study of somaclonal variation in wheat for the...
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STUDY OF SOMACLONAL VARIATION IN WHEAT FOR THE INDUCTION OF SALINITY TOLERANCE
By
Saleem Akhtar
Roll No. 12
Thesis submitted in partial fulfillment of the requirements for the degree of
DOCTOR of PHILOSOPHY
In
BOTANY
DEPARTMENT OF BOTANY
GC UNIVERSITY, FAISALABAD
March 2011
Declaration The work reported in this thesis was carried out by me under
the supervision of Dr. Mubashir Niaz, Department of Botany, GC
University, Faisalabad, Pakistan.
I hereby declare that the “Study of somaclonal variation in
wheat for the induction of salinity tolerance” and the contents of
thesis are the product of my own research and no part has been
copied from any published source (except the references, standard
mathematical or genetic models / equations/ formulas / protocols
etc.). I further declare this work has not been submitted for award of
any other degree / diploma. The University may take action if the
information provided is found inaccurate at any stage.
Signature of the student Saleem Akhtar
Registration No.: 2005-GCUF-10-113
CERTIFICATE BY THE RESEARCH SUPERVISOR I certify that the contents and form of thesis submitted by
Mr. Saleem Akhtar Roll No.12 has been found satisfactory and
according to the prescribed format. I recommend it be processed for
evaluation by the external examiner for the award of degree.
Signature Name : Dr. Mubashir Niaz Stamp
Chairperson
Dean, Faculty of Science and Technology, GC University, Faisalabad
SUPERVISORY COMMITTEE
Chairman ____________________________ (Dr. Mubashir Niaz) Member ____________________________ (Dr. Muhammad Iqbal) Member ____________________________ (Dr. Naeem Iqbal)
SCRUTINIZING COMMITTEE
1. Dr. Naeem Iqbal _______________________ (Convenar)
2. Dr. Muhammad Iqbal _______________________ (Member)
3. Dr. Syed Hammad Raza _______________________ (Member)
CONTENTS
CHAPTER TITLE PAGE
SUMMARY i
ACKNOWLEDGEMENTS v
1 INTRODUCTION 1
2 REVIEW OF LITERATURE 9
3 MATERIALS AND METHODS 52
4 RESULTS 59
5 DISCUSSION 106
REFERENCES 113
APPENDICES
LIST OF TABLES
Table No TITLE PAGE
1. CALLUS INDUCTION FREQUENCY OF 50 WHEAT VARIETIES, USING MATURE EMBRYO AS EX PLANT SOURCE UNDER DIFFERENT 2,4-D, LEVELS
61
2. RESPONSE OF WHEAT GENOTYPES TO CALLOGENESIS UNDER SALT STRESS
65
3. SALINITY EFFECT ON RFGR OF WHEAT CALLUS 71
4. NECROTIC PERCENTAGE IN CALLUS IN WHEAT GENOTYPES UNDER SALT STRESS.
72
5. REGENERATION POTENTIAL OF 20 WHEAT VARIETIES UNDER SALT STRESS (NO. OF PLANTS)
74
6. FRESH SHOOT WEIGHT OF WHEAT SOMACLONES AS AFFECTED BY SALINITY
78
7. SALINITY EFFECT ON DRY SHOOT WEIGHT OF WHEAT SOMACLONES
79
8. SALINITY EFFECT ON FRESH ROOT WEIGHT OF WHEAT SOMACLONES
81
9. SALINITY EFFECT ON DRY ROOT WEIGHT OF WHEAT SOMACLONES
82
10. EFFECT OF SALINITY ON SODIUM CONCENTRATION (mol m-3) OF 10 WHEAT VARIETIES
84
11. EFFECT OF SALINITY ON K+ CONCENTRATION (mol m-3) IN LEAF SAP OF 10 WHEAT VARIETIES AT DIFFERENT SALT LEVELS
86
12. K+/Na+ OF WHEAT SOMACLONAL AS AFFECTED BY SALINITY
87
13. EFFECT OF SALINITY ON NET PHOTO SYNTHESIS(PN) IN 10 WHEAT SOMACLONES
89
14. STOMATAL CONDUCTANCE (GS) IN10 WHEAT SOMACLONES AS AFFECTED BY SALINITY
90
15. EFFECT OF SALINITY ON PLANT HEIGHT IN 10 WHEAT SOMACLONES ALONGWITH PARENTS
94
16. SALINITY EFFECT ON NO. OF TILLERS IN WHEAT SOMACLONAL
96
17. EFFECT OF SALINITY ON NO. OF SPIKES IN 10 WHEAT SOMACLONES ALONGWITH PARENTS
98
18. SALINITY EFFECT ON NO. OF SPIKELETS IN 10 WHEAT SOMACLONES ALONGWITH PARENTS
100
19. EFFECT OF SALINTY ON 100 GRAIN WEIGHT (G) OF 10 WHEAT SOMACLONES ALONGWITH PARENTS
102
20. YIELD/PLANT (G) OF 10 WHEAT SOMACLONES ALONGWITH PARENTS AS AFFECTED BY SALINITY
104
i
SUMMARY
Selection of favorable somaclonal variants from callus culture are helping
tools to common breeding for the development of stress tolerant plants (Larkin
and Scowcroft, 1981; Dix, 1993; Ashraf, 1994). The inclusion of a variety in
selection program depends on its response to in vitro culture, especially to callus
formation and embryogenic callus production. In fact, for numerous genotypes,
results showed that plant genotypes differed in in vitro culture response (Abe
and Futsuhara, 1984; Mikami and Mirodjagh, 1999).
Salinity is a main factor hampering the crop production in arid and semi-
arid lands in world (Ashraf, 1994). Plant tissue culture methods provide a
promising approach to develop salt resistant plants. In vitro selection of salt
tolerant cultivars and regenerated plants has been reported in different crops
such as rice, barley, wheat and sunflower (Lutts et al; 1999; Sibi and Fakiri,
2000, Barakat and Abdel-Latif, 1996, and Alvarez et al., 2003). This concludes
that tissue culture techniques can be helpful to improve salt tolerance of different
plants.
Initially fifty wheat varieties were screened for their callus induction
potential under four levels of 2,4-D, i.e., 1, 2, 3 and 4 mgL-1. Uqab-2002 and AS-
2000 proved better with 90% callus induction frequency while ufaq-2002 and V-
4189 produced 85 and 80% callus induction frequency respectively. As far as the
dose of 2,4-D is concerned, maximum callus was obtained at the level of 3 mgL-1
2,4-D. Twenty genotypes i.e. (Bhakhar-2002, V-03079 and V-04189 LU-26S,
ii
Pothowar, Punjab-76, Barani-83, Kohinoor-83, Faisalabad-85, Chakwal-86
Pasban-90, Inqulab-91, Punjab-96, Uqab-2000, Chenab-70, Iqbal-2000, AS-
2000, Ufaq-2002, SA-42 and Parwaz-94,) having callus induction frequency of
70% or more were further tested for callus formation under four salinity levels,
i.e., 0,50,100 and 150mM NaCl + 3 mgL-1 2,4-D. Salinity adversely affected the
callus weight and Relative Fresh Weight Growth of callus. As the salinity
increased callus masses, Relative Fresh Weight Growth (RFWG) and necrotic
percentage in callus was decreased. Almost all the twenty genotypes produced
the callus of varying degree with and without salt. Among these genotypes V-
04189 produced good callus at 50mM salinity levels, LU-26S at 100mM level
while Uqab-2000 produced maximum calls at 150mM NaCl level. Ten wheat
genotypes, i.e., Inqulab-91, Uqab-2000, Chenab-70, AS-2000, and Bhakhar-
2002, Ufaq-2002, Parwaz-94, LU-26S, Punjab-76, Pasban-90 produce granular
and morphogenic callus under salt stress condition, were evaluated for RFWG of
callus under same salinity level. Salinity again reduced the relative fresh weight
growth. The maximum reduction was noted in genotype AS-2002 while Ufaq-
2002 showed minimum reduction in RFWG.
The 20 wheat genotypes which gave good callus formation under salinity
levels were evaluated for their regeneration potential under the same salinity
levels. Once again the effect of salinity was quite dominant on the production of
plantlets. Maximum (48) shoots were developed by LU-26S followed by Pasban-
90(40) while Ufaq-2002 and AS-2000 produced 31 shoots each .These
iii
genotypes developed roots on rooting medium and turned into complete plants.
Then these plants were grown to maturity in sand culture to obtain R0 seed.
Various ions, when present in supraoptimal levels in the medium show
differential effects on the overall plant or cell growth. It is therefore, imperative to
separate toxicity effects of various ions for improvement programs. Among all the
ions, Na+ and Cl- are more damaging to growth of wheat, the seed of wheat
somaclones were sown in plastic buckets having in net house of Agricultural
Biotechnology Research Institute Faisalabad and different parameters at
seedling stage were studied. The growing plants were irrigated at regular interval
of one week with full Hoagland’s nutrient solutions. The plants were subjected to
0,50,100,150 mM levels of salinity using NaCl. The data for various
morphological, physiological and biochemical attributes were recorded
Growths attributes such as root / shoot fresh and dry weight, root / shoot
length, photosynthesis rate and stomatal conductance revealed a significant
decrease under saline conditions in all the somaclones of wheat genotypes.
Salinity also decreased the total yield. Adverse impact of salt stress on growth of
wheat was associated due to increased osmotic potential which in turn was
associated with more concentration of Na+ and less concentration of K+. High
NaCl concentration proved to be toxic for the growth of all the wheat variants. In
the parameters studies, response was similar except minor variation among
genotypes.
The so developed somaclones of various genotypes along with their
mother plants were sown in pots having artificially developed four levels of
iv
salinity, i.e., 0, 4, 8 and 12 dSm-1using NaCl salt to study and compare
agronomic traits among themselves. Here again salinity decreased plant height,
No. of tillers, spikes, spikelets, 100 grain weight and yield per plant. Maximum
100grain weight was observed in Bhakhar-2000(P) followed by AS-2000(P) as
compared to other somaclones/genotypes of wheat. Bhakhar-2000(S) was also
at par with its parent Bhakhar-2002(P). At EC level of 4dSm-1, Inqulab-91(P),
Inqulab-91(S) and Pasban-90(P) were salt tolerant. At EC 8dSm-1, LU-26(P) was
noted as the best tolerant while at EC12dSm-1 Pasban-90(P) was superior to all
other genotypes. As for as yield per plant is concerned somaclones of Ufaq-
2002, AS-2000, Bhakhar-2002 at 4, Punjab-96, Punjab-76, AS-2000, Bhakhar-
2000 at 8 and Punjab-96, Punjab-76, Chenab-70 were found to be salt tolerant
at 12dSm-1 salinity level in yield per plant.
In conclusion, salinity adversely effects the plant growth, ion accumulation,
photosynthetic activities and total yield. NaCl salinity increases Na+ concentration
and decreases K+ concentration. However, somaclones developed having salt
tolerance at different salinity levels can be sown in marginal lands where no other
crop is grown.
v
ACKNOWLEDGEMENTS
I am extremely grateful to Almighty Allah, who enabled me to complete this study.
I express my gratitude to my supervisor, Dr. Mubashir Niaz Chairman,
Department of Botany for his guidance, kindness and helpful suggestions during
the research work. His concerned supervision and generous response to my
difficulties during the research work will never be forgotten.
I offer my thanks to Mr. Makhdoom Hussain, Director Wheat Research
Institute Faisalabad and Dr. Muhammad Zaffar Iqbal, Director Agricultural
Biotechnology Research Institute AARI, Faisalabad for their affectionate
supervision, valuable advices and help in planning and execution of the present
studies and providing all the facilities during the course of my research pursuits.
Without their sincere inspiration and keen interest, the completion of this work
would have been difficult.
I would like to express my deep sense of indebtedness to:
Dr. Iftikhar Ali (late), Dr. Muhammad Iqbal and Dr. Naeem Iqbal for their
cooperation, valuable suggestions during research and for reviewing the
thesis.
Dr. Sajid-ur-Rahman, Economic Botanist and Mr. Muhammad Asif,
Assistant Research Officer for their help extended in the compilation of the
thesis.
Finally I wish to thank my family member for their prayers for my success.
(SALEEM AKHTAR)
1
CHAPTER 1
INTRODUCTION
Salinity in agricultural land is a major problem in many countries of the world.
The extent, distribution and nature of the salt-affected lands of the world is very
imperfectly known for most countries due to lack of standardization of
characterization criteria. About over 800 million hectares of soil in the world are
suffered from both salinity and sodicity (Munns, 2005). Abiotic stresses such as
salinity, water logging, temperature etc. have largely affected crop production at
global level (Taiz and Zeiger, 1998). Among these abiotic stresses salinity is major
factor that effect the plant growth (Russ et al., 2000). The salt effected soils in
Pakistan are 6.3 million hectares (Anonymous, 2003) that include about 2.4 million
hectares in Punjab, 2.1 million hectares in Sindh, 0.5 million hectares in K.P. and
about 1.3 million hectares in the Baluchistan. Out of which 3369.7 thousand
hectares has become totally unproductive due to soil salinity and the productive
lands are still continuously going out of cultivation due to salinity problem (Chaudary,
2001). The presence of high salt concentration and water logging of soils are serious
problems affecting the agricultural production in Pakistan reducing yield by about
25% (Sandhu and Qureshi, 1986). Since many of these soils are beyond the reach
of conventional reclamation techniques, either for economic reasons or for lack of
2
fresh water. A major scientific thrust has been aimed at developing suitable salt, and
water logging tolerant crops to bring these lands into agricultural productivity
( Hollaender, 1979 ). In this respect, an understanding of the plant responses to
various stresses and the mechanisms that make some species or genotypes more
tolerant than others seems essential. The accumulation of toxic ions in the
rhizosphere initially causes injury to plant roots and their presence in aerial parts
cause damage to plant metabolism by reducing growth and yield ( Francois and
Maas, 1999 ). Salt tolerance is a complex phenomenon which varies not only among
species but also depends upon the environmental conditions perpetual irrigation
without proper drainage have caused large scale soils salinity. Salinity may arise as
a result of poor drainage, use of poor quality water, redistribution of salts in soil
profiles, shallow water table and lack of proper soil and water management practices
( Ansari et al., 1998 ). In sodic soils as a rule not only the high salt concentration but
also pH is the stress factor, particularly in cases where a higher contents of sodium
carbonate in the solid and liquid phase is present. The high pH hinders the life
function of crops and limits their productivity. Sodic soils have pH value and highly
exchangeable Na+ and/or often barren sodic soils having poor physical conditions
that badly affect the water and air movement in soils. Sodicity causes impaired
plant growth ( Pessarakli and Szaboles, 1999 ). Salinity can increase concentration
of certain ions that have an inhibitory effect on plant metabolism. It can adversely
effect soil structure such as soil permeability and soil aeration, or diminish physico-
chemical effect ( Evangelous and McDonald,1999).
Man is dependent on field crops for food. In Asia only about 8% of diet is from
3
animal protein and more than 75% comes from cereals, wheat (Triticum aestivum L.)
as it is known today evolved through inter crossing of its related wild ancestors.
Unlike many cereals, wheat is mainly food rather than a food crop ( Stephen and
Carter, 1980 ). The effect of NaCl salinity is very harmful for wheat as well as for
other crops. Salinity suppresses plant growth and suppressions increases as the salt
concentration increases (Sudyova et al., 2002).
Wheat is one of the major staple foods in world. In Pakistan it is grown on an
area of 8.41 million hectares with an annual production of 21.74 million tons
(Anonymous, 2008-9) which is very low compared to world production. It’s
production potential in our country is limited due to various environmental stresses,
particularly salt stress. High population growth rate demands higher yield of wheat in
this country. At present deficit in production/demand ratio is compensated with the
import which is an additional burden to an already meager economy. Generally in
Pakistan, wheat crop is sown from the month of October to end of December, but
the suitable time for sowing is the month of November. Harvesting is done in April
and May. According to Martin & Leonard (1963 ) there are many species of Triticum.
There are three main groups are diploids, tetraploids and hexapliods having 14, 28,
and 42 chromosomes respectively. Wheat plant is exposed to many diseases, insect
pests attack and many abiotic stresses including salt stress which may cause
reduction in yield. The production profile of wheat from salt effected areas is
extremely poor. The yield in these areas can be increased considerably through the
development of salt tolerant wheat varieties. However, consistent efforts are
required to boost in crop production in these lands.
