw= 99 dSm-1and then maintained with further
salinity increment However weight of fruits (fresh and dry) and total fruits per plant were
linearly decreased with increasing medium salt concentrations A decrease in different phases
of reproductive growth like flowering fertilization fruit setting yield and quality of seeds etc
are reported to be seriously affected at different level of salinity by various workers (Lumis et
al 1973 Waisel 1991 Shannon et al 1994 Tayyab et al 2016) Cole and Mclead (1985)
and Howie and Lloyd (1989) reported severe effects of different salinity treatments on
flowering intensity fruit setting and number of fruits of Citrus senensis Walker et al (1979)
also reported reduction in the fruit weight during early ripening stage of Psidium guajava
Decrease in fruit diameter of strawberries (Fragaria times ananassa) has been reported with
salinity (Ehlig and Bernstein 1958)
In this study photosynthetic pigments of C carandas were decreased with salinity and
this decrease was more sever at Eciw = 129 dSm-1sea salt salinity (Figure 31) Such a decline
in amount of leaf pigments across different salinity regimes was also reported in cotton
(Ahmed and Abdullah 1979) Pea (Hernandez et al 1995 and Hernandez et al 1999) Vicia
128
faba (Gadallah 1999) Mulberry genotype (Agastian et al 2000) and B parviflora (Parida et
al 2004)
Leaf sugars and protein were decreased in both salinity levels (Figure 32) which could
be attributed to inhibition in transport of photosynthetic product (Levit 1980) Decrease
synthesis and mobilization of glucose fructose and sucrose has been demonstrated in number
of plants growing under salt stress (Kerepesi and Galiba 2000) Inhibition in the protein and
nucleic acid synthesis in Pisum sativum and Tamarix tetragyna plants were also reported by
Bar-Nun and Poljahoff-Mayber (1977) Melander and Harvath (1977) suggested that salt
induced reduction in protein is due to increase in protein hydrolysis
A significant increase in leaves phenol with increase in salinity (Figure 32) was
observed in present investigation was also demonstrated previously in Achilleacollina (Giorgi
et al 2009) Lactuca sativa (Kim et al 2008) and B parviflora (Parida et al 2004)
Inspite of over irrigation of saline water and maintaining leaching fraction of about
40 in drum pots accumulation of salts in rhizosphere soil was not completely avoided which
was evident in the differences between ECiw and ECe values (Table 31) Deposition of salts
in rhizosphere soil interferer absorption of minerals in plants For instance leaf Na+ content
of C carandas was significantly increased while K+ decreased with increasing soil salinity
(Figure 33) Over accumulation of toxic ions disturbed plant water status which directly
affects plant growth (Flowers et al 1977 Greenway and Munns 1980) A negative
relationship between Na+ and K+ concentration in roots and leaves of guava was also reported
by Ferreira et al (2001) Increase in Na+ content decreased K+ availability and K+Na+ ratio
in Vicia taba (Gadallah 1999) and also affect the uptake of other essential minerals in
Casurina equsetifolia (Dutt et al 1991)
Carissa carandas found to be a good tolerant to salinity and drought and it can produce
edible fruits from marginal lands of arid areas Fruits of this species can be consumed in a raw
form as well as in industrial products like pickles jams jellies and marmalades
129
4 Conclusions
In the light of above mentioned investigations it appears that pre-soaking treatment of Cajanus
cajan seeds has initiated metabolic processes at faster rate earlier which has helped seeds to
start germinative metabolism prior to be effected by toxic Na+ ions at higher salinities Cajanus
cajan and Ziziphus mauritiana were found to be the good companions for intercropping These
species synergistically enhanced the growth and biochemical performance of each other by
improving fertility of marginal land and maintaining harmony among different physiological
parameters which was missing in their sole crop Their intercropping could produce fodder
and delicious fruits even from under moderately saline substrate up to profitable extant
Carissa carandas also tolerated low and moderately salinities well by adjusting proper
regulation of physiological and biochemical parameters of growth It can provide protein rich
edible fruits jams jellies and pickles of commercial importance for benefit of poor farmer
from moderately saline barren land
130
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Waisel Y (1991) The glands of Tamarix aphylla a system for salt recreation or for carbon
concentration Physiol Plant 83 506ndash510
Walker RR PE Kriedemann and DH Maggs (1979) Growth leaf physiology and fruit
development in salt-stressed guavas Aus J of Agric Res 30 477 ndash 488
Wardill T J GC Graham M Zalucki WA Palmer J Playford and KD Scott (2006)
The importance of species identity in the biocontrol process identifying the subspecies