4
It is very difficult, time consuming and tedious to breed salinity tolerant
varieties through conventional means. Where as exploitation of somaclonal
variation in plants for the development of salt tolerant cultivars through tissue
culturing is advantageous over conventional methods. Exploitation of somaclonal
variation is an efficient and potent method for producing novel and useful varieties
(Larkin and Scowcroft, 1981).
Characteristic symptoms of salinity are damage to vegetative growth in
herbaceous and woody plants including a slowing of leaf and shoot extension, low
rate of dry matter accumulation by aerial tissues and conspicuous yellowing of older
(lower)leaves suggesting premature senescence and wilting. Further leaf opening
and leaf shedding are typical responses of some species to salinity stress ( Drew,
1981 ). Sodium chloride significantly reduces root and shoot fresh and dry masses,
root length and shoot length (Zoppo et al., 1999). Growing leaf cells maintain their
turgor pressure in response to the salt stress by accumulating the osmotically active
solutes ( Humayun et al., 1992 ).
There are significant difference in salinity tolerance of plants and the way in
which they partition excess of salts in various parts to sustain growth. The
depressing effect of salinity on root growth is generally less severe than effect on
growth of aerial components (Stem, leaves, and fruits). Root growth is almost always
less effected than shoot growth by increasing soil salinity, so root shoot ratio
generally increases ( Munns and Termaat,1986). Soil and water salinity directly
affects the wheat production. The reduction in growth of many crops plants by
salinity may be due to its effect on dry matter production, ionic relations, water
5
potential, physiological disorders, metabolic reactions or a combination of all these
factors. ( Ashraf et al.,1991; Ashraf and Khan 1993; Ashraf and Khan, 1994 ).
Two approaches are being used to tackle soil stress problem. The first is to
amend the soil conditions to favour crop plants. A variety of approaches can be used
to manage these salt affected soils through well established techniques such as
provision of adequate drainage and use of amendments are available for this
purpose, yet due to limitation of poor quality irrigation water, high cost of
amendments and low soil permeability. The second one is to use the genetic ability
of plants for their adaptability to adverse soil conditions. The first approach is a long
term process and a few success has been seen in our country even after spending
Rs.21 billion up to 1988 ( Aslam et al., 1993B ). However the later is short term
strategy and includes the crop cultivation on the salt effected fields. To employ this
approach an understanding of the plant responses to salinity and its mechanism
that, make some species/genotypes more tolerant than others are essential. Various
varieties of wheat do not equally respond to soil salinity. Some varieties have been
observed very sensitive to soil salinity while others have shown high salt tolerance (
Ashraf and Khanum, 1997 ). Thus there is a need to screen out or evolve wheat
cultivars suitable for cultivation in saline areas. During the last few years, “in-vitro”
selection for salt tolerance has been adopted as a technological solution to salinity
problem. ( Blumwald and Poole, 1987, Khan and Qureshi,1992 ). Cell lines of the
major crops having desirable characters, such as salt resistance, can provide the
base for the regeneration of plants and provide an insight into the mechanism of
tolerance at cellular level.
6
In vitro somatic cell and tissue culture technologies have been developed to
assist plant breeding. During the last few years tissue culture methods have been
emerged that can be under taken to improve the field crops. The cell, tissue and
anther culture as tools to use in the betterment of crop plants has now been
recognized (Green, 1977; Vasil, 1987 ). The regeneration of a complete plant is
possible today from major cereal crops; such as bread wheat, maize, rice, and
barley ( Redway et al., 1990; Vasil et al., 1990; Duncan et al., 1985; Yamada et al.,
1986; and Luhrs & Lorz, 1987 ).
The first successful tissue culture in cereal from endosperm was done by La
Rhu in 1949.. Gamborg & Eleveigh (1968) produced solution cultures of wheat using
a medium comprising of mineral salts supplemented with sucrose, vitamin B and
2,4-D. Shimada et al. (1969) established callus and single cell cultures in wheat.
Since the first report on immature embryo culture of maize (Green and Phillips,
1975) extensive documented reports in a whole range of species ( Larkin and
Scowcroft, 1981) have shown undesirable genetic and cytogenetic variation between
regenerated plants and the starting material. However, some work gave the details
on the production of agriculturally useful in vitro selected traits of agronomic values
(Sibi and Fakiri, 2000 and Alveraz et al.,2003). Regenerated plants varied for a wide
range of agro-morphological characters. The variability associated with cell and
tissue culturing has been somaclonal variation (Larkin and Scowcroft, 1981). They
further told that it might be due to the reflection of heterogeneity between the cells
and explants tissue, or a simple representation of spontaneous mutation, and are
due to the activation by cultural environmental transposition of genetic materials.
7
Clonal variation had been demonstrated many times in cereals and forage
crop for both physiological/biochemical and structural/morphological characteristics.
These changes are typically undesirable, but the occasional appearance of traits not
found in existing population more than compensates for the large populations of
unusable regenerants. Much of the literature simply reports variation following
cloning in tissue culture with little attempt to determine the importance of the
variation or its heritability. Limited research has been conducted to compare
mutation breeding with chemical mutagens or irradiation to generation of variation
through tissue culture. Clonal variation is most useful for breeding programmes
where the desired trait are not known naturally form available germplasm or is not
readily available. Examples are salt tolerance, tolerance to mineral toxicities,
tolerance to various chemicals including herbicides, tolerance to cold or heat,
tolerance to certain pests and pathogens, and variation in useful physiological
characteristics. Much of the useful of clonal variation is that the variations are
generated under in vitro conditions, which allows for in vitro selection among millions
of cells for specific characters. Large scale field screening of plants and progeny is
usually limited by available space and labour. Clonal variation in general also has
less deleterious effects on mutated plants than chemical mutation or irradiation.
Clonal variation also can be combined with the use of chemical mutagens in culture
or irradiation of explant materials before placing in tissue culture. The amount of
colonal variation among culture varieties and lines within a crop varies greatly, with
some germ plasm highly variable and some tissue culture stable. High variability in
tissue culture appears to be a heritable trait (Ahloowalia, 1982).The amount of clonal
8
variation in a crop should be greatly increased by including highly variable germ
plasm in crosses, by culturing explants from F1 or F2 plants from these crosses, and
by including chemical mutagens in the tissue culture medium or by irradiating
explants before plating.
This study is mainly is related to understand the possible useful mechanisms
which may be found in both wheat and wheat-somaclones that may be exploited to
facilitate the process of breeding or salinity tolerance. The major objectives of the
study were:
1. Exploitation of somaclonal variation for creation of genetic variability and
induction of salt tolerance.
2. Determination of salt tolerance mechanism (s) in newly developed
somaclones.
3. Development of multiple salt tolerant variants for making suitable cultivars in
all kinds of salt affected areas.
The thesis being presented is written in accordance with the rules of the G.C.
University. Faisalabad and divided / subdivided in various sections which sometimes
appear repetitive. This has done with the hope that it makes the thesis better
understandable for the readers.
9
CHAPTER 2
REVIEW OF LITERATURE
The response of plants to soil salinity is of great concern to the
Agriculturists. To meet the food requirements of the human beings, problem of
salinity become more severe every year as the non-saline soils and non-saline
waters are being exploited more intensively. Further, extension of agriculture is
imperative through the cultivation on saline soils using irrigation water with
relatively higher soluble salts and sodium absorption ratio (SAR).
Salts may affect plant growth directly through disturbing the water and
nutrient balance of soils and plants, causing specific ion effects and indirectly
through detereorating the soils for plant growth.
Two techniques have been adopted to tackle soil salinity problem. The
first one is to amend the soil conditions to favour the plant growth. The second
one is to use the genetic potential of plants to adopt adverse soil conditions. The
former is a lengthy process and a very few results have been obtained due to
either for economic reasons i.e. high cost of amendments or for lack of the
availability of fresh water. However the latter is short term strategy to exploit the
genetic potential through soma clonal variation in plants for the development of
salt tolerant cultivars through tissue culture (Gandonou et al., 2005).
10
2.1 Effect of salinity on growth parameters and yield components
Salinity is the most important environmental factor that leads to severe
crop loss, especially in the arid and semi-arid lands. The inherent roots of
adaptability to salinity tolerance lie deep in the evolution of the flora of salt
affected areas. Even then the majority of the plants are sensitive to salts,
particularly those which cannot tolerate the saline conditions permanently.
Salt tolerance is a complex phenomenon which varies not only among
species but also depends upon the environmental conditions, for example,
temperature, relative humidity, air pollution, fertility, salinity and water availability
( Maas, 1986 ).
Pessarakli et al., (1991) reported that dry matter yield and number of tillers
decreased with increasing salinity in both wheat and barley. Wheat was more
severely affected than barley. Grieve et al. (1992) reported that number of tillers
per plant , spikelets per panicle and number of seeds per penicle decreased in
wheat as the salinity increased.
Khan et al. (1992) studied the salt tolerance of eight wheat varieties under
sodic soil field conditions. It was observed that number of spikelets per spike,
1000 grain weight, straw and grain yield decreased significantly with increasing
salinity/ sodicity levels.
Begum et al. (1992) found that NaCI stress consistently decreased
the rate of germination in wheat. Due to increase in salinity accumulation of Na+
and Cl- was increased and K+ accumulation was decreased in germinating seeds.
11
Parveen and Qureshi (1992) imposed incremental NaCI stress to seven
days old seedlings of wheat in order to determine the toxicity levels of Na + and
Cl- in the leaf sap of two wheat varieties Pb-85 (sensitive) and LU-26S (tolerant).
Concentrations of Na+ and Cl- present in the leaf giving 50% reduction in fresh
weight of shoot and root were tentatively taken as toxic levels for respective ions.
Accordingly the toxic level of Na+ in both varieties were around 250 m mol kg-1,
whereas corresponding value for Cl- was about 300 m mol kg-l indicating that Na+
was probably more toxic than Cl- in wheat.
Liu and Liu (1993) studied the effect of NaCl stress in barley and found
that increasing NaCl concentration resulted in increased root dry weight but
shoot dry weight decreased.
Deleon et al. (1995) introduced a rapid in vitro screening of bread wheat
with various levels of salts. After eight days seedlings were evaluated for height,
root length and root number. The test showed tolerance to NaCl at concentration
of 50mm and 100mM.
Ashraf and Leary (1996) also reported decreased in all growth parameters
.i.e. height of stem, number of grain, length of ear, biomass weight / plant in
wheat as the salinity levels increased.
Hamada (1996) demonstrated that wheat growth in pots in 2: 1 clay: sand
mixture subjected to different salinity levels (0-160 mM NaCl) and moisture
contents (20-80%). Growth was decreased by increasing salinity and decreasing
soil water. Root and shoot water contents were mostly unaffected. NaCI up to 80
12
mM increased the net photosynthetic rate (PN) while it was decreased by the
highest salinity level and the most severe drought. The content of Na+ in the
shoots and roots of wheat increased as the salinity increased.
Grattan and Grieve (1999) suggested that crop performance may be
suffered due to nutritional disorders induced by salinity. These disorders occurs
by the effect of salinity on minerals availability, competitive ionic uptake, transport
/ partitioning within the plant cell. Salinity can affects the nutrient uptake such as
Na +, reducing K+ uptake or by Cl- reducing N03- uptake. Salt stress can also
caused a combination of complex interactions that affected plant metabolism,
susceptibility to injury or internal nutrient requirement.
Qing and Cheng (1999) investigated the differences in Na+, K+
accumulation between the mutant and the wild wheat (Triticum aestivum L ). The
wild type accumulated more in the root and leaf than the mutant under NaCl
stress. There in Na+ deposited in leaf was more significant than the root. The
mutant had a less accumulation rate of Na + than the wild type during the stress.
K+ contents in the leaves and roots of both cultivars reduced severely when
subjected to NaCl stress, but the content in the leaf and root of the wild type were
lesser than the mutant one.. The Na+ distribution in the seedlings of the mutant
and the wild type differed significantly.. When subjected to salt stress for 96 h,
the quantity of the accumulated Na+ in root was 44.3% of the total Na+ per
seedling of the mutant, whereas it was 24.3% in the wild type, which likely
resulted from the reduction of Na+ transfer from roots to shoots in the mutant.
13
Schachtman and Liu (1999) concluded that potassium uptake is essential
for plant nurishment but under the problem soils sodium has a competition with
potassium for uptake across the plasma membrane of plant cells. This happens
in high Na+: K+ ratios and the plant growth is reduced and become toxic.
Akhtar et al. (2000) conducted a pot experiment to determine the effect of
salinity and waterlogging on five wheat varieties. The experiment was comprised
of four treatments,.i.e.,NaCI altered nutrient uptake, due to an accumulation of
Na + and Cl- contents both in shoots and roots. In particular, Adamello showed
higher decrease in the Ca2+ of roots and the K+ of shoots and a higher increase
in the Na + of shoots. Even though both cultivars reacted to the stress, cv.
Adamello was more sensitive to NaCI.
Naseem et al. (2000) conducted a solution culture experiment in a green
house to screen wheat genotypes against salinity. The experiment was
conducted by growing forty wheat genotypes. There were three treatments viz.
control (non saline), 100 and 200 mol m-3 NaCI. Salinity was imposed gradually
and plants were harvested after forty days stress. An increase in salinity reduced
the vegetative growth significantly. Genotypic BWN-75 proved to be tolerant at
both the stress levels due to exclusion of Na + and Cl-, as it maintained lower Na
+ and Cl- in its leaves. The genotypes PARC-N1, PARC-N2 and Bakhtawar were
also classified as tolerant and attributed to better management of these ions.
Iqbal et al., (2001) observed that the contents of sodium and chloride in
leaf sap increased while that potassium ions decreased under salinity as
compared to control. Among the genotypes, Pb-25, Pb-28, SARC-6, KLR-I-4 and
14
Bakhtawar stopped the uptake of Na+ and favoured K+ and thus maintained high
K+: Na+ ratio.
Alem et al. (2002) cultivated seven varieties of both bread and durum
wheat each in three different places in the area of Errachidia (southeastern
Morocco). These were differed by the degree of salinity in irrigation water.
Results obtained showed that the reduction in leaf area was major mechanism
that made it possible to tolerate the effects of the reduction in the availability of
water under saline conditions in Bread wheat, that in turn, limited the reduction
in leaf area.
James et al. (2002) grew two tetraploid wheat genotypes (Wollaroi and
Line 455) that differed in the degree of salt induced leaf injury in 150 mM NaC1
for 4 weeks, to determine the characters that affected tolerance to higher internal
salt concentration. Shoot biomass of both genotypes was substantially reduced
by the increase in salinity, but genotypic differences were seen only after 3
weeks, when durum cultivar Wollaroi exhibited greater leaf injury and a larger
decrease in biomass than line 455. Salinity caused a greater reduction in
stomatal conductance of both varieties. The most responsive indicator of salinity
stress was g(s), followed by CO2 assimilation.
Rivelli et al. (2002) conducted an experiment by using four wheat lines
with different values Na + exclusion to find out that whether less Na + uptake had
an ill effect on water relations or growth rates under saline conditions. Plants
were grown hydroponically with and without150 molm-3 NaCl, and samples were
collected for the measurements of water relations, biomass, stomatal
15
conductance, and ion accumulation. After 4 weeks salt, decreased the biomass
in all varieties up to 50%, while the effect of salinity on relative growth rate
confined largely to the first week. Little difference was noticed between
genotypes in the effect of salinity on water relations, as indicated by their relative
water content and estimated turgor. This concluded that selecting lines with less
Na + accumulation for the purpose to improve salt tolerance is unlikely to
introduce limitations for osmotic adjustment.
Rajpar and Sial (2002) reported that ten hexaploid wheat cultivars, namely
Tonic, LU-26S, Q-19, 'KRLl-4, Kharchia-65, SARC-l, PAK-81, KTDH-19,
Cadenza and NIAB-20, were grown up to flag leaf stage in NaCl solution at
120mol/m3. Q-19 was significantly shorter, without tillers and having significantly
less shoot dry weight than the other cultivars. Kharchia-65, KTDH-19 and
Cadenza were taller, with higher shoot dry weight and had more tillers and main
stem leaves than most of the cultivars. Compared to other cultivars, poor
performance of Q-19 was associated with higher Na + concentration and less K+/
Na + ratio. Better performance of Kharchia was associated with less Na + and Cl-
concentration and high K+ /Na+ ratio t in the flag leaf sap, however, this trend was
not observed in KTDH-I9 and Cadenza.
Akram et al. (2002) studied the effects of various levels of salinity (10,15,
or 20 dSm-l) on the yield and yield components of salt-tolerant, medium tolerance
and susceptible wheat varieties in a pot experiment. Salinity reduced the spike
length, spikelets , grains per spikelet, grain weight, and yield per plant. The
susceptible variety was the most adversely affected.
16
Munns et al. (2003) conducted salt tolerance studies in the genus Triticum
and found that it is related with less contents of Na+ in leaves. Durum and other
tetraploid wheats generally have greater accumulation of Na+ as compare to
bread wheat, and are salt sensitive, but a durum wheat landrance line 146 was
found to havig exceptionally lesser leaf Na+contents. Generations were
developed by crossing 149 with high Na+ accumulation variety Tamaroi, as well
as between 149 and a durum landrace with greater Na + accumulation line 141.
The third leaf of parent varieties, F 1, F2, and low and high-selected F2 and F3
progenies were taken to determine Na+ uptake when grown in 150 mM NaCl.
Sodium contents were significantly less in the lower Na+ uptake Line 149
compared with high Na+ uptake Tamaroi (5-fold higher Na+ accumulation) and
Line 141 (7 -fold greater Na + accumulation). There was no inherent effect on Na
+ accumulation.
Hussain et al. (2003) studied six durum wheat lines with different sodium
concentrations in leaves to determine the effects of sodium exclusion on leaf
length and biomass production under saline soil. Leaf chlorophyll content, plan
height, ion concentration and dry matter were studied at 3 salinity levels (1, 75,
and 150 mM NaCl, . Yield and yield components were recorded on 2 different
groups of genotypes. Low Na + content lines showed much higher chlorophyll
retention than those of higher Na+ lines, the initiation of leaf senescence being
extended for a week or more under the low Na+ genotypes. The was greater
difference was recorted at 75 mM NaCl. At ear emergence, the salinity effects on
biomass were less in the low Na + than in the higher Na+ genotypes at 75 mM
17
NaCI, but no difference was observed between groups at 150 mM NaCI. At the
mature stage, salinity showed a similar trend on biomass of both genotypes at
both 75 and 150 mM NaCI. Grain yield at 150 mM NaCI was also reduced
equally in both the genotypes, being only 12% of controls. But , at 75 mM NaCI
level, there was a significant yield difference between these genotypes; yield
having high Na + genotype was only 70% of the control, whereas yield in
genotype having less Na+ was 88% of controls. The higher yield of in low Na +
genotype was because of more number of grains and grain weight in the tiller .
Iqbal (2003) carried out a pot study to see the effect of two salt species,
i.e., NaCI and Na2 SO4 on growth and physiology of spring wheat. Na+
concentration levels in both the salt species were 0, 50, 100 and 200 mM. A
significant difference was noticed in case of osmotic pressure , which is
indicates that NaCI had a greater effect on osmotic pressure. There was no
significant effect of two salt species on K+, Na + contents and K+ /Na + ratio. A
significant increase in osmotic pressure and Na+ content in the sap was
observed with the increase in Na+ concentrations whereas leaf area, K+ contents
and K+ /Na+ ratio decreased with as the Na + concentration increased.
Munns and James (2003) followed rapid and effective greenhouse
screening techniques that CaC identify genetic variation in salinity tolerance. A
set of previously unexplored tetraploid wheat genotypes, from five subspecies of
Triticum turgidum, were used in a case study for developing and validating
glasshouse screening techniques for selecting for physiologically based traits
that confer salinity tolerance. Short-term experiments (l week) measuring either
18
biomass or leaf elongation rates revealed large decreases in growth rate due to
the osmotic effect of the salt, but little genotypic differences, although there were
genotypic differences in long-term experiments. Specific traits were assessed.
Na + exclusion correlated well with salinity tolerance in the durum subspecies,
and K+ 1 Na + discrimination correlated to a lesser degree.
Saqib et al. (2004) conducted a pot trial using sandy clay loam soil during
2001-2002 to examine the interactive effects of soil compaction; salinity and
waterlogging on grain yield and yield parameters in two wheat (Triticum
aestivum) genotypes (Aqaab and l\1H-97). Compaction was done at 10%
moisture level by dropping 5 kg weight, controlled by a tripod stand for 20 times
from 0.6m height on a wooden block placed inside the pots filled with. Soil bulk
density of non-compact and compact treatments was acheived as 1.21 and 1.65
Mg m3, respectively. The required salinity level (15dS m-I) was developed
artificially by mixing the required amount of NaCl in soil before filling in the pots.
Mean reduction in grain yield was 44% under non-compact saline conditions
against 76% in the compact saline conditions. Similarly, the reduction about 20%
more for 100 grain weight and shoot length, 30% more for number of spikelets
per spike, 37% more for number of tillers per plant, and 32% more for straw
weight in compact saline treatment than in non-compact saline treatment was
noticed. Reduction of 36% in grain yield was caused by compaction alone.
Poustini and Siosemardeh (2004) used 30 genotypes of wheat (Triticum
aestivum L.) to check the leaf and grain K+/Na+ ratios and their ion selectivity
responses under NaCI salinity in a pot experiment. Significant correlations was
19
found between grain dry matter and some ionic traits which indicated that, in
spite of the adverse effects of the higher leaf Na+ contents on grain dry matter,
the mechanisms involved in the rate and/or duration of grain filling may be more
effetivet in terms of salt tolerance where grain yield is concerned. The earlier 11
cultivars could therefore be considered as the most completely salt resistant
cultivars. In genotype Kharchia-65, salinity did not affect the grain K+/Na+ ratio
and ion selectivity, but the grain filling period and harvest index of it were
reduced under saline conditions.
Ali et al. (2005) tested the wheat varieties of different origin including
commercial varieties of different provinces of Pakistan i.e., Punjab, KP and Sindh
under different salinity levels in laboratory as well as in saline field conditions in
diverse ecological zones. firstly, 16 genotypes were tested for germination at 6
different salinity levels ranging from 0-25 dS m-I (2, 5, 10, 15, 20, 25) EC = dS
m-1. Then, out of 16 genotypes, 11 were studied for the relative growth rate
under different levels of salinity and after their study in the laboratory, 9
genotypes were selected to test in the naturally saline areas of Punjab province.
in germination test, the varieties viz. Pasban-90, Sarsabz, Bakhtawar, 93032 and
933118 were less affected than other cultivars. On overall yield performance
basia, the Sarsabz cultivar produced the greater seed yield (4.37 t/ha). By These
findings, it is indicated that the genotypes viz. Sarsabz, Bakhtawar and Pasban-
90 are better resistant to saline environment as compared to other cultivars.
Katerji et at. (2005) studied that salt resistant variety Cham-I, developed
by ICARDA, produced a greater grain yield than the less salt tolerant cultivar
20
Haurani, in a lysimeter experiment but the main parameters responsible for the
pasta quality were declined considerably. Three irrigation water of different
qualities were used .i.e; fresh water as a control with EC of 0.9 dSm-1 and two
saline waters with the EC of 4 and 8 dSm-l, developed by mixing equivalent
amounts of NaCI and CaCl2 to fresh water. Salinity had a little positive effect on
the grain quality of variety Cham-l , but Haurani variety showed no salinity effect
on the grain quality.
EI-Hendawy et al. (2005) conducted a pot experiment by using thirteen
wheat genotypes grown in soil under different four salinity levels (control, 5, 10
and 15 dS m-I). At vegetative stage number of tillers, number of leaf and leaf area
per plant were recorded. Dry weight per plant at vegetative, reproductive and
maturity stages were calculated while yield components of main spike and total
grain yield at maturity stage were determined. The results revealed that number
of tiller was affected greater by salinity than the leaf number and leaf area at the
vegetative stage. Salinity decreased the dry matter per plant significantly at all
the growth stages. Number of spikelet on the main stem were decreased much
more with the increase in salinity than spike length, number of grain and 1000
grain weight at maturity stage.
Houshmand et al. (2005) conducted experiment by using eight durum
wheat cultivars including three salt-resistant and one salt-sensitive variety. The
study was done under both the saline and non-saline field conditions as well as
under glasshouse condition with saline solution culture of 0 (control), 75 and 150
mol m-3 NaCl concentrations. Data on days to heading, days to maturity, plant
21
height, number of grains per spike, grain weight per spike, 1000 grain weight,
number of spikes , grain yield and harvest index were recorded in the field
conditions. While plant dry weight, Na+, K+ and Ca+2 accumulated in the
hydroponically grown seedlings were measured after 20 days of salinity
application. In the field conditions, salinity significantly reduced (P < 0.0 I)
means of all the traits averaged on eight tested wheat genotypes.
Basal et al. (2006) reported that increasing soil salinity is becoming a
serious problem in the agriculture areas of over the world wide. The object of this
research was to find out salt-tolerant CRS accession (s) that would serve as
parental material for further salt-tolerant (M-9044-0031, M-9044-0061, M-9044-
0140 and M-9044-0150) and three putative salt-sensitive CRS accessions (M-
9044-0060, M-8744-0091 and M-8744-0175) plus Acala 1517-77, Deltapine 50,
TAM 94 /L-25 Stonville-453 and Nazilli-84 were exposed to two different salt
concentrations (125 and 250 mol m-3 NaC1)and control in completely
randomized design with ten replications. The significant differences were found
among the cotton genotypes for reduction in shoot dry weight (SDW), total dry
weight (TDW), leaf area (LA) and plant water content (PWC), SDW, TDW and
PWC were correlated positively regardless of treatment, indicating that either trait
could be used as a selection criterion. TAM94L-25, M-9044-0060, M-8744-0091
and M-9044-0140 may provide parental material for salt tolerance in upland
cotton breeding.
Hu et al. (2006) Examined the short term effects of salinity and drought
on nutrient imbalance in wheat seedlings. Wheat was sown in the greenhouse in
22
soil under saline and drought conditions for 26 days after the sowing. After
harvesting, it was observed that the reduction in plant height, accumulative
evapo transpiration, and shoot biomass under drought was quite similar to under
salinity as compared to control plants.
Eker et al. (2006) tested salt tolerance of 19 hybrid maize (Zea mays L.)
cultivars in nutrient solution culture at early growth stage under controlled
conditions. For the salinity stress treatment, NaCl was applied to the nutrient
solution at a concentration of 250 mM for 6 days before the harvesting. The
harvesting of plants wase done after the 17 days of growth and assesed for the
shoot and root dry matter production, severe leaf damage .i.e; necrotic patches
on older leaves, and the potassium (K+), sodium (Na+) and calcium (Ca+2)
contents in the roots and shoots. There were great differences among the
varieties in response to the NaCI treatment. The development time and severity
of leaf symptoms caused by 250 mM NaCI were different in different varieties.
On the base severity in leaf symptoms, the genotypes Maverik and C. 7993 were
found as the most resistant and sensitive varieties, respectively. Decreases in
the shoot dry matter production as a due to the NaCI treatment were gtreater
than the reduction in root growth. There was also a profound genotypic variation
in the concentrations of K\ Ca+2 and Na + in roots and particularly in shoots as
well. The higher salt tolerance in maize varieties based on the severity of leaf
symptoms was due to with significantly lower Na+ concentrations in shoots. The
K+/ Na+ and Ca2+/ Na+ ratios were also significantly greater in most of the
resistant varieties. The results show the extent of a large varietal variation in
23
tolerance to NaCI stress in maize that should be utlized in breeding programs
with the aim to develop maize cultivars with high NaCl tolerance during the early
stage of growth. Among the ions determined, shoot Na+ contents were a reliable
screening parameter in sorting varieties for their tolerance to salinity stress.
Ruan et al. (2007) conducted an experiment to study the differential
effects of less salinity on the growth and ion concentrations in the mainstem and
the subtillers of wheat crop; two spring wheat (Triticum aestivum L.) cultivars
(sakha 8 and Thasos) were sown in a greenhouse in soil with and without
salinity. The results told that dry weight and leaf area in the subtillers (T1 and T2)
at day 55th after sowing were greatly reduced by increase in salinity. Salinity
affected the subtiller growth more while the main stem was less affected during
the vegetative stages of growth. . Both the wheat cultivars were more sensitive to
salinity stress at vegetative stages than to the reproductive stages. The salt
resistant cultivar sakha 8 is characterized by the exclusion of sodium ions in the
leaves. Thus, under moderately saline conditions the more reduction in subtillers
may be resulted from an ionic imbalance in the salt-tolerant variety Sakha 8 and
from sodium toxicity in the salt-sensitive cultivar.
Thalj and Shalaldeh (2007) conducted an experiment to screen wheat and
barley genotypes for salinity resistant, 10 bread wheat, 12 durum wheat and II
barley genotypes were planted under saline conditions. Salinity was ranged
(20.6-21.9 and 4.5-5.5 dS m-1) for both soil and water, respectively. In wheat
genotypes potassium (K+) content, (K+/Na+) and Sodium (Na+) have showed a
strong positive correlation with seed yield in wheat genotypes, respectively
24
whereas, chloride (Cl) showed a strong negative correlation with the seed yield.
In barley genotypes, phosphorus and potassium have a strong negative
correlation with the biological yield respectively and with the straw yield,
respectively at the three leaf stage.
Adiloglu et al. (2007) determined the effects of different levels (0, 15, 30
and 60 mM) of NaCl and KCI salt on seedling growth and some biological
indexes of wheat, shoot height, stem diameter, leaves number of plant, fresh
weight of shoot and dry matter weight index were investigated. The results
showed that biological index of wheat decreased with increasing salt in
comparison to control. The adverse affect of NaCl on wheat plant was obtained
higher than that of KCI. This should carefully be considered if wheat is grown
under saline soil condition
Caterina and Giuliani (2007) conducted an experiment to study the effects
of saline irrigations on grain yield and quality of sunflower. A pot experiment was
conducted for over two crop seasons on two hybrids, a standard one (Carlos)
and a high one in oil contents (Tenor) exposed to five salinity levels of irrigation
water (0.6, 3, 6, 9 and 12 dS m-I). Soil salinity was maintained for the entire crop
cycle, and leaf ion content was monitored after maturity. Tenor cultivar
accumulated higher Na+ and Mg+2 content with lower K+ values. There were no
difference between the two hybrids for CI- content. A progressive increase in leaf
Na+, K+ and Na+ /K+ ratio with increasing salinity level was noticed. Seed weight
per head, 1000 achene's weight, and number of seeds per plant and oil yield
significantly decreased as the salt stress increased in both hybrids. The
25
percentage in seed yield decrease was greater per unit increase in EC irrigation
water. As the oil fatty acid composition is concerned, the main significant
differences as a result of salinity stress were a progressive increase in oleic acid
content, from 82.2% to 86.7% fin Tenor and from 21.8% to 27.3% for Carlos
genotype, which was associated with a decrease in linoleic acid content, .i.e;
from 5.9% to 3% for Tenor and from 66% to 61.3% for Carlos line. These results
confirmed the possible suppression of oleate desaturase under salt affect.
Turhan et al. (2008) conducted a trial to study the effects of salinity on
physiological features of sunflower (Helianthus annuus L.). Three NaCl
concentrations were used .i.e; 0.5, 1 and 1.5% NaCl. The data revealed that
plant growth was decreased with the increase in levels of NaCl. Chlorophyll
concentration and Normalized Difference Vegetation Index (NDVI) were
determined in the plants by a using m spectrorediometer. A significant (r2 =
0.76) correlation between chlorophyll and NDVI values was found.
Saqib et al. (2008) Studied the interaction between the leaf ionic
composition and grain yield and yield parameters of wheat under salinity x
sodicity and salinity alone. Experiment was carried out in soil culture in pots filled
with soil. Three treatments including control (ECe 2.6 dS m" I and SAR 4.53),
salinity (ECe 15 dS m" 1 and SAR 9.56), and salinity x sodicity (ECe 15 dS m" 1
and SAR 35) were applied. They showed that in sensitive line salinity alone
decreased less grain yield as compare to the combine effect of salinity and
sodicity.
Zheng et al. (2008) performed an experiment to investigate relieving
26
senescence potential in the new genotypes. They compared two cultivars for gas
exchanges and related physiological parameters of winter wheat, DK961 (salt-
tolerant) and IN 17 (salt-sensitive) under the different salinity levels of NaCI
concentrations. During the whole reproductive period, decrease in the leaf area
index, leaf area duration, leaf relative water content, net photosynthetic rate,
stomatal conductance, pigment contents, ions contents, and dry matter
accumulation of spikes was noted in both genotypes with the increase in salt
concentrations. However, the salt-resistant lines exhibited no significant
reductions in these parameters as compared to the control (0.3% and 0.5% salt
concentrations).