of Acacia nilotica (Leguminosae Mimosoideae) by genetic distance and the
implications for biological control J Biogeograph 32 2145-2159
White PJ and DJ Greenwood (2013) Properties and management of cationic elements
for crop growth Soil Cond Plant Growth 160-194
White PJ and DJ Greenwood (2013b) Properties and management of cationic elements
for crop growth In PJ Gregory and S Nortcliff (Eds) Soil conditions and plant
growth Oxford UK Blackwell Publishing pp 160ndash194
165
White PJ TS George PJ Gregory AG Bengough PD Hallett and
BM McKenzie (2013a) Matching roots to their environment Ann Bot 112 207ndash
222
Willey RW (1979) Intercropping its Importance and Research Needs Part 1 Competition
and Yield Advantages Vol-32
Wojtkowski PA (2006) Introduction to agroecology principles and practices Food
Products Press
Wright IJ and M Westoby (1999) Differences in seedling growth behaviour among species
trait correlations across species and trait shifts along nutrient compared to rainfall
gradients J Ecol 87 85ndash97
World Bank (2006) Managing food price risks and instability in an environment of market
liberalization World Bank Washington DC
Xu BC FM Li and L Shan (2008) Switchgrass and milkvetch intercropping under 2 1
row-replacement in semiarid region northwest China Aboveground biomass and
water use efficiency Eur J Agron 28 485-492
Yamoah CF AA Agboola and GF Wilson (1986) Nutrient contribution and maize
performance in alley cropping systems Agrofor Sys 4 247ndash254
Yaragattikar AT and CJ Itnal (2003) Studies on Ber Based Intercropping Systems in the
Northern Dry Zone of Karnataka Karnataka J Agric Sci 16 22-25
Yang B Xin N Li and JG Wang (2010) Planting modes of intercroped seed-using
watermelon and jujube in dry sandy land Nonwood Forest Res 3 026
Yang J W Zheng Y Tian Y Wu and D Zhou (2011) Effects of various mixed salt-
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166
Yelton MM SS Yang SA Edie and ST Lim (1983) Characterization of an effective
salt-tolerant fast-growing strain of Rhizobium japonicum J Gen Microbiol 129
1537ndash1547
Zada K S Ahmad and MS Nazar (1988) Land equivalent ratios relative yields and
relative yield totals of intercropped maize and soyabean Pak J Agric Res 9 453-
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Zahran HH (1991) Conditions for successful Rhizobium-legume symbiosis in saline
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Zahran HH (1999) Rhizobium-legume symbiosis and nitrogen fixation under severe
conditions and in an arid climate Microbiol Mol Biol Rev 63 968-989
Zahran HH and JI Sprent (1986) Effects of sodium chloride and polyethylene glycol on
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Zegada-Lizarazu W Y Izumi and M Iijima (2006) Water competition of intercropped
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Zhang F J Shen J Zhang Y Zuo L Li and X Chen (2010) Chapter one-rhizosphere
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167
Zhu J-K (2001) Plant salt tolerance Trends Plant Sci 6 66-71
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168
6 THESIS APENDECES
Appendix-I One way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution of seed germination of pre-soaked seeds of C cajan in non-saline water prior to germination under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Mean
germination rate
(GR)
Salinity treatment 4422 20 221133 21015 0000
Error 441949 42 10522
Total 4864 62
Mean germination
velocity (GV)
Salinity treatment 418813 20 20941 51836 0000
Error 169671 42 40398
Total 588484 62
Mean
germination
time (GT)
Salinity treatment 0271 20 0013 8922 0000
Error 0064 42 0002
Total 0335 62
Mean germination
Index (GI)
Salinity treatment 4422 20 221133 21015 0000
Error 441949 42 10523
Total 4864607 62
Final
germination
(FG)
Salinity treatment 32107 20 1605397 25285 0000
Error 2666 42 63492
Total 34774 62
Appendix-II Two way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution of seed germination of pre-soaked seeds of C cajan in non-saline water prior to germination under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Germination percentage per
day
Salinity treatment 509583 20 25479 19187 0000
Time 53156 9 5906 4663 0002
Salinity treatment times time 251743 180 1398576 1053 ns
Error 531130 400 1327825
Total 1375283 629
Germination
rate per day
Salinity treatment
Time 761502 9 84611 83129 0000
Salinity treatment times time 442265 20 22113 24630 0000
Error 359117 400 0898
Total 2108622 629
Appendix-III One way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution of seed
germination of pre-soaked seeds of C cajan in respective saline water prior to germination under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Final mean germination
velocity (GV)
Salinity treatment 0538 6 0089 35585 0000
Error 0035 14 0003
Total 0573
Final mean
germination time (GT)
Salinity treatment 20862 6 3477 26256 0000
Error 1854 14 0132
Total 22716 20
Final mean germination
index (GI)
Salinity treatment 110514 6 18419 190215 0000