It is learnt from the literature that plant height and other agronomic traits
like growth and yield parameters are greatly decreased as the root zone salinity
level increased this study was conducted to create genetic diversity through
tissue culture for salt tolerance and other growth and yield characteristics so that
it may be used for breeding more productive varieties for salt-affected soils.
2.1.1. Photosynthesis and stomatal conductance
The rate of photosynthesis, CO2 assimilation is normally reduced due to
salinity. Perhaps this reduction is due to reduction in stomatal conductance (
Sharma, 1996 ) and consequent non availability of CO2 for carboxylaton. Nosn-
stomatal restriction in photosynthesis, caused due to the direct effects of NaCl on
photosynthetic equipment independent of stomatal closure, for several plant
species has also been observed in both halophyte and non-halophyte plants
(Brugnoli and Lauteri, 1991 ).
27
Brugnoli and Lauteri (1991) reported a decline in CO2 assimilation rate
when bean and cotton plants were sown under salinity and it is direct effect of
stress on net photosynthesis. The inhibition in photosynthetic capacity caused by
salts is apparently happens due to occurrence of patchy photosynthesis.
Seemann and Critchley (1985) indicated effective compartmentation in
that, r Phaseolus Vulgaris, broke down in plants grown under higher salt
oncentration and the Cl- was essentially needed in the chloroplast-cytoplasm and
for the compartmentation in vacuole which might have reduced the efficiency if
RuBP carboxylase in vivo.
Sultana et al. (2000) evaluated the affect of salinity on rice and found that
reduction in photosynthetic rate in the salinized plants which not depended only
in the reduction of available CO2 by stomatal closing but also depended on
combine effects of leaf water and osmotic potential, transpiration rate, stomatal
conductance and other chemical constituents like photosynthetic pigments,
soluble proteins and carbohydrates.
Ashraf and Parveen (2002) examined the response of two spring wheat
cultivars, salt tolerant SARC-1 and salt sensitive Potohar to different levels of
NaCl. SARC-1 had higher stomatal conductance (gS), net photosynthetic
rate(PN) and transpiration rate than cv. Potohar at the vegetative stage, but the
cultivars did not differ significantly in water-use efficiemcy (PN/E). at the grain
development stage, SARC-1 had significantly higher Pn and gs in the flag leaf
than cv. Potohar under salinity.
28
Ismail (2003) studied the effect of different concentrations of salinity (NaCl
up to 250 mM) on three wheat varieties for germination, dry matter production
and some relevant metabolic parameters and found increasing salinity resulted in
a significant decrease in water content and higher levels inhibited photosynthetic
activity. K+/Na+ ratio decreased significantly with the rise in salinity.
Iqbal (2003) conducted a hydroponic experiment to find out the effect of
salinity in wheat crop using four salinity levels 0, 50, 100 and 150 mM NaCl. They
found that net photosynthesis (Pn) was not significantly affected by salinity during
the morning. During noon and afternoon, Pn was significantly greater at 150 mM
NaCl level than the 50 mM NaCllevel whereas the difference between other
treatments was not significant. Pn was lower at 50 mM NaCl than in other
treatments at all sampling times. Stomatal conductance (gs) was more during the
morning than the noon and afternoon as well..
Sharma et al. (2005) studied the effect of different salinity levels on two
wheat genotypes k-65 and HD-2329 in a pot experiment and found that, stomatal
conductance (g S), transpiration rate (E) and the net photosynthesis rate (PN)
were reduced with the addition of salt. The reduction was more in HD-2329 than
that of K-65. The sodium and potassium concentrations were greater in the roots
and leaves of K-65 as compare to HD-2329. Thus at cellular level K-65 has
proved salt tolerance by adjusting PN, E, gS, and K deposition in leaves along the
excessive production of antioxidative enzyme activities (SOD and POX).
Zhang and Xing (2008) determined the damage to photosynthesis caused
by salt in Arabidopsis seedlings and reported that delayed fluorescence (DF)
29
intensity and net photosynthesis rate (Pn) were decreased in with the same way
as increasing NaCl or sorbitol concentration. Seedlings Incubated under 200 mM
NaCl gained a quike and reversible decline and subsequent lower and
irreversible loss in both the Pn and DF intensity and it is ion specific.
Li et al. (2008) found out the differences in stomatal photosynthetic
characteristics of five diploidy wheat species and pointed out that the net
photosynthesis rate showed significant correlation with stomatal conductivity, and
the changes in intercellular CO2 content showed the opposite trend to the
changes in stomatal limitation, indicating that the stomatal conductivity is the
main factor limiting the photosynthetic rate.
It is quite clear from above review that salinity affects the stomatal
conductance and net photosynthesis. It is observed the both factors are less
affected in the morning time. In some cases net photosynthesis was greater at
higher salinity levels than normal soils.
2.2. Somaclonal variation for genetic variability.
Wheat breeding by traditional means has been in practice for
centuries, but no success has been found for a breakthrough to cope with the
world’s demand. The varieties thus evolved do not last longer. Long term
objectives can not be achieved unless more genetic variability is generated. In
vitro technology complements the conventional means of wheat breeding in
generating diversity when both are combined with each other. During last
decades, achievements made for the betterment and utilization of in vitro
techniques in wheat development has been enormous especially for the
30
induction of genetic variability, production of haploid and wide hybridization in
incompatible crosses. Problem of the depletion of genetic resources combined
with losses of native germplasm has lead to disappearance of genetic diversity.
So biotechnological approaches are being evaluated to induce genetic variability
and conservation of germplasm. In vitro culture exhibit somaclonal variation and
generated plants show heritable variation (Ahloowalia, 1982).
It has been concluded by several scientists that tissue culture is a novel
source to generate heritable genetic variability (Bhaskaran and Smith, 1990 and
Larkin and Scowcroft, 1981). Several changes are involved in this system, i.e.,
callus induction and proliferation, organogenesis, plant regeneration and field
testing. Substantial genotypic differences have been reported at each step
(Ahloowalia, 1982).
The exploiting of in vitro selection and transformation are all depended on
the regeneration of wheat plants from callus. Wheat embryo culture technique is
being used widely for plant regeneration (Bhaskaran and Smith, 1990).
Regeneration in plant from embryo culture calli in wheat crop is a step oriented
method comprising of callus formation, callus multiplication, organogenesis and
plant development (Mohammad,1993). Low concentration of auxin results in
better organogenesis and plant regeneration (Sharma et al., 1981).
2.2.1. Use of somaclonal variation for development of salt tolerant plants.
31
In vitro somatic cell and tissue culture techniques have been developed to
assist plant breeding. Exploitation of somaclonal variation in plants for the
development of salt tolerant cultivars through tissue culture is advantageous over
conventional techniques which is an efficient and potent method for producing
novel and useful varieties ( Larkin and Scoweroft, 1981; Gondonou et al., 2005 ).
La Rue (1949) conducted successfully tissue culture for the first time in cereal
crops by using endosperm. Gamborg and Eveleigh (1968) successfully produced
suspension cultures of wheat and Shimada et al. (1969) reported callus culture
and cell culture in the wheat crop. The literature on use of somaclonal variation
for salinity tolerance in wheat and other crops is reviewed as under.
Kishore (1985) investigated the effect of salt on callus culture of Oryza
sativa L. and found that free praline, Na+, ,Cl- and Ca+2 were significantly
increased with the increase in NaCl level. While Yang et. al., (1990) told that in
Sorghum NaCl stress reduced the relative growth of callus and the Na+ /K+ ratio
was lower in salt tolerant callus while the accumulation of Na+ and K+ was more
in salt tolerant than in less tolerant callus.
Subbashini nd Reddy (1990) studied the rice in relation to salt stress.
They found that there is a significant effect of salt stress on rice at callus level. It
significantly reduced callus growth and K+ contents, and increased Na+ , Cl- and
paraline contents.
Trividi et al. (1991) maintained cultured of different wheat varieties on
media containing NaCl and found that under stress, the callus derived from
sensitive lines showed smaller reduction in growth rate and high intracellular K+
32
than from sensitive one. Under low level of salinity the growth of resistant
cultivars was unaffected as compare to sensitive one.
Jia et al. (1993) selected a stable NaCl tolerant variant callus line of
Setaria italica. Which produced numerous regenerated plants from tolerant calli
and found that these plants grew well in medium containing 0.6-0.8 % NaCl.
Gulati and Jaiwal (1994) exposed the salt resistant callus of Vigna radiata on
medium having stress treatment, i-e., 100 mM NaCl and noted that resistant
callus retained higher level of K+ than the sensitive one. They suggested that
higher internal K+ contents impart resistance to salt stress.
Lu and Jia (1994) treated the embryogenesis callus of pearl millet initiated
from young inflorescence segments with NaCl and found that tolerant lines
showed good resistance to stress caused by NaCl. Poustini and Baker (1994)
wheat cv. Sholeh (tolerant to salinity) and Inia-66 (susceptible) were grown at 0,
2.5 or 5 g NaCl/litre. CO2 uptake by Sholeh was decreased only at the highest
salinity level. Decreases in CO2 uptake were paralleled by reductions in
transpiration and stomatal conductance in both cultivars, and it is suggested that
the change in stomatal resistance in saline conditions may be responsible for
reduced photosynthesis.
He and Yu (1995) found that with increasing level of NaCl , the salt
tolerant callus contained more soluble sugars, reduced water content and had
higher Na+ and K+ contents.
Diaz-de-Leon et al. (1995) screened excised mature embryos of 14
cultivars of Triticum aestivum for salt tolerance on MS medium fortified with 30 g /
33
litres of sucrose and 50, 100 and 150 mM NaCl. After 8 days, the developed
seedlings were evaluated for height, root length and root number. Cultivars were
classified as tolerant (0-35% inhibition compared with control values), moderately
tolerant (36-68% inhibition) or sensitive (69-100% inhibition). In relation to height
and root length, the cultivars Kharchia and Shorawaki were tolerant at 150 mM
NaCl. NaCl did not affect root number significantly. Anions (in the presence of
Na+) and cations (in the presence of Cl-) distinctly affected some parameters.
Poustini (1995) performed a pot experiments, 2 wheat cultivars (one salt-
resistant) were watered with solutions containing 0, 2.5 or 5 g NaCl/litre from 4
weeks after sowing. Salinity decreased the number of tillers bearing spikes and
relative growth rate but did not affect net assimilation rate. Flag leaf water
potential was decreased and Na+ and K+ contents were increased by salinity. It is
concluded that the effects of salts on growth and yield parameters of wheat are
due to its effect on plant ionic status and not on the rate of carbon uptake.
Barakat and Latif (1995) exposed the embryogenic calli isolated from
immature embryos of 4 wheat cultivars to in vitro selection techniques for salt
tolerance. Investigatios were done to see the effect of NaCl on selected and
unselected cell lines. The relative growth weight of callus decreased while dry
weight increased as the salinity increased in both cell lines. The selected cell
lines gave significantly higher relative growth responses compared with the
unselected cell lines across all medium protocols and in all cultivars. Na+ and Cl-
content of both cell lines increased with increasing salinity, while K+ content
decreased. The selected cell lines of each cultivar produced significantly higher
34
amounts of Na+, K+ and Cl- than the unselected callus lines at most salinity
levels.
Barakat and latif (1996) isolated embryogenesis calli from immature
embryo of four wheat varieties and subjected them to three in vitro techniques for
salt tolerance and investigated the impact NaCl on the selected and unselected
cell lines which indicated that the relative growth rate of callus was decreased as
the level of NaCl increased in both callus lines. The sodium and chloride content
of both lines were increased with increasing salinity level while K+ content was
decreased.
Lutts et al., (1996) exposed the embryo derived callus of rice cultivars
with different in salt tolerance ability to various concentration of NaCl and
reported that higher level of NaCl induced inhibition in growth, Na+ and Cl-
accumulation as well as internal osmotic potential.
Moraru et al. (1996) studied the response of Calluses derived from
immature embryos of 3 genotypes cultured in vitro in the presence of 4 g and 6 g
NaCl/litre. Mean callus weight was determined before and after treatment. Turda
81 showed the least reduction in callus weight due to NaCl treatment (33% at 4 g
and 18.57% at 6g). The other 2 genotypes, AF93-2 & Fundulea 29, showed
reductions of 52-84 %.
Ozgen et al. (1996) used mature and immature embryos of seven wheat
cultivars of durum wheat on MS medium fortified with 2, 4-D. They observed low
frequency of callus formation in mature embryos with higher regeneration
capacity as compare to immature embryos. Regeneration through embryo culture
35
was influenced by culturing medium and varietal genetic factors which can be
controlled and environmental factors which can not be controlled
Poustini and Salmasi (1997) sown two wheat cultivars, Sholeh ( known to
be salt tolerant ) and Inia 66, in the greenhouse, with 3 salinity levels (0, 2.5 and
5 g NaCl /litre) being applied 4 weeks after sowing. Both cultivars showed
significant reductions in total DM production and significant increases in DM
remobilization under saline conditions. Sholeh performed better in terms of
vegetative growth than Inia 66, whereas the reverse was true for reproductive
growth and grain production. Inia 66 showed higher grain dry weight and harvest
index than Sholeh under salinity, due to a reduction in the grain filling period.
Barakat and Haris (1998) coducted a pot experiment in sandy soil to
evaluate 7, 11 and 9 somaclonal progeny of the respective wheat cultivars Giza
157, Yecora Rojo and West Bred 911 grown under 4 levels of salt stress (1.5, 4,
6 and 8 dS m-1). Grain yield, number of spikes, 100-grain weight, plant height
and number of days to heading were significantly influenced by both irrigation
water salinity and somaclonal genotype. Additionally, spike length in Yecora Rojo
and West Bred 911 and grain weight per spike in West Bred 911 somaclones
were significantly affected by both factors. The interaction between the 2 factors
was found significant for 100 grain weight, spike length, crop height and number
of days to tillering for all 3 cultivars. Moreover, significant interaction was found
for number of grains per spike and the grain yield only in West Bred 911
somaclones, and for number of spikes only in Yecora Rojo and West Bred 911
somaclones. The reductions noticed in grain yield were 58.2, 62.6, and 64.7% for
36
Giza 157, Yecora Rojo and West Bred 911, respectively, corresponding to an
increase in water salinity from 1.5 to 8 dS m-1. The correlations between yield
and the other yield parameters and among yield components were highly
significant and clearly observed in Yecora Rojo and West Bred 911 lines.
Correlations were of a lower magnitude in Giza 157 lines. Under salinity stress,
the somaclones GS1, GS2 and GS7 of Giza 157, YS1, YS4, YS5 and YS6 of
Yecora Rojo and WS3, WS6, WS7 and WS8 of West Bred 911 gave better grain
yields and were superior for most of the other agronomic traits compared to their
parents. These results show that it is possible to produce salt tolerant
somaclones by regenerating plants from calli cultured under salt stress
conditions.
Kishor (1998) studied the effect of salt on the callus culture of Oryza sativa
L. and found that free praline, Na+ , K+ and Ca++ were significantly increased
with the increasing level of NaCl. Ochatt et. al. (1998) exposed the salt resistant
potato callus on various levels of NaCl (60-450 m M ) and recorded a higher
callus fresh weight at 120 / or 150 mM concentration of NaCl.
Taghvaii et al. (1998) studied embryogenic callus formation of different
explants (inflorescence, immature embryo, mature embryo and primary roots) in
six spring and winter wheat cultivars and effect of differet doses of 2,4-D on
callus initiation in vitro. Significant differences were found between rate of callus
initiation of immature embryos and concentrations of 2,4-D among different
cultivars, but differences between callus initiation of inflorescence and mature
embryos were not significant. The highest amount of callus formed and the
37
amount of embryogenic callus in all cultivars were produced from immature
embryos and inflorescences. However the highest regeneration rate was
achieved from 2,4-D-free medium, and increase of NaCl concentrations in
medium, caused in reduction of callus growth.
Kopertekh and Butenko (1998) selected wheat Lines resistant to salinity .
All the lines exceeded the parental controls in terms of biomass increase under
salt stress conditions.
Arzani and Mirodjagh (1999) evaluated 28 cultivars for callus production
under saline stress using immature embryo culture. Morphogenic calli were
exposed to different concentrations of NaCl (0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8 and
2.1% w/v) added to the culture medium during two subsequent subcultures (4
weeks each). Comparison of cultivars for callus induction from immature
embryos was based on callus induction frequency and fresh weight growth of
callus (FWG). While, for salt tolerance, the relative fresh weight growth (RFWG)
and necrosis percent in callus were used. There were significant differences
among cultivars for potential of regeneration from immature embryos, and
Shahivandi, a native durum wheat cultivar originating from western Iran, was
superior among the cultivars tested. FWG distinguished cultivars better than
callus induction frequency did for callus induction evaluation. Hence, a range of
FWG from 1.23 to 14.65 g was observed in Mexical-75 and Omrabi-5 cultivars,
respectively. Growing calli derived from cultivars PI40100' and Dipper 6 showed
superiority for tolerating salinity under in vitro conditions.
38
Mirodjaghe et al. (1999) investigated salt tolerance of 28 cultivars of
durum wheat using in vitro technique. Calli of immature were exposed 8 NaCl
levels including 0, 0.3, 0.6, 0.9, 1.2, 1.5 and 1.8% (w/v). Assessment of calli was
conducted after 0, 8 and 16 days of subculture in the NaCl-containing medium.
Callus growth rate, relative fresh weight growth rate of callus and necrosis
percentages of callus were measured. Relative fresh weight growth rate of callus
was most reliable. PI40100 and Dipper-6 were had the best in vitro salt
tolerance.
El-Bahr et al. (2000) developed an in vitro procedure for the selection and
characterization of salt tolerant Egyptian wheat cultivars.Callus cultures
established from mature embryos of cultivars Giza-164 and Sakha-8 were
cultured using Linsmaier and Skoog's media fortified with 2 mg 2,4-D/litre were
subcultured on the same medium with NaCl (0, 2000, 4000, 6000, 8000 and
10000 ppm) for a total of 12 weeks. Callus growth (measured as fresh weight)
and the level of salt tolerance decreased with the increase in salinity. In general,
higher growth rates, but lower salt tolerance, were recorded for the calluses of
Giza-164 than those of Sakha-8. The proline content of Sakha-8 callus tissues
increased at 6000 ppm NaCl. In Giza-164, the corresponding increase was
observed at 8000 ppm. This was attributed to the higher proline level in the
calluses of Sakha-8 when cultured in saline treatments. The increase in salinity
raised the sodium level, but lowered the potassium and calcium contents, in the
calluses of both cultivars. Plantlets were obtained from Giza-164 and Sakha-8
callus tissues tolerant to 2000 and 6000 ppm NaCl, respectively. In general,
39
calluses of Sakha-8 regenerated into plantlets more efficiently than those of
Giza-164.
Ozalp et al. (2000) investigated the in vitro photosystem II (PS-II) activity
measurements for screening hexaploid wheat (Triticum aestivum) cultivars for
NaCl tolerance by studying their responses under stress and control (without
NaCl) conditions. One from the four lines under study was 'Kharchia' known as
high salt tolerant. Wheat seedlings were sown hydroponically in environmental
controlled chambers and subjected to differennt range of NaCl concentrations
(0.034, 0.17, 0.68 or 3.42 M) for a 1, 3 or 5-day period. The doses of NaCl were
applied at the proper time so that all plants were 10 days old at harvesting stage
Cell membrane stability (CMS) as recorded by a conductivity method. The PS-II
activity values were affected badly by NaCl levels and duration of treatment. By
these both methods clear the difference between salt-sensitive and salt-tolerant
cultivars. Statistical analysis explainedthat PS-II activity and CMS measurements
were well correlated (r=0.7589) It was suggested that PS-II activity can be used
as an extra screening toal besides CMS to determine salt tolerance of wheat.
Almansouri et al. (2000) studied the behavior of embryo derived calli from
three durum wheat varieties in the presence of NaCl (0-300 mM ) . the observed
that in all treatments callus growth (fresh and dry weight of callus) was not
affected in stress sensitive calli. Salt stress increased the K+ concentrations at
the callus level in all the cultivars.
Rus et al. (2000) studied the changes in the salt responses created
through long term callus cultures of cultivated tomato species ( Lycopersoin
40
esculent Mill. ). They found that callus relative growth rate and cell volumewas
tented to decreas with subculturing under both control and saline media (100
mMol/L NaCl ), although reduction in growth was much higher under stressful
medium.
Abdel-Hady et al. (2001) produced salt tolerant callus culture of three
barley (Hordeum vulgare) cutivars from immature embryo on MS medium
supplemented with different NaCL levels (4000, 8000 or 12000 ppm). They found
that growth rate of callus decreased as the salinity levels increased in the culture
medium. Callus growth rate was significantly different among tested cultivars
.
Almansouri et al. (2001) studied the deleterious effects of NaCl and PEG
on isolated wheat embryos and whole seeds germinated onto an in vitro
Linsmaier and Skoog (LS) medium and concluded that stress inhibition of
germination could not be attributed to an inhibition of mobilization of reserves and
that the major effect of PEG occurred through inhibition of water uptake. The
detrimental effects of NaCl can be linked to long term effects of accumulated
toxic ions. The behavior of the three cultivars during germination did not fully
reflect their mean level of putative stress resistance in field conditions, and
germination is, therefore, not recommended as a reliable selection criterion for
breeding purposes.
Bezerra et al. (2001) investigated the impact of different NaCl
concentrations on embryogenic calli of maiz. However, at the highest
concentration tested (150-200 mM/L), lower growth as well as lower praline
41
accumulation was observed. They reported reduction in growth at high
concentration of NaCl in the medium.
Poustini and Ciocemardeh (2001) conducted a pot experiment to
determine the impact of salt stress on the ion distribution patterns in three wheat
(Triticum aestivum) cultivars. Sodium chloride treatments (0.0, 2.5 and 5.0 g
NaCl/litre water) were applied to cultivars Inia-66, Tabasi and Sholeh. Na+
content of the plants increased under salt-stressed conditions, especially during
the grain filling period, while that of the K+ decreased under the same conditions.
The selective system of K+ over Na+ from roots to stems showed almost no
resistance to salt stress, but those from stems to the leaves and from stems to
the grains showed significantly higher efficiencies, resulting in higher K+/Na+
ratios in the grains and leaves under salt-stressed conditions. In the susceptible
Inia-66 cultivar, a higher K+/Na+ ratios were observed from roots to stems, but at
the starting point of the leaves and grains, the two salt-resistant cultivars showed
higher efficiencies for K+/Na+ selective system. It is concluded that the selective
system in ion transport between different plant parts may be considered as a salt
resistance index, which is more effective between stems and grains.
EL-Bahr et al. (2001) developed in vitro procedure for the selection and
characterization of salt tolerant Egyptian wheat cultivars and found that callus
growth (measured as fresh weight) decreased with the increase in salinity. In
general, higher growth rates, but lower salt tolerance, were recorded for calli
production. Plantlets were obtained from callus tissues tolerant to 2000 and 6000
ppm NaCl, respectively. A less genetic depended in vitro regeneration method
42
able to produce multiple shoot clusters and whole plants in four different wheat
cultivars were reported by Ahmad et al. (2002). All four genotypes were positively
responsive to shoot multiplication depending upon media composition.
Sudyova et al. (2002) tested embryogenic calluses derived from immature
embryos of three wheat (Triticum aestivum) cultivars and two triticale
(Triticosecale) genotypes for salt resistance in vitro. The 5 genotypes were: Ilona,
Hana, 8167, Ra-Tc-15 and Lasko. Tested callus culture medium was fortified
with 3, 6, and 9 g L-1 of NaCl. To weigh whole calluses in aseptic conditions as a
non destructive method was used to calculate a weight increase of callus. Ilona
showed the relative fresh weight gain of the control and tested calluses (0 vs. 9 g
L-1 NaCl) at nearly same level (25.30% vs. 20.30%). The growing calluses
obtained from wheat Ilona were found to have the highest salt resistant. After sub
cultivation on regenerating rooting medium, there were produced 9 plants, which
were cultivated to maturity. The highest salt-sensitivity was observed for in vitro
Lasko and R-Tc-15 triticale calluses. The relative fresh weight gain of callus and
regeneration potential decreased as the concentration of NaCl increased in both
triticale genotypes. The regeneration of tested Ra-Tc-15 triticale calluses was not
successful among the highest concentration of NaCl in medium (9 g l-1).
Callusing on the medium containing 12.5 mmol NaCl grew slower than grown on
medium with out added NaCl ( Tanvir et al., 2002 ).
Javed (2002A) determined the effect of salinity stress on the growth and
ion deposition in the One-month-old calluses of wheat cultivars LU-26S and
Potohar in MS liquid medium were subjected to different salt concentrations (100
43
or 200 mol m-3) to assess the effects of salinity stress on the growth and ion
accumulation in the calluses. The growth rate of calluses decreased with
increasing salt concentrations, with Potahar recording a higher growth rate
reduction than LU-26S. Callus Na+ and Cl- increased, whereas K+ increased with
increasing salt concentrations, with LU-26S recording the highest Na+ and Cl-
increment and Potohar recording the highest K+ reduction. Callus K+/Na+ ratio in
both cultivars decreased with increasing salt concentrations.
One-month-old calluses of wheat cultivars LU-26S and Potohar in MS
liquid medium were also subjected to different salt concentrations (100 or 200
mol m-3 ) by Javed (2002B) to assess the effects of salinity on the accumulation
of organic solutes in the calluses. Callus dry weight increased with increasing salt
concentrations, with LU-26S recording higher increments in dry weight compared
to Potohar. The total soluble proteins and carbohydrates, and free amino acids in
the calluses of both cultivars increased with increasing salt concentrations, with
LU-26S recording higher increments in the parameters measured compared to
Potohar.
Javed (2002C) studied one-month-old calluses of wheat cultivars LU-26S
and Potohar in MS liquid medium subjected to different salt concentrations (100
or 200 mol m-3) to assess the effects of salinity on the callus water relations. The
relative water content (RWC) of both calluses decreased with increasing salt
concentrations, with Potohar recording higher RWC reductions than LU-26S. On
the other hand, water (WP) and osmotic potential (OP), as well as turgor
potential (TP) of both calluses increased with increasing salt concentrations, with
44
Potohar recording higher WP and LU-26S recording higher OP and TP
increments.
Oudija and Ismaili (2002) tested two durum wheat genotypes (Karim and
Sebou) and two soft wheat (Sais and Merchouch) cultivars, in vitro for salt
tolerance. Salt treatments were applied during the callus initiating stage. With the
increase in salt concentration up to 10 g litre-1 resulted in a gradual decrease of
zygote germination (P_0.0001), callus embryogenesis (P_0.0001) and
callogenesis rates (P_0.0001). However, in culture media with high salt
concentration (15 and 20 g/L-1), the callus embryogenesis rate remained high.
Salt affected regeneration even when, at this stage, the culture medium was
deprived of salt. Indeed, calluses which had been initiated in 20 salt g L-1, had
the lowest regeneration rate. This concentration resulted in worthless albino
seedlings.
Muranakae et al. (2002) found out the impact of NaCl treatment on the
photosynthetic machanism in wheat (Triticum aestivum L.) cultivars differed in
salt tolerance by comparison with iso-osmotic PEG treatment. Both cultivars
similarly reduced the photosystem 2 (PS2) energy conversion efficiency
(PHIPS2) rapidly when plants were imposed to a 100 mM NaCl solution, though
no decline was detected under the iso-osmotic PEG treatment. There was no
correlation between the reduction of the leaf relative water content (RWC) and
the PHIPS2 in the two iso-osmotic stress treatments. In contrast, a decline of
PHIPS2 along with the increasing of the leaf sodium content over 4% dry matter
was detected under the NaCl treatment, while no such correlation was observed
45
with other cations. The recovery of PHIPS2 after photoinhibitory irradiation was
repressed by the NaCl treatment as the increase of the duration of the treatment.
Norin 61 subjected to the 100 mM NaCl treatment for 10 d showed a decline of
the PHIPS2 after 1 h moderate irradiation of 400 µmol m-2 s-1 PPFD. Thus the
concentrated Na+ within a leaf under salinity treatments the stability of PS2
functions may decrease and lead to photochemical inactivation.
Poustini (2002) conducted a greenhouse study to determine the impact of
salinity stress in 30 wheat cultivars (including salt-tolerant cultivars Kharchia-66,
Mahooti, Alvand, Roshan, Sorkh-tokhm, Sholeh, Tabasi, Kavir and Mahdavi).
Tap water (EC=0.6 dSm-1) was used as the control, and increasing
concentrations of NaCl in water up to an EC of 16 dS/m were given as salt
treatments. Based on the grain and shoot dry weights, seventeen cultivars
exhibited absolute salt tolerance. The significant correlations between the grain
and shoot dry weights, and grain filling period under saline conditions indicated
that extending the grain filling period, which resulted to a higher grain dry weight,
highly and positively affected the salt tolerance. The relative salt tolerance was
highest in Tabasi and Sholeh, and lowest in cultivars Atrak and Ghods. There
were some other cultivars, although not known as salt-tolerant, that showed
relative salt tolerance based on either one or both the grain and shoot growth
rates (e.g. cultivar Bessotoon). Leaf burn, particularly at 98 days after sowing,
can be taken as a rapid visual indicator of salt tolerance in wheat cultivars.
Ashraf and Shabaz (2003) screened twenty five cultivars of early CIMMYT
hexaploid wheat were evaluated for salt tolerance in a green house experiment
46
using photosynthetic capacity and water relation parameters as selection criteria.
Under salt stress (150 mM NaCl) the cultivars Frontana, Norin-10, Mayo-54,
Noreste-66, and Yaktana-54 excelled all other genotypes in shoot dry mass,
while Na(20)TPP, Inia-66, Penjamo-62, Frontana, Siete Cerros, and Jaral-66 in
grain yield per plant in both absolute and relative (percent of control) terms.
Although net photosynthetic rate (PN) declined in all cultivars due to salt stress, it
was not helpful in discriminating among cultivars according to salt tolerance.
Similarly, no positive relationships of salt tolerance of the cultivars with stomatal
conductance, leaf water potential, or turgor pressure were found. Every cultivar
used its own specific mechanism to tolerate salt stress. However, a large amount
of variation in salt tolerance observed in 25 early CIMMYT wheat cultivars can be
of considerable practical value for improving salt tolerance in the existing
commercial hexaploid wheats.
Tomar and Punia (2003) cultured mature embryos and seeds of wheat
cultivars C 306, HD 2329, UP 2338, PBW 343 and WH 533 on MS medium
supplemented with 0, 0.5, 1, 1.5 or 2% NaCl to determine a suitable medium for
in vitro salt-tolerance screening in wheat. Callus growth in WH 533 and HD 2329
was observed up to 1% NaCl concentration, whereas callus growth in UP 2338
was observed only up to 0.5% NaCl concentration. Callus growth in C 306 and
PBW 343 cultured in MS medium supplemented with NaCl was not observed.
Seed germination of PBW 343 and UP 2338 was recorded only up to 1.5% NaCl
concentration, whereas those of cultivars C 306, HD 2329 and WH 533 were
recorded up to 2% NaCl concentration.
47
Javaid-Akhtar et al. (2003) coducted a hydroponics study to determine
the salt tolerance of 20 wheat genotypes under three salinity levels .i; control,
100 and 200 mM. Seven-day-old seedlings were transplanted in thermopore
sheets with holes floating on 1/2 strength Hoagland's solution (200 litres) and
assessed after 20 days. Classification criteria was formulated according to the
salt tolerance of the genotypes based on biomass production . Salt tolerant
cultivars included 8244, 8659, 8730, B2-57 and B2-5711. Less salt tolerant
genotypes were 8290,8284, 8602-1, B4-5711, 8706-1, 8784, 8670, and 8638.
The sensitive genotypes were 8699, 5038, 8750, 8757, B4-92, B2-5713, and B2-
5734.
Zair et al.(2003) quantified somatic embryogenesis through callus culture
under salt stress conditions for 8 wheat genotypes currently cultivated in
Morocco. The genotypes were classed according to the mean number of somatic
embryos formed per immature embryo and regenerated plants per 100 explants
under saline conditions. Regenerated plants from control callus (R0-0) and callus
initiated on 10 g L-1 NaCl (R0-10) did not show significant differences concerning
plant height, spike length and grain number per ear but, the R0 plants remained
less developed than parent plants. When irrigated with a solution containing
more than 20 g L-1 NaCl, the seeds of line to Te derived from R0-10 regenerated
plants showed the best elongation of roots and coleoptiles. Moreover, a
chlorophyll fluorescence test exhibited a clear improvement in salt tolerance of
R1-10 plants at four to five-leaf stage, as compared to R1-0 plants. It is
48
concluded that plant regeneration from callus masses on high NaCl levels may
be a valid method of selection for salt tolerance.