Error 1356 14 0097
Total 111869 20
Final
germination percentage (GP)
Salinity treatment 6857 6 1142857 40 0000
Error 400 14 28571
Total 7257 20
Appendix-IV Two way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution of seed
germination of pre-soaked seeds of C cajan in respective saline water prior to germination under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Germination percentage per
day
Salinity treatment 86644 6 14440816 505428 0000
Time 23378 6 3896 136373 0000
Salinity treatment times time 2717 36 75472 2641 0001
Error 2800 98 28571
Total 115540 146
Germination rate
per day
Salinity treatment 117386 6 19564 360762 0000
Time 128408 6 21401 394636 0000
Salinity treatment times time 58747 36 1632 30091 0000
Error 5314 98 0054
Total 309855 146
169
Appendix-V One way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution on seedling
emergence and height of germinating seeds of C cajan under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Seedling height of C cajan
Salinity treatment 200822 5 40056 169666 0000
Error 2833 12 0236
Total 203115 17
Seedling
emergence of C cajan
Salinity treatment 24805 6 4134 6381 000
Error 9070 14 647867
Total 33875 20
Appendix-VI Two way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution on growth and
development of C cajan in lysemeter (Drum pot) under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Plant height of
C cajan
Salinity treatment 261079 5 52215 720259 0000
Time 126015 8 15751 132488 0000
Salinity treatment times time 76778 40 1919 16144 0000
Error 11413 96 118893
Total 477028 161
Appendix-VII One way ANOVA for completely randomized design for effect of irrigation water of different sea salt solution on growth
and development of C cajan in lysemeter (Drum pot) under various salinity regimes
Variables Source Sum of Squares df Mean Square F-value P
Number of
Flowers of C
cajan
Salinity treatment 3932 3 131075 39719 0000
Error 264 8 33
Total 419625 11
Number of pods
of C cajan
Salinity treatment 1473 3 491 23105 0000
Error 170 8 2125
Total 1643 11
Number of
seedspod of C cajan
Salinity treatment 3 3 1
Error 0 8 0
Total 3 11
Number of seeds plant of
C cajan
Salinity treatment 19332 3 6444 45621 0000
Error 1130 8 14125
Total 20462 11
Weight of
seeds plant of C cajan
Salinity treatment 592976 3 197658 85572 0000
Error 18478 8 2309
Total 611455 11
Chlorophyll a
of C cajan
Salinity treatment 0117 3 0039 81241 0000
Error 0004 8 0000
Total 0121 11
Chlorophyll b
of C cajan
Salinity treatment 0004 3 0001 15222 0001
Error 0001 8 0000
Total 0005 11
Total chlorophyll of
C cajan
Salinity treatment 0160 3 0053 164401 0000
Error 0002 8 0000
Total 0162 11
Chlorophyll a b
ratio of C cajan
Salinity treatment 242 3 0806 9327 0005
Error 0692 8 0086
Total 3112 11
Carotenoids of
C cajan
Salinity treatment 0015 3 0005 4510 0039
Error 0009 8 0001
Total 0025 11
Soluble sugars
of C cajan
Salinity treatment 0043 3 0014 6515 0015
Error 00178 8 0002
Total 0061 11
Insoluble
sugars of C
cajan
Salinity treatment 0118 3 0039 36262 0000
Error 0008 8 0001
Total 0127 11
Total sugars of
C cajan
Salinity treatment 0019 3 0006 4239 0045
Error 0012 8 0001
Total 0031 11
Protein of C cajan
Salinity treatment 0212 3 0070 15735 0001
Error 0036 8 0004
Total 0248 11
170
Appendix-VIII One way ANOVA for completely randomized design for range of salt tolerance of nitrogen fixing symbiotic bacteria
associated with root of C cajan
Variables Source Sum of Squares df Mean Square F-value P
Nodule
associated
Rhizobial colonies of C
cajan
Salinity treatment 35927 2 17963 229402 0000
Error 1409 18 0078
Total 37337 20
Appendix-IX Two way ANOVA for completely randomized design for growth and development of Z mauritiana in large size clay pot being irrigated with water of two different sea salt concentration
Variables Source Sum of Squares df Mean Square F-value P
Height of
Z mauritiana
Time 91030 2 45515 839 0000
Salinity treatment 3268 2 1634 10 0000
Time times Salinity treatment 1533 4 383 238 ns
Error 6751 42 161
Total 104554 71
Number of
branches of
Z mauritiana
Time 25525 2 127625 25333 0000
Salinity treatment 86333 2 43166 11038 0000
Time times Salinity treatment 27416 4 6854 1752 ns
Error 16425 42 3910
Total 6575 71
Number of
flowers of
Z mauritiana
Time 73506 2 36753 167777 0000
Salinity treatment 12133 2 6066 25061 0000
Time times Salinity treatment 27824 4 6956 28736 0000
Error 10166 42 242063
Total 127759 71
Fresh weight of
Shoot of
Z mauritiana
Time 3056862 2 1528431 340777 0000
Salinity treatment 107829 2 53914 12020 0000
Time times Salinity treatment 51303 4 12825 2859 0031
Error 251167 56 4485
Total 3515820 71
Dry weight of Shoot of
Z mauritiana
Time 784079 2 392039 338932 0000
Salinity treatment 26344 2 13172 11387 0000
Time times Salinity treatment 13042 4 3260 2818 0033
Error 64774 56 1156690
Total 913855 71
Succulence of
Z mauritiana
Time 0002 2 0001 0214 ns
Salinity treatment 0006 2 0003 0682 ns
Time times Salinity treatment 0007 4 0002 0406 ns
Error 0199 45 0004
Total 51705 54
Spacific shoot
length of Z mauritiana
Time 0000 2 914 0176 0000
Salinity treatment 0002 2 0001 2096 ns
Time times Salinity treatment 0003 4 0001 1445 ns
Error 0023 45 0001
Total 6413 54
Moisture
contents of Z mauritiana
Time 1264 2 0632 0243 ns
Salinity treatment 3603 2 1801 0691 ns
Time times Salinity treatment 4172 4 1043 0400 ns
Error 117146 45 2603
Total 131675 54
Relative growth
rate of Z mauritiana
Time 1584206 1 1584206 532968 ns
Salinity treatment 18921 2 9460 3183 ns
Time times Salinity treatment 61624 2 30812 10366 0000
Error 89172 30 2972
Total 4034 36
Appendix-X One way ANOVA for completely randomized design for growth and development of Z mauritiana in large size clay pot
being irrigated with water of two different sea salt concentration
Variables Source Sum of Squares df Mean Square F-value P
Chlorophyll a
of Z mauritiana
Salinity treatment 0004 2 0002 7546 0003
Error 0006 21 0000
Total 0010 23
Chlorophyll b of Z mauritiana
Salinity treatment 0037 2 0018 4892 0018
Error 0080 21 0003
Total 0117 23
171
Total
chlorophyll of
Z mauritiana
Salinity treatment 0144 2 0072 39317 0000
Error 0038 21 0002
Total 0182 23
Chlorophyll ab ratio of
Z mauritiana
Salinity treatment 1499 2 0749 33416 0000
Error 0471 21 0022
Total 1969 23
Total soluble
sugars of
Z mauritiana
Salinity treatment 378271 2 189135 36792 0000
Error 107952 21 5140
Total 486223 23
Total protein contents of
Z mauritiana
Salinity treatment 133006 2 66502 5861 0009
Error 238268 21 11346
Total 371274 23
Appendix-XI Three way ANOVA for split-split plot design for physiological investigations on growth of Z mauritiana and C cajan in
drum pot being irrigated with water of sea salt concentration at two irrigation intervals
Variables Source Sum of Squares df Mean Square F-value P
Height of
Z mauritiana
Time 4499 2 2249 28888 0004
Crop 448028 1 448028 2208 ns
Irrigation intervals 2523 1 2523 2774 ns
Time times Crop 928088 2 464044 2288 ns
Time times irrigation interval 1120400 2 560200 0615 ns
Crop times irrigation interval 2690151 1 2690 2957 ns
Time times Crop times irrigation interval 171927 2 85963 0094 ns
Error 10916 12 909732
Total 35
Canopy volume of Z mauritiana
Time 7943 2 3971 6554 ns
Crop 0382 1 0382 0579 ns
Irrigation intervals 0068 1 0069 0103 ns
Time times Crop 0265 2 0133 0201 ns
Time times irrigation interval 1142 2 0571 0852 ns
Crop times irrigation interval 0722 1 0722 1077 ns
Time times Crop times irrigation interval 1998 2 0999 1491 ns
Error 8043 12 0670
Total 29439 35
Appendix-XII Two way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C
cajan in drum pot being irrigated with water of sea salt concentration at two irrigation intervals
Variables Source Sum of Squares df Mean Square F-value P
Plant length of
Z mauritiana
Crop 2986 1 2986 75322 0000
Irrigation interval 2986 1 2986 75322 0000
Crop times Irrigation interval 15336 1 153367 3868 ns
Error 317166 8 39645
Total 292428 12
Shoot length of
Z mauritiana
Crop 1069741 1 1069741 30890 0000
Irrigation interval 1069741 1 1069741 30890 0000
Crop times Irrigation interval 253001 1 253001 73058 0026
Error 27704 8 3463
Total 103376 12
Root length of
Z mauritiana
Crop 19763 1 19763 2671 ns
Irrigation interval 481333 1 481333 65059 0000
Crop times Irrigation interval 800333 1 800333 108177 0000
Error 59186 8 7398
Total 49165 12
Main branches
of Z mauritiana
Crop 33333 1 33333 5797 0042
Irrigation interval 48 1 48 8347 0020
Crop times Irrigation interval 0333 1 0333 0057 ns
Error 46 8 575
Total 2888 12
Lateral
branches of Z mauritiana
Crop 1344083 1 1344083 41356 0000
Irrigation interval 54675 1 54675 16823 0000
Crop times Irrigation interval 784083 1 784083 24125 0000
Error 26 8 325
Total 22465 12
Leaf numbers of
Z mauritiana
Crop 22465 12 98283 96482 0000
Irrigation interval 25025 1 25025 24566 0001
Crop times Irrigation interval 11907 1 11907 11688 0009
Error 8149 8 1018667
172
Total 2037850 12
Shootroot ratio
of Z mauritiana
Crop 0027 1 0027 1842 ns
Irrigation interval 0001 1 0001 0097 ns
Crop times Irrigation interval 0825 1 0825 54909 0000
Error 0120 8 0015
Total 27776 12
Plant fresh
weight of Z mauritiana
Crop 398107 1 398107 577818 0000
Irrigation interval 139514 1 139514 20249 0000
Crop times Irrigation interval 146898 1 146898 21321 0000
Error 5511 8 688982
Total 7248659 12
Plant dry weight of Z mauritiana
Crop 87808 1 87808 471436 0000
Irrigation interval 57893 1 57893 31082 0000
Crop times Irrigation interval 61132 1 61132 32821 0000
Error 14900 8 186257
Total 1875710 12
Stem fresh
weight of
Z mauritiana
Crop 46687 1 46687 227539 0000
Irrigation interval 17933 1 17933 87402 0000
Crop times Irrigation interval 20180 1 20180 98351 0000
Error 16414 8 205185
Total 1718530 12
Root fresh weight of
Z mauritiana
Crop 58450 1 58450 2295 0000
Irrigation interval 42186 1 42186 165641 0000
Crop times Irrigation interval 37307 1 37307 146487 0000
Error 203746 8 25468
Total 357145 12
Leaf fresh weight of
Z mauritiana
Crop 29970 1 29970 19089 0000