Dang and Lang (2003) used induced mutation to exploit genetic variability
in rice cell culture to obtain salt resistant plants through in vitro selection and
found that optimum medium was well responsive to callus production in Doc Do
and Pokkali than Trang Thai Lan and KDM105 with MS+2,4-D(2mgL-1) to
embryogenic callus formation ability. Parts of the calli were transferred to NaCl
supplemented medium in an attempt to produce physiological variants from
somaclones and assesed some physiological aspects of cellular adoption in
response to salinity among cultivars selected in Soc nau, Trang Thai Lan,
AS996, these increased the %age of calli showing regeneration by 90.79%,
75.00%, 73.08%, and68.75%, respectively.
Shariatpanahi and Dabirashrafi (2003) used somaclonal variation to
produce high-yielding salt tolerant lines in barley. Embryo derived calluses were
cultured on e MS medium fortified with various doses of 2,4-D and then shifted
to the medium containing NaCl for plantlet regeneration. The %age of callus,
root and shoot were analysed and found that the genotypic effect on callus
induction was significant while the mature embryo produced the lowest callus
induction.
Khan et al. (2004) studied somaclones of sugarcane cv. CP-43/33 in the
field on saline sodic soil, and salt tolerance in these plants were evaluated based
on growth and quality parameters. The somaclones performed better than the
source plant in term of tillers per plant, stem height, number of nodes per stem.
49
Yadav et al. (2004) performed a experiment to study callus growth, to
select salt-tolerant cell lines and to regenerate whole plant from selected/non-
selected cell lines in wheat in seven genotype using NaCl @ 4,12,20,28,36& 44
dSm-1 and found that callus growth on salt-containing media was slow compared
to untreated medium. There was a continuous decrease in relative callus growth
under NaCl salinity. Evaluation of regenerated plants showed somaclonal
variation for spike length, spikelet number and grain number.
Ghannaddha et al. (2005) studies the relationship between different traits
and salt tolerance in bread wheat through tissue culture and observed that with
the increase of salt the callus size was decreased and Na+ contents of the callus
increased. Genotypes and genotypes x salt effects were significant for k+ and
K+/Na+. Similar findings were observed by Patade et. al., (2008).
Gandonou et al. (2005) studied three sugarcane varieties for in vitro salt
screening to callus induction and embryogenic callus production. The culivars
responded well to callus induction with a %age of about 82, 84 and 100% for
CP70-321, NCo310 and CP65-357, respectively. The high percentages of
embryogenic callus obtained for three varieties indicated that these lines have a
high ability for embryogenic callus production. Relative fresh weight growth of
callus was about 1.07, 1.282,and 0.925 for CP70-321, NCo310 and CP65-357
respectively. NaCl impact resulted in calli necrosis and a reduction in their
growth. However, growing calli obtained from varieties CP70-321 and NCo310
exhibited less necrosis percentage and less relative fresh weight growth
50
reduction under salt stress. They proved to be more salt tolerant in vitro than
CP65-357.
Lu, et al. (2007) established a protocol for in vitro induction of salinity
tolerant somaclonal variation, calli of tripod bermudagrass cv. TifEagle were
cultured in suspension medium. To create somaclonal variation the, calli were
subcultured for 18 months and were then subjected to three-round selection for
salt-tolerant calli by culturing on medium containing 0.3 M NaCl for ten days
followed by a recovery for two weeks. The so survived calli were regenerated on
regeneration medium containing 0.1M NaCl. Three somaclonal variant lines (2,
71, and 77) were collected and analyzed. The selected somaclonal variants
showed higher relative growth and less injury than Tifeagle under salinity stress,
indicating that they enhanced salt tolerance. In addition, they have relatively
more water content and low electrolyte leakage than Tifeagle by withholding the
irrigation, indicated that they also increased drought tolerance. The line 71 had a
higher K+/Na+ ratio under salt stress conditions, indicated that different
mechanisms for salinity resistance might exist in these three variants.
Keeping in view above information and conclusions, this study was
planned to create genetic variability and develop new cultivars of wheat by using
tissue culture techniques through callus culture for cultivation in the marginal
lands where no other crop is sown successfully. As wheat breeding by traditional
means has been in practice for centuries, but nosuccess has been made for a
breakthrough to cope with world’s demand. The varieties thus developed do not
last long. Long term objectives can be achieved unless more genetic variability is
51
generated. In vitro technology complements the conventional means of whet
breeding in generating diversity when both are combined with each other. So it is
concluded that tissue culture is a novel source to generate heritable genetic
variability to develop salt tolerant plants of wheat genotypes.
52
CHAPTER 3
MATERIALS AND METHODS
The Research pursuits on “Study of Somaclonal variation in wheat for the
induction of salinity tolerance” reported in this thesis were conducted at the
Department of Botany, Government College University, Faisalabad and partially
at the Agricultural Biotechnology Research Institute AARI, Faisalabad, Pakistan
during the year 2005-2009. This study was initiated to exploit somaclonal
variation and to benefit from its potential for wheat improvement under salt
affected soils.
GROWTH CONDITIONS AND EXPERIMENTAL TECHNIQUES
Growth conditions and techniques used in these studies in most of the
experiments are described in this chapter; other more specific to individual
experiments will be presented in the relevant chapters.
Seed source
Fifty wheat genotypes including those known salt tolerant varieties i.e. LU-
26S and Pasban-90 were obtained from Wheat research Institute, Agric.
Biotechnology Research Institute, ARRI, Faisalabad and National Agric.
Research Centre, Islamabad (Annexure-1).
53
Callus initiation
Mature seeds /mature embryo of 50 wheat genotypes ( Triticum aestivum
L) were sterilized with ethanol (100 %) followed by 0.01 % HgCl2 with
subsequent shaking for 30 seconds. Then applied three rinsing of autoclaved
distilled water under aseptic conditions. Afterwards seeds were cultured in
18x150 mm test tubes containing solidified MS medium (Murashige and Skoog,
1962) (Annexure-2) supplemented with 4 levels of 2,4-D (1, 2 ,3, and 4 mgL-1) to
induce callus & at least 10 seeds per genotype (one seed per test tube).
Prior to autoclaving, pH of the medium was maintained to 5.8 poured in
test tubes of 18x150 mm and then autoclaved at 15 psi pressure at 121o C for 30
minutes. Then the material was placed in incubation room at 28+-2oC with 12
hours photoperiod illuminated with florescent light to produced 2500 lux. Callus
induction frequency at different levels of 2, 4-D was recorded at an interval of 15
and 45 days. The responsive varieties (having 50 or > 50% callus frequency)
were subjected to different levels of salinity for further studies (Annexture-3).
The following laboratory parameters were recorded:
Callus induction
Fresh weight growth of callus(FWG)
Relative fresh weight growth(RFWG)
Frequency of necrotic calli
Regeneration
54
Plantlet production
The callus induction/ fresh weight growth of callus and frequency of
necrotic calli was calculated on percentage basis.
Relative fresh weight growth Rate
Fresh weight of the calli after harvesting from the saline medium was
determined with the help of electronic sartorious research balance (Model R-160)
and their fresh relative growth rate were calculated with the following formula:
In (FW)-In (IW).
Salinity treatment
The responsive wheat genotypes were cultured on MS medium
supplemented with 2,4-D @ 3 mgL-1 and four salinity levels to induce salt
tolerance. There were four treatments including control, and each treatment was
replicated ten times. 100 mg calli were used in each treatment. The following
treatments were studied during the course of this research work.
So Control S1 50 mM NaCl S2 100 mM NaCl S3 150 mM NaCl
55
Regeneration of plantlets
Surviving calli under salt stress were shifted to regeneration medium for
the development of plantlets. MS basal medium with out any addition of 2,4-D
was used for the regeneration of plants under same salinity levels ( Hussain et
al., 2001). The plantlets developed by this method were shifted to the pots filled
with sand and gravel under the same salinity levels under controlled conditions
up to maturity to produced Ro seeds. Then R1 plants along with parents were
treated with salt to assess their extent of salinity tolerance after following Arzani
and Mirodjagh, 1999.
The following parameters were studied at seedling stage.
Shoot fresh weight
Shoot dry weight
Root fresh weight
Root dry weight
Na+ concentration in plants
K+ concentration in plants
K+/Na+ ratio in plants
To obtain shoot dry weight, the shoots were stored in paper bags. First
these were air dried and then were oven dried at 65 +- 5oC till the weight
was constant.
56
Phenotypic measurements
On the maturity of crop, data regarding vegetative growth and yield
components were also recorded.
Plant height
Tiller/plant
Number of spike/plant
100-grain weight
Number of spikelets
Yield/plant
To study the yield components somaclones of different wheat genotypes
were grown along with their parent on artificially salinized soils in pots. The salt
levels were developed in the following way:
Original EC of the soil = A
EC to be deliverd = B
Difference of EC = B-A = C
T.S.S. = C x 10 = D meq/l
The meq/L was converted in to mg/100 g by using following relationships.
D x Eq.wt./1000 x S.P = mg/100g.
57
Plant Analysis
Plant sample collection
After uprooting the plants, soil was removed carefully from the roots. Roots
and shoots were separated and the latter were dried with tissue paper. Fresh
weight of shoots and roots was recorded immediately. The youngest fully
expanded leaf was detached, washed with distilled water, blotted and stored in
1.5 cm3 polypropylene micro centrifuge tube at freezing temperature. The leaf
was used later for tissue sap extraction. Fresh weight of shoot was again
recorded after removing the leaf and the sample was preserved in the paper bag.
To obtain dry weight, the plant shoot was dried at 70oC till the weight was
constant. Calculations were made to get the dry weight of shoot including the leaf
separated for tissue sap extraction.
Extraction of tissue sap
For the extraction of sap the plant material was frozen in 1.5 cm3 micro
centrifuge tubes, thawed and crushed by using a metal rod with a tapered end.
Small holes were made in the cap and in the base of the tube and the sap were
centrifuged out into a second centrifuged tube at about 6000xg. After further
centrifugation of second tube was done at 9000xg for 3 minutes, the sap was
diluted for the estimation for inorganic ions by following Gorham et al. (1984).
58
Determination of sodium and potassium
The tissue sap was diluted as required by adding distilled water and
sodium and potassium estimation was done by using Flame photometer with the
help of standard solutions of NaCl and KCl prepared from reagent grade salts
(Method 10a and 11a of USDA Hand book 60: Annonymous, 1954).
Physiological parameters
The following physiological data were also recorded:
Stomatal conductance
Stomatal conductance of the fully expanded leaf was measured with IRGA
(Infera red gas analyzer) from 10:30 a.m. to 1:0 p.m.
Net Photosynthesis rate
The net photosynthesis rate was also measured by IRGA (Infra red gas
analyzer).Analytical set of conditions was molar air flow per unit leaf was 335
mMm-1 S-1, atmos pressure 99.61 (Pa) PAR on leaf surface 1250 µmol m-2 S-1,
CO2 concentration 357 umol m-1 and ambient temperature for control plant was
28oC.
Statistical Analysis
Data of the experiment was subjected to statistical analysis using
completely randomized design (C.R.D.) in factorial arrangement (Steel and
Torrie, 1980) using M STAT computer soft ware. The significance among mean
and interaction values was determined by using LSD test.
59
CHAPTER 4
RESULTS
Preliminary screening of wheat genotypes for callogenesis.
Plant tissue culture has provided plant breeders with certain techniques for
creating genetic variability without involving sexual crossing. Callus cells under go
genetic changes during callogenesis and which after regeneration give rise to plantlets
genetically variable (Larkin and Scoworoft, 1981). Mature seed / embryo of fifty wheat
genotypes were cultured on solidified MS medium fortified with 4 levels of 2,4-D, i.e.,
1,2,3, and 4 mgL-1 in 18 x 150 mm test tubes (1 seed / tube). Ten replication were
followed in each genotypes for each 2,4-D levels. The rest of the procedure is
mentioned in the chapter Material and Methods. The callus initiation was recorded after
30 days of culture. After 45 days of culture callus frequency / fresh growth was recorded
(Table-1)
Responses of genotypes to callogenesis
The data reveals that all the genotypes were different from each other in
response to callogenesis. Callus induction frequency ranged from 25% to 90%. Among
the varieties maximum callus (90%) was produce by variety Uqab-2000 and AS-2002
followed by Ufaq-2002 and V-4189 with callus score of 85% and 80% respectively.
60
Minimum callus (25%) was noticed in Barani-70 and WL-711 with the callus induction
potential of 30%. All other varieties responded with ranges between these varieties.
All the wheat genotypes produced the callus at all the 2, 4-D levels. Good callus
production was recorded at the level of 3mg and 2mgL-1 2, 4-D and it was less in 1mg
and 4mg per litre. The callus produced at 3mgL-1 was more granular and morphogenic
in nature.
Interaction between varieties x 2, 4-D showed different % age of callus at
different 2,4-D levels. It ranges from 20-100 %. Ufaq-2002, SA-42, Barani-83, Uqab-
2000 and AS-2000 showed higher callus induction frequency. Less callus (20%) was
produced by Barani-70, WL-711 and V-04112. Ufaq-2002 produce maximum callus at
3mgL-12,4-D while AS-2000 at 2mgL-1. SA-42, Barni-83 and Uqab-2000 gave maximum
response at 4mgl-1 2, 4-D levels.
The objective of this study was to obtain preliminary information about callus
formation potential of 50 wheat genotypes which was done successfully. Genotypes
showing better callus response were studied further for regeneration and somaclonal
variation under 4 salinity levels. 20 genotypes that produced 70% or more callus
induction frequency i.e., Ufaq-2002, SA-42, Parwaz-94, LU-26S, Pothowar, Punjab-76,
Barani-83, Kohinoor-83, Faisalabad-85, Chakwal-86, Pasban-90, Inqulab-91, Punjab-
96, Uqab-2000, Chenab-70, Iqbal-2000, AS-2000, Bhakhar-2002, V-03079 and V-
04189 were tested for somaclonal variation study under salt stress conditions.
61
Table.1: Callus Induction frequency of 50 wheat varieties, using mature embryo as ex plant source under different 2,4-D, levels Name of Variety 2,4-D, levels Mean
1mg L-1 2mgL-1 3mgL-1 4mgL-1 Ufaq-2002 60 90 100 90 85 Mexi Pak 30 30 40 40 35 Chenab-70 40 40 50 50 45 Barani-70 20 30 30 20 25 SA-42 60 60 90 100 77.5 Blue Silver 60 60 60 60 60 Lyall Pur-73 50 40 50 50 47.5 Parwaz-94 70 70 70 70 70 PARI-73 60 60 60 60 60 Sandal 30 50 50 50 45 SA-75 50 60 50 60 55 LU-26 S 70 70 80 80 75 WL-711 30 30 40 20 30 Punjab-81 60 60 70 60 62.5 Pak-81 60 60 70 70 65 Pothowar 50 90 80 80 75 Punjab-76 60 70 90 80 75 Barani-83 60 60 80 100 75 Kohinoor-83 80 80 70 70 75 Fsd-85 70 70 80 70 72.5 Chakwal-86 60 80 80 70 72.5 Shalimar-86 50 70 70 60 62.5 Rohtas-90 60 60 70 60 62.5 Pasban-90 60 60 90 90 75 Inqlab-91 70 80 80 80 77.5 Shahkar-95 70 70 70 60 67.5 Punjab-96 60 60 90 80 72.5 MH-97 50 60 60 60 57.5 Kohistan-97 60 60 60 60 60 Uqab-2000 80 90 90 100 90 Chenab-70 80 80 80 70 77.5 Iqbal-2000 70 80 80 80 77.5 SH-2000 60 70 60 60 62.5 AS-2000 80 100 90 90 90 Sehar-2006 70 70 70 60 67.5 Shafaq-2006 60 70 60 60 62.5 Fareed-2000 50 60 60 60 57.5 Bhakar-2002 60 80 80 90 77.5 GA-2002 60 70 60 50 60 V-03094 40 50 50 50 47.5 V-03079 70 80 80 80 77.5 V-04188 30 70 40 40 45 V-04189 70 80 90 80 80 V-03138 50 60 50 50 52.5 V-04040 50 50 50 50 50 V-04067 30 60 60 50 50 V-04112 20 60 50 50 45 V-04171 30 50 60 60 50 Fsd-83 50 70 70 70 65 Punjab-85 60 60 70 60 62.5 Mean 55.6 64.8 67.6 65.22
62
Effect of salinity on callogenesis and organogenesis in 20 wheat varieties It is very difficult and time consuming to develop salinity tolerant varieties
through traditional breeding techniques. Whereas exploitation of somaclonal
variation in plants to develop salt tolerant cultivars is an efficient and potent
methods for producing novel and useful varieties (Gandonou et. al; 2005). The first
study in Pakistan on callogenesis and organogenesis in wheat under salt stress was
carried out by Mahmood and Quraishi in 1983 .Moreover, the study was restricted to
callogenesis. The present piece of study has, infact, broadened the scope of
somaclonal variation and investigations have been made on the impact of various
doses of NaCl salinity in callus formation and regeneration in 20 wheat varieties.