Irrigation interval 117018 1 1170187 7453 0025
Crop times Irrigation interval 2310 1 2310 14714 0004
Error 125596 8 15699
Total 699711 12
Stem dry weight
of Z mauritiana
Crop 13587 1 13587 216591 0000
Irrigation interval 11856 1 11856 18899 0000
Crop times Irrigation interval 6787763 1 6787 108197 0000
Error 50188 8 62735
Total 4689795 12
Root dry weight
of Z mauritiana
Crop 1358787 1 13587 216591 0000
Irrigation interval 1497427 1 14974 118615 0000
Crop times Irrigation interval 128773 1 12877 1020052 0000
Error 100993 8 12624
Total 124421 12
Leaf dry weight
of Z mauritiana
Crop 2374 1 2374 135380 0000
Irrigation interval 8748 1 8748 4987 ns
Crop times Irrigation interval 26403 1 2640 150539 0000
Error 140313 8 17539
Total 127170 12
Plant moisture of Z mauritiana
Crop 22082 1 22082 5608 0045
Irrigation interval 38702 1 38702 9830 0013
Crop times Irrigation interval 44406 1 44406 11279 0009
Error 31496 8 3937
Total 29872 12
Stem moisture of Z mauritiana
Crop 0005 1 0005 0000 ns
Irrigation interval 110663 1 110663 12023 0008
Crop times Irrigation interval 0897 1 0897 0097 ns
Error 73633 8 9204
Total 28532 12
Root moisture of Z mauritiana
Crop 235266 1 235266 16502 0003
Irrigation interval 3923 1 3923 0275 ns
Crop times Irrigation interval 0856 1 0856 0060 ns
Error 114051 8 14256
Total 17572 12
Leaf moisture
of Z mauritiana
Crop 130413 1 130413 47746 0000
Irrigation interval 22256 1 22256 8148 0021
Crop times Irrigation interval 210662 1 210662 77127 0000
Error 21850 8 2731
Total 38888 12
173
Relative growth
rate of Z mauritiana
Crop 0000 1 0000 287467 0000
Irrigation interval 0000 1 0000 164217 0000
Crop times Irrigation interval 0000 1 0000 179626 0000
Error 0000 8 0000
Total 0009 12
Relative water
contents of Z
mauritiana
Crop 37381 1 37381 1380 ns
Irrigation interval 49871 1 49871 1841 ns
Crop times Irrigation interval 13496 1 13496 0498 ns
Error 216649 8 27081
Total 50855 12
Chlorophyll a of Z mauritiana
Crop 0103 1 0103 32466 0000
Irrigation interval 0003 1 0003 1075 ns
Crop times Irrigation interval 0000 1 0000 0187 ns
Error 0025 8 0003
Total 1498 12
Chlorophyll b
of Z mauritiana
Crop 0027 1 0027 196164 0000
Irrigation interval 0002 1 0002 15656 0004
Crop times Irrigation interval 0006 1 0006 45063 0000
Error 0001 8 0000
Total 0456 12
Total chlorophyll
of Z mauritiana
Crop 0257 1 0257 53469 0000
Irrigation interval 0001 1 0001 0315 ns
Crop times Irrigation interval 0002 1 0002 0442 ns
Error 0038 8 0004
Total 3736 12
Chlorophyll a b ratio of
Z mauritiana
Crop 0002 1 0002 0028 ns
Irrigation interval 0169 1 0169 1696 ns
Crop times Irrigation interval 1064 1 1064 10643 0011
Error 0799 8 0099
Total 43067 12
Carotenoids of
Z mauritiana
Crop 0018 1 0018 42747 0000
Irrigation interval 0002 1 0002 5298 0050
Crop times Irrigation interval 0003 1 0003 8118 0021
Error 0003 8 0000
Total 0451 12
Phenol of
Z mauritiana
Crop 24641 1 24641 13168 000
Irrigation interval 5078 1 5078 2714 ns
Crop times Irrigation interval 10339 1 10339 5525 0046
Error 14969 8 1871
Total 6289 12
Proline of Z mauritiana
Crop 0001 1 0001 52288 0000
Irrigation interval 0000 1 0000 6972 0029
Crop times Irrigation interval 0000 1 0000 0358 ns
Error 0000 8 0000
Total 0005 12
Protein of Z mauritiana
Crop 200001 1 200001 296 ns
Irrigation interval 69264 1 69264 102 ns
Crop times Irrigation interval 4453 1 4453 006 ns
Error 540367 8 67545
Total 814086 11
CAT enzyme of
Z mauritiana
Crop 74171 1 74171 11404 0009
Irrigation interval 299930 1 299930 46117 0000
Crop times Irrigation interval 15336 1 15336 2358 ns
Error 52029 8 65036
Total 441467 11
APX enzyme of
Z mauritiana
Crop 191918 1 191918 6693 0032
Irrigation interval 4665 1 4665 162723 0000
Crop times Irrigation interval 336912 1 336912 11750 0009
Error 229383 8 28672
Total 5423 11
GPX enzyme of
Z mauritiana
Crop 0000 1 0000 0020 ns
Irrigation interval 0103 1 0103 5893 0041
Crop times Irrigation interval 0109 1 0109 6220 0037
Error 0140 8 0017
Total 0353 11
SOD enzyme Crop 8471 1 8471 1364 ns
174
of
Z mauritiana
Irrigation interval 6220 1 6220 1001 ns
Crop times Irrigation interval 21142 1 21142 3405 ns
Error 49664 8 6208
Total 85498 11
NR enzyme of
Z mauritiana
Crop 7520 1 75208333333 37253364154 0003
Irrigation interval 1360 1 1360 6737 0318
Crop times Irrigation interval 0016 1 0016 0079 ns
Error 1615 8 0201
Total 10512 11
Nitrate of
Z mauritiana
Crop 003 1 003 3028 ns
Irrigation interval 0018 1 0018 1831 ns
Crop times Irrigation interval 0003 1 0003 0336 ns
Error 0079 8 0009
Total 0130 11
Appendix-XIII Three way ANOVA for split-split design for physiological investigations on growth of Z mauritiana and C cajan in drum
pot being irrigated with water of sea salt concentration at two irrigation intervals
Variables Source Sum of Squares df Mean Square F-value P
Height of
C cajan
Time 14990 2 7495 235059 0000
Crop 7848 1 7848 42235 0000
Irrigation intervals 749056 1 749056 9676 0009
Time times Crop 2638 2 1319140 7098 00262
Time times irrigation interval 309932 2 154966 2001 ns
Crop times irrigation interval 9127 1 9127 0117 ns
Time times Crop times irrigation interval 31974 2 15987 0206 ns
Error 928935 12 77411
Total 29065 35
Apendix-XIV Two way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C
cajan in drum pot being irrigated with water of sea salt concentration at two irrigation intervals
Variables Source Sum of Squares df Mean Square F-value P
Plant length of C cajan
Crop 1056563 1 1056563 12331 0007
Irrigation interval 21675 1 21675 2529 ns
Crop times Irrigation interval 137363 1 137363 1603 ns
Error 68544 8 8568
Total 334030 12
Shoot length of C cajan
Crop 808520 1 808520 36580 0000
Irrigation interval 165020 1 165020 7466 0025
Crop times Irrigation interval 285187 1 285187 12902 0007
Error 17682 8 22102
Total 224013 12
Root length of C cajan
Crop 16567 1 16567 0674 ns
Irrigation interval 3520 1 3520 0143 ns
Crop times Irrigation interval 26700 1 26700 1087 ns
Error 196453 8 24556
Total 11133 12
Main branches
of C cajan
Crop 80083 1 80083 64066 0000
Irrigation interval 10083 1 10083 8066 0021
Crop times Irrigation interval 075 1 075 06 ns
Error 10 8 125
Total 335 12
Letral branches
of C cajan
Crop 0 1 0
Irrigation interval 0 1 0
Crop times Irrigation interval 0 1 0
Error 0 8 0
Total 0 12
Leaf numbers
of C cajan
Crop 1776333 1 1776333 16679 0003
Irrigation interval 972 1 972 9126 0016
Crop times Irrigation interval 176333 1 17633 1655 0234
Error 852 8 1065
Total 22342 12
Shootroot ratio of C cajan
Crop 0385 1 0385 0638 0447
Irrigation interval 0007 1 0007 0011 0916
Crop times Irrigation interval 2669 1 2669 4424 0068
Error 4825 8 0603
Total 264061 12
Crop 76816 1 76816 7494853 0025
175
Plant fresh
weight of
C cajan
Irrigation interval 730236 1 730236 7124832 0028
Crop times Irrigation interval 266869 1 266869 2603812 0145
Error 81993 8 102491
Total 25941 12
Plant dry weight of C cajan
Crop 38270 1 38270 1150145 0009
Irrigation interval 53046 1 53046 15942 0003
Crop times Irrigation interval 20202 1 20202 6071 0039
Error 26619 8 3327
Total 4150 12
Stem fresh weight of
C cajan
Crop 16100 1 16100 1462 ns
Irrigation interval 9900 1 9900 0899 ns
Crop times Irrigation interval 00675 1 0067 0006 ns
Error 8806 8 11007
Total 3318 12
Root fresh weight of
C cajan
Crop 0190 1 0190 0248 ns
Irrigation interval 27331 1 27331 35753 0000
Crop times Irrigation interval 2698 1 2698 3529 0097
Error 6115 8 0764
Total 432050 12
Leaf fresh
weight of C cajan
Crop 541363 1 541363 13825 0005
Irrigation interval 347763 1 347763 8881 0017
Crop times Irrigation interval 208333 1 208333 5320 0049
Error 313246 8 39155
Total 7236 12
Stem dry weight
of C cajan
Crop 10323 1 10323 11530 0009
Irrigation interval 0452 1 0452 0505 ns
Crop times Irrigation interval 0232 1 0232 0259 ns
Error 7162 8 0895
Total 125151 12
Root dry weight
of C cajan
Crop 0007 1 0007 012 ns
Irrigation interval 0607 1 0607 972 0014
Crop times Irrigation interval 0367 1 0367 588 0041
Error 05 8 0062
Total 3515 12
Leaf dry weight
of C cajan
Crop 9363 1 9363 15649 0004
Irrigation interval 34003 1 3400 5683 0000
Crop times Irrigation interval 11603 1 11603 19392 0002
Error 4786 8 0598
Total 95072 12
Plant moisture of C cajan
Crop 199182 1 19918 6011 0039
Irrigation interval 272215 1 27221 8215 0020
Crop times Irrigation interval 76654 1 76654 2313 0166755
Error 265079 8 33134
Total 38272 12
Stem moisture
of C cajan
Crop 100814 1 10081 3290 0107246
Irrigation interval 53460 1 53460 1744 0223065
Crop times Irrigation interval 19778 1 1977 0645 0444938
Error 245119 8 30639
Total 31036 12
Root moisture
of C cajan
Crop 26266 1 26266 1389 ns
Irrigation interval 223809 1 223809 11836 0008
Crop times Irrigation interval 0097 1 0097 0005 ns
Error 151272 8 18909
Total 58346 12
Leaf moisture
of C cajan
Crop 2623 1 2623 39350 0000
Irrigation interval 1765 1 1765 26477 0000
Crop times Irrigation interval 1425 1 1425452 21378 0001
Error 533411 8 66676
Total 36263 12
Relative growth
rate of C cajan
Crop 0000 1 0000 17924 0002
Irrigation interval 0000 1 0000 21296 0001
Crop times Irrigation interval 0000 1 0000 88141 0017
Error 0000 8 0000
Total
Crop 256935 1 256935 1560 ns
Irrigation interval 268827 1 26882 1633 ns
176
Electrolyte
leakage of C
cajan
Crop times Irrigation interval 30379 1 30379 0184 ns
Error 1316923 8 16461
Total 50381 12
Chlorophyll a
of C cajan
Crop 0101 1 0101 7957 0022
Irrigation interval 0062 1 0062 4893 ns
Crop times Irrigation interval 0199 1 0199 15600 0004
Error 0102 8 0012
Total 5060 12
Chlorophyll b
of C cajan
Crop 0017 1 0017 7758 0023
Irrigation interval 0027 1 0027 12389 0007
Crop times Irrigation interval 0056 1 0056 25313 0001
Error 0017 8 0002
Total 1727 12
Total
chlorophyll of C cajan
Crop 0178 1 0178 14819 0004
Irrigation interval 0198 1 0198 16520 0003
Crop times Irrigation interval 0509 1 0509 42379 0000
Error 0096 8 0012
Total 13217 12
Chlorophyll a b
ratio of C cajan
Crop 0065 1 0065 0691 ns
Irrigation interval 0033 1 0033 0357 ns
Crop times Irrigation interval 0016 1 0016 0173 ns
Error 0756 8 0094
Total 35143 12
Carotenoids of C cajan