The mature embryo / seed of 20 wheat varieties (Ufaq-2002, SA-42, Parwaz-94,
LU-26S, Pothowar, Punjab-76, Barani-83, Kohinoor-83, Faisalabad-85, Chakwal-86,
Pasban-90, Inqulab-91, Punjab-96, Uqab-2000, Chenab-70, Iqbal-2000, AS-2000,
Bhakhar-2002, V-03079 and V-04189) which gave good response to callogenesis in
the previous studies were cultured on MS medium + 3mgL-1 2,4-D supplemented
with 4 doses of NaCl salt i.e., 0,50,100 and 150mM NaCl according to the methods
described in chapter-2. 20 test tube of each varieties having one seed per tube
were cultured on each salt level.
Callogenesis under salt stress
Differences among genotypes for callus induction potential under salt stress
were quite evident from the results presented in Table-2. Almost all the genotypes
produced callus of varying degree with and with out salt. Comparison of the cultivars
indicated earlier, the callus initiation was high in case of control (0 NaCl) and callus
63
weights ranged from 0.05169 g (Kohinoor-83) to 0.10 g (Uqab-2000), at 50 mM. The
range was from 0.039g (Pothowar) to 0.090 (LU-26S), at 100mM from 0.0280
(Faisalabad-85) to 0.0794g (LU-26S) at 150mM NaCl. The data led to the conclusion
that increase of salt in culture medium directly affect the production of callus in all
the genotypes. The callus growth at higher salt level was very low as compared to
control. Mean values showed that genotypes LU-26S proved to be more salt tolerant
followed by Uqab-2000, Inqulab-91 and V-04179 and Kohinoor-83 was the least
responsive to callus initiation. Computation of mean callus values of wheat cultivars
under cumulative salt stress given in Fig-III. Analysis of variance showed that the
differences among treatment means, genotypes mean and genotypes x treatment
induction was non significant. 10 genotypes, i.e., Ufaq-2002, Parwaz-94, LU-26S,
Punjab-76, Pasban-90, Inqulab-91, Uqab-2000, Chenab-70, AS-2000, and Bhakhar-
2002, produced granular, compact, Shiny and morphogenetic calli under salt stress
and were further valuated for relative fresh weight growth on the basis of callus
characteristics and shape. The rest ten varieties, i.e., SA-42, Pothowar, Barani-83,
Kohinoor-83, Faisalabad-85, Chakwal-86, Punjab-96, Iqbal-2000, V-03079 and V-
04189 produce necrotic calli (Table.7). The callus produced by these varieties was
loose, watery in nature and in dry form. The 10 wheat varieties (Ufaq-2002, Parwaz-
94, LU-26S, Punjab-76, Pasban-90, Inqulab-91, Uqab-2000, Chenab-70, AS-2000,
and Bhakhar-2002) that produced good granular calli under salt stress were
evaluated for relative fresh growth rate of callus under the same salinity levels.
64
Relative fresh growth rate of callus
The relative fresh weight growth of calli (Table.3) in 10 wheat varieties Ufaq-
2002, Parwaz-94, LU-26S, Punjab-76, Pasban-90, Inqulab-91, Uqab-2000, Chenab-
70, AS-2000, and Bhakhar-2002, were calculated as per formula given in chapter-3
(page-54).
Analysis of variance showed that treatment means, genotypes means and
interaction between treatment and genotype to relative fresh weight growth of callus
were non significant. Although interaction was not significant, genotype Punjab-76
showed more reduction than other varieties at all the three salinity levels, i.e., 0,
50,100 and 150 mM NaCl while variety AS-2000 reduced callus weight at 150 mM
NaCl. A trend of reduction in relative fresh growth rate with increase in salinity was
observed. Maximum callus reduction was recorded at salt level 150mM (0.1684)
followed by 100 (0.1947), 50 (0.2065) and 0 mM NaCl (0.2016)(Table-3).
Mean differences in varieties described that maximum reduction was
observed in AS-2002 (0.1831) followed by Punjab-76 (0.1884) and Chenab-70
(0.1894) respectively while minimum reduction in relative fresh weight growth was
recorded in Ufaq-2002 (0.2085). The other varieties reduced relative fresh weight
growth in between this range.
All the 10 varieties were evaluated for their regeneration potential under the
same salinity levels.
65
Table.2: Response of wheat genotypes to callogenesis under salt stress Name of Varity 0 mMNaCl 50
mMNaCl 100 mMNaCl 150 mMNaCl Mean
Ufaq-2002 0.093375 0.08926 0.079783 0.072508 0.0837315 SA-42 0.099794 0.089914 0.08417 0.074737 0.08715375Parwaz-94 0.08423 0.08049 0.07268 0.05818 0.073895 LU-26S 0.07787 0.09993 0.09037 0.0794 0.0868925 Pothowar 0.06097 0.05776 0.03905 0.003862 0.0404105 Punjab-76 0.09277 0.06374 0.07456 0.06535 0.074105 Barani-83 0.080697 0.07781 0.06727 0.05983 0.07140175Kohinoor-83 0.053406 0.05169 0.04368 0.03588 0.046164 Faisalabad-85 0.07109 0.06683 0.04842 0.02832 0.053665 Chakwal-86 0.05755 0.05655 0.05345 0.04386 0.0528525 Pasban-90 0.09962 0.09587 0.08306 0.06871 0.086815 Inqulab-91 0.1058 0.09415 0.08546 0.07123 0.08916 Punjab-96 0.05286 0.0525 0.04661 0.04257 0.048635 Uqab-2000 0.11169 0.10137 0.08889 0.07987 0.095455 Chenab-70 0.08561 0.08558 0.07824 0.07288 0.0805775 Iqbal-2000 0.0857 0.08551 0.07553 0.0713 0.07951 AS-2000 0.1007 0.09648 0.07912 0.07568 0.087995 Bhakhar-2000 0.10075 0.09425 0.08206 0.07784 0.088725 V-03079 0.10695 0.09598 0.08092 0.0781 0.0904875 V-04189 0.1014 0.09628 0.07989 0.07798 0.0888875 Mean 0.0861416 0.0815972 0.07166065 0.06190435
Analysis of variance of wheat genotypes to callogenesis under salt stress SOV DOF SS MS F Replication 9 0.008 0.001 6.0231 Salinity 3 0.124 0.041 280.4685N.S Varieties 19 0.474 0.025 169.0280N.S Salinity x Variety 57 0.214 0.004 25.4007N.S Error 711 0.105 0.000 Total 799
66
Callogenesis, Morphogenesis and Plantlet development in Wheat
67
Callogenesis in different Wheat varieties
68
Morphogenesis in Wheat
69
Development of Plantlets
70
Somaclones of different wheat varieties in pots
71
Table.3: Salinity effect RFGR of wheat Callus(mg) Name of Varity 0 mMNaCl 50
mMNaCl 100 mMNaCl 150 mMNaCl Mean
Ufaq-2002 0.2304 0.2206 0.2058 0.1774 0.20855 Parwaz-94 0.2280 0.2144 0.2042 0.175 0.2054 LU-26S 0.2248 0.2104 0.2028 0.1754 0.20335 Punjab-76 0.2068 0.1938 0.1840 0.1690 0.1884 Pasban-90 0.2244 0.2142 0.1946 0.1798 0.20325 Inqulab-91 0.2310 0.2058 0.1964 0.1742 0.20185 Uqab-2000 0.2234 0.1950 0.1886 0.1532 0.19005 Chenab-70 0.2122 0.1972 0.1934 0.1550 0.18945 AS-2000 0.2078 0.1978 0.1770 0.1500 0.18315 Bhakhar-2002 0.2272 0.2162 0.2002 0.1752 0.2047 Mean 0.2216 0.20654 0.1947 0.16842 , : Analysis of variance of RFGR of wheat callus under salt stress SOV DOF SS MS F Replication 4 0.000 0.00 2.1509 Salinity 3 0.076 0.025 500.6676N.S Varieties 9 0.015 0.002 32.3505N.S Salinity x Variety
27 0.003 0.000 2.3596N.S
Error 156 0.008 0.00 Total 199 0.102 0.00 Necrotic percentage
Data regarding necrotic calli produced under salt stress is presented in
Table.4. Salinity increased the necrotic % age in calli of wheat genotypes. As the
salinity increased, necrotic % age also increased. Among the genotypes, it is clear
that maximum necrotic % age was observed in Pothowar (35.5) followed by Punjab-
76 and Chakwal-86 with the % age of 34.5 and 34.0, respectively. While it was low
in Ufaq-2002 (4.5%). Maximum % age of necrosis (27.55) was recorded at the
salinity level of 150mM NaCl. The minimum value (15.6%) was noticed where no salt
was applied.
72
Interaction between S x V showed that all the genotypes produced necrotic in
callus masses at higher salinity levels, while it was low under control conditions.
Table.4: Necrotic percentage in callus in wheat genotypes under salt stress. Name of Varity 0 mMNaCl 50
mMNaCl 100 mMNaCl 150 mMNaCl Mean
Ufaq-2002 2 3 3 10 4.5 SA-42 20 25 26 40 27.75 Parwaz-94 5 7 7 12 7.75 LU-26S 5 5 8 10 7.0 Pothowar 30 36 36 40 35.50 Punjab-76 8 10 10 19 11.75 Barani-83 25 38 36 38 34.5 Kohinoor-83 38 30 30 42 33.75 Faisalabad-85 30 30 32 40 33.0 Chakwal-86 30 33 33 40 34.0 Pasban-90 6 7 8 12 8.2 Inqulab-91 4 9 9 16 9.5 Punjab-96 16 28 30 36 27.5 Uqab-2000 6 6 7 21 10.0 Chenab-70 7 7 7 20 10.25 Iqbal-2000 20 22 20 33 23.75 AS-2000 8 10 10 20 12.0 Bhakhar-2000 7 7 8 20 10.5 V-03079 30 30 32 40 33.0 V-04189 30 30 33 42 33.7 Mean 15.6 18.65 19.25 27.55 - Regeneration Studies in 20 Wheat Varieties under Salt Stress
This experiments were aimed at production of callus derived plants of
varieties previously screened for callogenesis under salt stress. Calli developed after
4 week of seed culture were proliferation by sub culturing 4-5 times on the same
salinity levels + 3mg L-1 2,4-D. After proliferation, calli of 20 wheat varieties (Ufaq-
2002, SA-42, Parwaz-94, LU-26S, Pothowar, Punjab-76, Barani-83, Kohinoor-83,
Faisalabad-85, Chakwal-86, Pasban-90, Inqulab-91, Punjab-96, Uqab-2000,
73
Chenab-70, Iqbal-2000, AS-2000, Bhakhar-2002, V-03079 and V-04189) were
subjected to regeneration under the same salinity levels, i.e., 0,50,100 and 150mM
NaCl. The regeneration media was comprised of MS media with out having any
auxin (2,4-D) 10 test tubes of each varieties were maintain in each somaclone
under the control environmental conditions.
The response of genotypes to plantlet development under 4 levels of NaCl is
presented in Table-5. Range of plantlets developed varies from 1 to 48 plants
among all the varieties. Maximum shoots (48) were developed by LU-26S followed
by Pasban-90(40), Ufaq-2002(31), Inqulab-91(29) and Uqab-2000(26). Minimum
shoot developed in SA-42(1).Total number of plants ranged 1 to 48. shoot
development was suppressed with increase in salinity that indicated the deleterious
effect of NaCl on morphogenetic process in wheat.
74
Table.5.Regeneration Potential of 20 wheat varieties under salt stress (No. of plants) Name of Varity 0 mMNaCl 50
mMNaCl 100 mMNaCl 150 mMNaCl Total
Ufaq-2002 15 10 4 2 31 SA-42 1 0 0 0 1 Parwaz-94 7 6 5 2 20 LU-26S 21 10 10 7 48 Pothowar 4 1 1 0 6 Punjab-76 9 3 2 1 15 Barani-83 2 0 0 0 2 Kohinoor-83 1 1 0 0 2 Faisalabad-85 4 2 0 0 6 Chakwal-86 7 1 0 0 8 Pasban-90 17 10 8 5 40 Inqulab-91 12 8 6 3 29 Punjab-96 4 1 1 0 6 Uqab-2000 13 7 5 1 26 Chenab-70 6 4 2 1 13 Iqbal-2000 6 2 2 0 10 AS-2000 12 9 7 3 31 Bhakhar-2000 10 6 6 2 24 V-03079 2 1 0 0 3 V-04189 1 1 1 0 3 Total 154 83 60 27 324
Maximum shoots developed(154) were observed where no salt was applied,
i.e., 0 mM NaCl followed by 83 and 60 at salinity levels 50 and 100mM NaCl. While
minimum plants (27) were produced at the higher salinity level of 150mM NaCl.
At control level LU-26S was at the top with maximum plant (21) while SA-42,
Kohinoor-83 and V-04189 were at the bottom with 1 plant each. At 50mM NaCl level
varieties LU-26S was also at the top at par with Pasban-90 having 10 plants each.
Minimum response to regeneration was exhibited by Pothowar at par with Kohinoor-
83, Chakwal-86, Punjab-96, V-03079 and V-04189 having 1 plant each. At 100 mM
NaCl LU-26-S once again showed best performance while SA-42, Barani-83,
75
Kohinoor-83, Faisalabad-83, Chakwal-86, and V-03079 failed to developed any
plant. At 150mM level of salinity 10 varieties SA-42, Pothawar, Barani-83, Kohinoor-
83, Faisalabad-85, Chakwal-86, Punjab-96, Iqbal-2000, V-03179 and V-04189 did
not produced any plant. While LU-26S also remained on the top at this salt level as
well.
Thus these developed plantlets were transferred on rooting medium salinized
with the same salinity levels (MS+1mgl-1 IAA). Plants of 10 wheat varieties (Ufaq-
2002, Parwaz-94, LU-26S, Punjab-76, Pasban-90, Inqulab-91, Uqab-2000, Chenab-
70, AS-2000, and Bhakhar-2002) developed roots and turned in to complete plants.
Then these complete plants having both shoots and roots were shifted to sand
culture in pots to obtain regenerator seed (R0). The R0 seeds of these varieties were
further evaluated for chemical analysis and yield components.
Plant analysis
There is no single selection criterion for salinity tolerance (Blum, 1988) that
has been tested through the process of selection with positive end results. Selection
for improvement of crop salt tolerance may be made at a number of stages in the life
cycle of a crop, although selection at seedling stage would clearly as easier and
more economical way. On the other hand, seedling or plant dry matter as an
expression of total growth is an important criterion in terms of over all yield under
stress conditions. It integrates various possible effect of response to salinity into one
measure of resistance. As root growth often expresses the relative resistance of a
76
plant to salt stress, it was also considered as a possible criterion of resistance to
salinity (Qureshi et el., 1990).
R0 seeds of 10 wheat somaclones i.e. (Ufaq-2002, Parwaz-94, LU-26S,
Punjab-76, Pasban-90, Inqulab-91, Uqab-2000, Chenab-70, AS-2000, and Bhakhar-
2002) were germinated and grown in plastic buckets containing vermiculite and
gravel (1:1 by volume) in a wire house to avoid the birds. 4 levels of salinity, non-
saline and saline (50, 100, and 150mM NaCl) were used. The control and saline
buckets were filled and drained daily. Full strength Hoagland nutrient solution were
used as the nutrient solution. The buckets were recycled with the same solution
throughout the week. Salt were mixed to the nutrient solution to develop the needed
concentration to final desired salinity levels.