Crop 0021 1 0021 19599 0002
Irrigation interval 0028 1 0028 26616 0000
Crop times Irrigation interval 0041 1 0041 38531 0000
Error 0008 8 0001
Total 1443 12
Phenol of C cajan
Crop 0799 1 0799 3171 ns
Irrigation interval 0040 1 0040 0159 ns
Crop times Irrigation interval 0911 1 0911 3617 ns
Error 2016 8 0252
Total 970313 12
Proline of C cajan
Crop 0008 1 0008 14867 0004
Irrigation interval 0019 1 0019 34536 0000
Crop times Irrigation interval 0008 1 0008 14969 0004
Error 0004 8 0000
Total 0155 12
Protein of C
cajan
Crop 116376 1 116376 3990 ns
Irrigation interval 434523 1 434524 14899 0048
Crop times Irrigation interval 33166 1 33166 1137 ns
Error 233303 8 29163
Total 817371 11
CAT enzyme
of C cajan
Crop 0249 1 0249 0121 ns
Irrigation interval 2803 1 2803 13702 ns
Crop times Irrigation interval 92392 1 9239 4517 ns
Error 16362 8 2045
Total 28654 11
APX enzyme
of C cajan
Crop 855939 1 855939 4073 ns
Irrigation interval 1078226 1 1078226 5130 ns
Crop times Irrigation interval 13522 1 13522 64349 000
Error 1681112 8 210139
Total 17137 11
GPX enzyme
of C cajan
Crop 0965 1 0965 9265 0160
Irrigation interval 1167 1 1167 11195 0101
Crop times Irrigation interval 0887 1 0887 8514 0194
Error 0833 8 0104
Total 3854 11
SOD enzyme
of C cajan
Crop 4125 1 4125 9731 0142
Irrigation interval 4865 1 4865 11477 0095
Crop times Irrigation interval 20421 1 20421 48172 0001
Error 3391 8 0423
Total 32804 11
Nitrate
reductase
enzyme
Crop 0053 1 0053 0034 ns
Irrigation interval 0001 1 0001 0000 ns
Crop times Irrigation interval 10329 1 10329 6650 0327
177
of C cajan Error 12424 8 1553
Total 22808 11
Nitrate of
C cajan
Crop 0039 1 0039 0576 ns
Irrigation interval 0083 1 0083 1222 ns
Crop times Irrigation interval 0003 1 0003 0005 ns
Error 0545 8 0068
Total 0668 11
Appendix-XV Two way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C
cajan intercropped on marginal land under field condition
Variables Source Sum of Squares df Mean Square F-value P
Height of Z mauritiana
Time 79704 3 26568 77303 0000
Treatment 979209 1 979209 4702 0455
Time times Treatment 756019 3 252006 1210 3381 ns
Error 3332 16 208259
Total 90366 39
Canopy volume of Z mauritiana
Time 1049 3 3498 115444 0000
Treatment 3509 1 3509 5966 0266
Time times Treatment 3374 3 1124 1911 1684 ns
Error 9413 16 5883
Total 1284 39
flowers numbers of Z
mauritiana
Time 1794893 3 598297 770043 0000
Treatment 19980 1 19980 10152 0057
Time times Treatment 21017 3 7005 3559 0381
Error 31488 16 1968
Total 1882468 39
Fruits numbers
of Z mauritiana
Time 324096 3 108032 297941 0000
Treatment 10824 1 10824 64081 0000
Time times Treatment 7141 3 2380 14093 0001
Error 2702 16 168913
Total 351833 39
Appendix-XVI One way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C cajan intercropped on marginal land under field condition
Variables Source Sum of Squares df Mean Square F-value P
Weight of ten
fruits (FW) of
Z mauritiana
Treatment 557113 1 557113 6663 0032
Error 668923 8 83615
Total 1226036 9
Weight of ten fruits (DW) of
Z mauritiana
Treatment 4356 1 4356 0321 ns
Error 10862 8 13577
Total 112976 9
diameter of fruit of Zmauritiana
Treatment 0534 1 0534 0946 ns
Error 4514 8 0564
Total 5048 9
Fruit weight per plant of
Z mauritiana
Treatment 0739 1 0739 4022 ns
Error 1471 8 0184
Total 2211 9
Fruit sugar
(soluble) of
Z mauritiana
Treatment 5041 1 5041 0081 ns
Error 497328 8 62166
Total 502369 9
Fruit sugar (extractable) of
Z mauritiana
Treatment 32041 1 32041 0424 ns
Error 604384 8 75548
Total 636425 9
Total fruit
sugars of Z mauritiana
Treatment 16 1 16 0780 ns
Error 164 8 205
Total 18 9
Chlorophyll a of
Z mauritiana
Treatment 0082 1 0082 1384 0020
Error 0024 4 0006
Total 0105 5
Chlorophyll b
of Z mauritiana
Treatment 0011 1 0011 8469 0043
Error 0005 4 0001
Total 0016 5
Total chlorophyll of
Z mauritiana
Treatment 0152 1 0152 11927 0025
Error 0051 4 0013
Total 0203 5
Treatment 0015 1 0015 0867 ns
Error 0067 4 0017
178
Chlorophyll a b
ratio of Z mauritiana
Total 0082 5
Carotinoids of Z mauritiana
Treatment 0011 1 0011 9719 0035
Error 0004 4 0001
Total 0015 5
Leaf protein of
Z mauritiana
Treatment 0106 1 0106 4 ns
Error 0106 4 0027
Total 0213 5
Leaf sugars
(soluble) of
Z mauritiana
Treatment 054 1 054 0025 ns
Error 848 4 212
Total 8534 5
Leaf sugars
(Extractable) of Z mauritiana
Treatment 486 1 486 8055 0046
Error 2413 4 0603
Total 7273 5
Total sugars in
leaf of Z
mauritiana
Treatment 216 1 216 0104 ns
Error 83333 4 20833
Total 85493 5
Leaf phenols of
Z mauritiana
Treatment 8166 1 8166 5665 ns
Error 5766 4 1442
Total 13933 5
Leaf nitrogen of Z mauritiana
Treatment 15 1 15 1939 ns
Error 3093 4 0773333
Total 4593 5
Soil nitrogen of
Z mauritiana
Treatment 0375 1 0375 21634 ns
Error 0693 4 0173
Total 1069 5
Appendix-XVII Two way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C
cajan intercropped on marginal land under field condition
Variables Source Sum of Squares df Mean Square F-value P
Height of Ccajan
Time 700196 2 350098 2716 0000
Treatment 594405 1 594405 16017 0000
Time times Treatment 488829 2 244415 6586 0004
Error 1001996 27 37111
Total 705495 59
Number of branches of
Ccajan
Time 8353 2 4176 1050050 0000
Treatment 24066 1 24066 18672 0000
Time times Treatment 24133 2 12066 9362 0000
Error 348 27 1288
Total 8572 59
Number of flowers of
Ccajan
Time 289297 2 144648 301277 0000
Treatment 365066 1 365066 0701 ns
Time times Treatment 730133 2 365066 0701 ns
Error 14059 27 520733
Total 317415 59
Number of pods
of Ccajan
Time 347682 2 173841 70559 0000
Treatment 159135 1 159135 1558 ns
Time times Treatment 8167 2 40835 0399 ns
Error 27574 27 1021276
Total 447407 59
Appendix-XVIII One way ANOVA for completely randomized design for physiological investigations on growth of Z mauritiana and C
cajan intercropped on marginal land under field condition
Variables Source Sum of Squares df Mean Square F-value P
Shoot weight
(FW) of
Ccajan
Treatment 0 1 0 0 ns
Error 87444 4 21861
Total 87444 5
Shoot weight
(RW) of Ccajan
Treatment 0 1 0 0 ns
Error 13808 4 3452
Total 13808 5
Number of
seeds of
Ccajan
Treatment 245 1 245 0005 ns
Error 940182 18 52232
Total 940427 19
Weight of seeds
of Ccajan
Treatment 02 1 02 0000 ns
Error 7585 18 421406
Total 7585 19
179
Chlorophyll a of
Ccajan
Treatment 0001 1 0001 5442 ns
Error 0001 4 0000
Total 0002 5
Chlorophyll b
of Ccajan
Treatment 0006 1 0006 9079 0039
Error 0002 4 0001
Total 0008 5
Total
chlorophyll of
Ccajan
Treatment 0017 1 0017 51558 0001
Error 0001 4 0000
Total 0019 5
Chlorophyll a b ratio of
Ccajan
Treatment 0183 1 0183 5532 ns
Error 0132 4 0033
Total 0316 5
Leaf protein of Ccajan
Treatment 0001 1 0001 0017 ns
Error 0228 4 0057
Total 0228 5
Leaf sugars of
Ccajan
Treatment 0015 1 0015 0003 ns
Error 1624 4 406
Total 16255 5
Leaf phenols of
Ccajan
Treatment 0201 1 0201 0140 ns
Error 5746 4 1436
Total 5948 5
Leaf nitrogen
of Ccajan
Treatment 1306 1 1306 3062 ns
Error 1706 4 04266
Total 3013 5
Appendix-XIX Two way ANOVA for completely randomized design for investigations on determining range of salt tolerance in Carissa
carandas
Variables Source Sum of Squares df Mean Square F-value P
Height of C carandas
Time 72042 5 14408 55957 0000
Salinity treatment 49345 2 24672 196775 0000
Time times Salinity treatment 16679 10 1667920 13302 000
Error 3009 24 125385
Total 143777 53
Volume of
canopy of
C carandas
Time 3329 4 0832 38126 000
Salinity treatment 1393 2 0696 67129 000
Time times Salinity treatment 0813 8 0102 9792 000
Error 0207 20 0010
Total 5969 44
Appendix-XX One way ANOVA for completely randomized design for investigations on determining range of salt tolerance in Carissa carandas
Variables Source Sum of Squares df Mean Square F-value P
Number of
flowers of C carandas
Salinity treatment 10288 2 5144194 1342937 0000
Error 229833 6 38305
Total 10518 8
Number of fruits of
C carandas
Salinity treatment 18000 2 9000 268215 0000
Error 201333 6 33555
Total 18201 8
Flower shedding
percentage of C carandas
Salinity treatment 1541647 2 770823 53455 0000
Error 86519 6 144199
Total 1628166 8
Weight of ten fruits (FW) of
C carandas
Salinity treatment 82632 2 41316 187678 0000
Error 1321 6 0220
Total 83953 8
Weight of ten
fruits (DW) of
C carandas
Salinity treatment 4355 2 2177 13753 0005
Error 095 6 0158
Total 5305 8
Fruits per plant
(FW) of
C carandas
Salinity treatment 133127 2 66563 278148 0000
Error 1435861 6 239310
Total 134563 8
Fruits per plant
(DW) of C carandas
Salinity treatment 8782 2 439117 117790 0000
Error 223677 6 37279
Total 9006 8
Size of fruits of C carandas
Salinity treatment 1301 2 0651 4125 ns
Error 0946 6 0158
Total 2248 8
Salinity treatment 5607 2 2804 17592 0003
180
Diameter of fruit
of C carandas
Error 0956 6 0159
Total 6563 8
Chlorophyll a of C carandas
Salinity treatment 0112 2 0056 119786 0000
Error 0003 6 0000
Total 0115 8
Chlorophyll b of
C carandas
Salinity treatment 0005 2 0002 434 0000
Error 0000 6 0000
Total 0005 8
Total chlorophyll of C carandas
Salinity treatment 0159 2 0079 104188 0000
Error 0005 6 0001
Total 0164 8
Chlorophyll a b
ratio of C carandas
Salinity treatment 9661 2 4831 324691 0000
Error 0089 6 0015
Total 9751 8
Carotenoids of C carandas
Salinity treatment 0029 2 0014 28822 0000
Error 0003 6 0001
Total 0032 8
Leaf Protein of
C carandas
Salinity treatment 2722 2 1361 98 0012
Error 0833 6 0138
Total 3555 8
Soluble sugar of
C carandas
Salinity treatment 234889 2 117444 12735 0006
Error 55333 6 9222
Total 290222 8
In soluble sugars
of C carandas
Salinity treatment 595395 2 297698 39094 0000
Error 45689 6 7615
Total 641085 8
Total sugar of
C carandas
Salinity treatment 1576898 2 788448 39201 0000
Error 120676 6 20113
Total 1697574 8
Phenols of C carandas
Salinity treatment 14675 2 7338 74202 0000
Error 0593 6 0099
Total 15268 8
Leaf Na+ of
C carandas
Salinity treatment 1346 2 673 673 0000
Error 6 6 1
Total 1352 8
Leaf K+ of C carandas
Salinity treatment 798 2 399 133 0000
Error 18 6 3
Total 816 8
Leaf K+ Na+
ratio of C carandas
Salinity treatment 0305 2 0153 654333 0000
Error 0001 6 0000
Total 0307 8
181
7 Publications