10 seeds of each somalones were grown in each treatment and were split in
to 5 replication each of 2 plants. Plants were harvested 7 weeks after sowing. Fresh
weight of shoot and root were recorded while the youngest expanded leaves were
preserved in the tubes at freezing temperature for the extraction of sap. K+ and Na+
were determined from the leaf sap while shoot and root dry weight were obtained
after drying at 70oC in an oven.
Shoot fresh weight
Data given in Table.6. indicate that salinity significantly decreased the shoot fresh
weight and at all salt levels comparison of means showed significant differences
among treatment, variety and salinity x varieties.
Maximum (9.94g) shoot fresh weight was observed in control followed by 50,
100 and 150mM NaCl and it differs significantly from all other treatments. Minimum
77
fresh shoot weight (5.06g) was observed under highest level of salt. Differences in
mean in varieties showed that maximum shoot fresh weight (10.20g) was recorded
in somaclones of Ufaq-2002 at par with Inqulab-91 followed by Parwaz-94 (8.90g)
minimum shoot fresh weight (5.75g) produced by somaclone of Punjab-76 the other
varieties responded in between these range.
Interaction between S x V was also significant. Somaclones of Ufaq-2002
gave maximum shoot fresh weight (14.26 and 13.4g) at control and 50 mM NaCl
while somaclone of Punjab-76 give minimum shoot fresh weight (6.52g) at these salt
levels. In case of 100 mM NaCl variety Inqulab-91 produced maximum shoot fresh
growth weight (8.98g) and LU-26S yielded low fresh weight of shoot i.e. 5.04 g. At
higher salinity level (150mMNaCl) Inqulab-91yielded maximum fresh shoot weight of
7.84 g and Parwaz-94 was at the lowest (3.46g). It is clear from presentation given
in Fig-VIII that Ufaq-2002 was more tolerant at salinity levels 0f 0 and 50mM salt
levels while Pasban-90, LU-26S, AS-2000 and Bakhar-2002 produced more shoot
fresh weight at 50mM NaCl than control (Wyn Jones,1985). It was probably because
of the fact that low amount of salts might have rather stimulating effect on biological
activities with in the soil , especially the micro-organisms. Another reason could be
that salts might have served as nutrients at low concentrations.
Shoot dry weight
A look at the data ( Table.7 ) revealed average shoot dry weight decreased
from 1.0906 in plants from control ( 0 NaCl ) to 0.4034 g. at highest salinity level 150
mM NaCl. Differences among varietal means were also varies significantly. The
highest dry shoot weight ( 0.9785 ) was obtained in LU-26S , followed by Ufaq-2002
78
(0.9445) , Parwaz-94 ( 0.814 ) and the least being in somaclone from Uqab-2000
(0.4395).
Salinity and varietals interaction also produced significant differences.
Evaluation of somaclones in salinity were negatively related with each other.
Minimum dry shoot weight (0.204 g) was found in Bhakhar-2002 at 150mM salt level
and maximum (1.402g) was in Ufaq-2002 at where the salts were not added.
Table.6: Fresh Shoot weight of wheat somaclones (g) as affected by salinity
Name of Varity 0 mMNaCl 50
mMNaCl 100 mMNaCl 150 mMNaCl Mean
Ufaq-2002 13.40b 14.26a 7.20m 5.92p 10.20A Parwaz-94 12.94c 13.90a 5.32qr 3.46v 8.905B LU-26S 9.04h 8.84hi 5.04r 3.98tu 6.725F Punjab-76 7.42lm 6.52o 5.22qr 3.84u 5.750G Pasban-90 9.62f 9.42fg 7.36lm 5.46q 7.965C Inqulab-91 11.32e 12.12d 8.98hi 7.84jk 10.06A Uqab-2000 8.14j 8.00jk 7.66kl 6.2op 7.500 CDE Chenab-70 9.18gh 8.64i 7.40lm 4.26t 7.370DE AS-2000 8.94hi 8.90f 7.84jk 4.98rs 7.665CD Bhakhar-2002 9.40fg 8.16j 6.924n 4.66s 7.285E Mean 9.940A 9.876A 6.902B 5.060C Mean sharing the same letter do not differ significantly at P = 5% LSD V = 0.55 S= 0.35 S x V = 0.35 ,
Analysis of variance of fresh shoot weight of wheat somaclones SOV DOF SS MS F Replication 4 88.012 22.007 27.9064 Salinity 3 870.889 290.296 368.1816** Varieties 9 355.953 39.550 50.1615** Salinity x Variety
27 278.930 10.331 13.1024*
Error 156 123.000 0.788 Total 199 1716.784
79
Table.7: Salinity effect on Dry Shoot weight of wheat somaclones(g)
Name of Varity 0 mMNaCl 50 mMNaCl
100 mMNaCl 150 mMNaCl Mean
Ufaq-2002 1.402 1.016 0.86 0.50 0.9445 Parwaz-94 1.09 0.876 0.786 0.504 0.814 LU-26S 1.47 0.932 0.798 0.714 0.9785 Punjab-76 0.844 0.83 0.652 0.498 0.706 Pasban-90 1.178 0.80 0.762 0.32 0.765 Inqulab-91 1.364 0.826 0.604 0.386 0.795 Uqab-2000 0.654 0.532 0.322 0.25 0.4395 Chenab-70 1.062 0.768 0.908 0.35 0.772 AS-2000 1.114 0.864 0.568 0.308 0.7135 Bhakhar-2002 0.728 0.624 0.292 0.204 0.462 Mean 1.0906A 0.8068B 0.6552C 0.4034D
Mean sharing the same letter do not differ significantly at P = 5% LSD V= 0.27
Analysis of variance of dry shoot weight of wheat somaclones
SOV DOF SS MS F Replication 4 3.764 0.941 19.4823 Salinity 3 11.283 3.761 77.8675** Varieties 9 4.914 0.546 11.3055** Salinity x Variety 27 1.802 0.067 1.3819* Error 156 7.535 0.048 Total 199 29.298 Root fresh weight
The data ( Table.8 ) show that the maximum root fresh weight (9.770g ) was
obtained at S1 ( 0 mM NaCl ) which decreased statistically as the salinity increased
and was minimum (5.332g ) from treatment S4 i-e. 150 mM NaCl. The relation
between salinity and fresh root weight is shown in the data.
Comparison of means among genotypes under controlled conditions
indicated that Ufaq-2002, Pasban-90, Parwaz-94 and Uqab-2000 had significantly
80
the highest root fresh weight as compare to other wheat somaclones while Punjab-
76 has the lowest value.
At salinity level 100 and 150 mM NaCl. Ufaq-2002 remained at the top while
Bhakkar-2002 and Punjab-76 were at the lowest respectively. Comparison of
genotypes on over all basis indicated that Ufaq-2002 had more root fresh weight
followed by Parwaz-94 and Inqulab-91 respectively while Punjab-76 had lowest
fresh root weight.
Root Dry Weight
A similar trend for root dry weight was observed. The data of root dry
weight presented is Table.9. Comparison of treatment means indicated that addition
of salt significantly decreased the root dry weight of wheat somaclones under all the
salinity levels. Highest dry root weight (0.7242g) at par with 0.7208g was found at 0
and 50mM NaCl respectively while it was 0.4602g at 100mM and 0.4058 at 150mM
salt level. The differences between the treatments were significant. Comparison of
different wheat somaclones revealed the highest root dry weight (1.133g) was
noticed in Ufaq-2002, while Punjab-76 yielded low dry root weight ( 0.3735 g ). At
50mM NaCl a slight increase in dry root weight was noted in Ufaq-2002 as
compared to the control (0 Mm NaCl) and Punjab-76 showed poor response(o.522g)
Ufaq -2002 also produced better root dry weight at salinity level of 100 and 150 mM
NaCl but it was less than the control. Somaclones of Punjab-76 were at the lowest in
dry root weight at both the higher levels of salt.
81
The interaction between treatments x somaclones also produced significant
Differences. Maximum root dry weight (1.266g) was recorded in Ufaq-2002
somaclones at 50 mM salt level while (0.228g) root dry weight was found in Chenab-
70 somaclones at the salinity level of 150 mM salt level.
Table.8: Salinity effect on Fresh Root Weight of wheat somaclones(g) Name of Varity 0 mMNaCl 50
mMNaCl 100 mMNaCl 150 mMNaCl Mean
Ufaq-2002 12.14a 10.46c 8.92hi 8.06k 9.785A Parwaz-94 11.3b 9.4fg 6.76m 6.2no 8.415B LU-26S 8.02k 7.28l 5.54p 4.84rs 6.420E Punjab-76 6.14o 8.08k 3.14w 4.02uv 5.345F Pasban-90 11.38b 9.18gh 5.28pq 4.18qr 7.745CD Inqulab-91 9.76de 9.72ef 7.12l 6.5mn 8.275BC Uqab-2000 11.3b 9.42efg 4.06uv 4.36tu 7.285D Chenab-70 9.24gh 8.9hi 4.48st 4.12uv 6.695E AS-2000 9.9d 9.72ef 5.3pq 5.52pq 7.660D Bhakhar-2002 8.8ij 8.52j 3.8v 4.52st 6.410E Mean 9.770A 9.068B 5.44k4C 5.332C Mean sharing the same letter do not differ significantly at P = 5% LSD= V = 0.54 S= 0.34 S x V = 0.34 , Analysis of variance of fresh root weight of wheat somaclones SOV DOF SS MS F Replication 4 52.454 13.114 17.4630 Salinity 3 825.082 275.027 366.2469** Varieties 9 286.887 31.876 42.4489** Salinity x Variety 27 103.579 3.836 5.1086* Error 156 117.146 0.751 Total 199 1385.148
82
Table.9: Salinity effect on dry root weight of wheat somaclones Name of Varity 0 mM NaCl 50mM
NaCl 100 mM NaCl
150 mM NaCl
Mean
Ufaq-2002 1.266a 1.326a 0.96b 0.94b 1.133A Parwaz-94 0.916b 0.792b 0.48b 0.424b 0.6560B LU-26S 0.67b 0.81b 0.45b 0.354b 0.5710B Punjab-76 0.424b 0.522b 0.26b 0.276b 0.3735B Pasban-90 0.766b 0.654b 0.434b 0.302b 0.5390B Inqulab-91 0.608b 0.682b 0.578b 0.468b 0.5850B Uqab-2000 0.746b 0.554b 0.326b 0.308b 0.5085B Chenab-70 0.604b 0.614b 0.362b 0.228b 0.452B AS-2000 0.66b 0.672b 0.418b 0.374b 0.5310B Bhakhar-2002 0.582b 0.5825b 0.30b 0.28b 0.4385B Mean 0.7242A 0.7208A 0.4602B 0.4058B Mean sharing the same letter do not differ significantly at P = 5% LSD= V = 3.05 S= 1.93 S x V = 1.93 , LSD = 3.059 LSD = 1.935 Analysis of variance of dry root weight of wheat somaclones SOV DOF SS MS F Replication 4 96.065 24.016 1.0268 Salinity 3 94.974 31.658 1.3535* Varieties 9 202.436 22.493 0.9616* Salinity x Variety 27 630.019 23.334 0.9976* Error 156 3698.837 23.390 Total 199 4672.330
83
Ionic concentration
Na+concentration in the leaf sap.
The data relating Na+ concentration in the sap of fully expanded leaf on
different wheat somaclones is presented in Table-10. On overall basis, youngest
expanded leaf had manyfold lower Na+ concentration in controlled treatment than the
salinity applications. Comparison of treatment means indicated non significant
differences in Na+ concentration under controlled conditions, but the addition of salt
significantly increase Na+ concentration under these conditions. Maximum Na+
(52.08mM )was recorded at higher salinity level .i.e. 150 mM NaCl followed by 20.81
and 7.52 at the level of 100 and 150bmM NaCl. The minimum Na+ concentration
(2.26) was observed at controlled level where no salt was applied.
Comparison of means revealed that there had a significant difference in Na+
ion concentration within genotypes. Somaclone AS- 2002 proved more salt sensitive
with the value of 27.87 mM Na+ concentration in the leaf followed by Parwaz-94 and
LU-26S with the value of 23.91 and 23.65 respectively less affected somaclones
was of variety Uqab-2000 with the mean values of 20.20 mM Na+ concentration
Interaction between the salinity and somaclones was also found statistically
significant. There was positive correlation between genotypes and salinity levels as
the salinity increased Na+ concentration also increased. Maximum Na+
concentration (70.04) was found for wheat somaclone AS- 2002 at 150 mM NaCl
level while minimum for LU- 26S at controlled level having no salt (Fig.XII)
84
Table.10: Effect of Salinity on Na+concentration (mol m-3) of 10 wheat varieties Name of Varity 0 mMNaCl 50
mMNaCl
100 mMNaCl 150 mMNaCl Mean
Ufaq-2002 1.64z 8.02u 19.88n 46.08g 18.91F
Parwaz-94 2.22yz 10.06qr 27.66j 55.68c 23.91B
LU-26S 1.44z 8.56st 24.76l 59.82b 23.65BC
Punjab-76 2.20yz 5.14v 14.88o 44.8h 16.77G
Pasban-90 2.30y 5.18v 14.34p 46.66f 17.14G
Inqulab-91 1.84z 3.44wx 10.35q 39.7i 13.83H
Uqab-2000 2.40y 7.82u 22.32m 48.6e8 20.20E
Chenab-70 2.96x 9.06s 25.94k 55.6d 22.94C
AS-2000 3.72w 9.82r 27.86j 70.04ac 27.86A
Bhakhar-2002 1.82z 8.10tu 20.08n 55.88 21.47D
Mean 2.262D 7.522C 20.81B 52.08A
Mean sharing the same letter do not differ significantly at P = 5% LSD= V = 0.79 S= 0.50 S x V = 0.50 ,
Analysis of variance of Na+ concentration of wheat somaclones SOV DOF SS MS F
Replication 4 18.445 4.614 2.8704
Salinity 3 74910.306 24970.102 15534.8038**
Varieties 9 3091.746 343.527 213.7208**
Salinity x Variety 27 2281.048 84.483 52.5681*
Error 156 250.749 1.607
Total 199 80552.304
85
K+ concentration in younger fully expanded leaf The effect of somaclones and salinity levels on K+ concentration in wheat
crop has been shown in Table.11. Analysis of variance indicates the somaclones,
salinity and there interaction decreased the K+ concentration in wheat plants
significantly. K+ concentration decreased with increasing soil salinity. K+ decreased
from 231.6 at controlled salt level to 133.7 mM at highest salinity level of 150 mM
NaCl. The observed decreased in K+ concentration with increasing salinity may be
due to competition among sodium and potassium because sodium antagnosis
potassium uptake due to which sodium partially can substitute potassium in certain
metabolic reactions at moderate salinity level.
Maximum K+ concentration (204 mM ) was found in Punjab-76 and minimum (183.9mM ) in the somaclone of Bhakhar-2002. The other genotypes responded in
between these two range. The resultant reduction in potassium may be the result of
growth dilution effect.
Interaction between genotypes and salinity was also observed statistically
significant. As salinity increase, K+ concentration decreased in a consistent pattern.
Maximum K+ concentration (238.0 mM) was found in Parwaz-94 at controlled salinity
level and minimum (115.4) for Ufaq-2002 at higher salinity level that is 150mM NaCl
the behaviour between genotype and salinity is presented in the data.
86
Table.11: EFFECT OF SALINITY ON K+ CONCENTRATION (Mol M-3) IN LEAF SAP OF 10 WHEAT VARIETIES AT DIFFERENT SALT LEVELS
Name of Varity 0 mMNaCl 50 mMNaCl
100 mMNaCl
150 mMNaCl
Mean
Ufaq-2002 237.2a 228.6d 208.2l 115.4v 196.900CDEParwaz-94 238.6a 217.8i 202.0m 142.6q 200.3B LU-26S 224.0fg 223.4g 211.4k 119.0t 194.4EF Punjab-76 235.6b 228.4d 214.40j 137.6r 204.0A Pasban-90 233.2c 210.4i 199.2m 137.0s 197.7BCD Inqulab-91 229.4d 215.4k 201.6n 141.6rs 193.9F Uqab-2000 226.0e 225.6j 210.2m 146.8q 196.1DEF Chenab-70 232.4c 220.2ef 194.8k 144.2p 203.6A AS-2000 237.8a 201.18h 195.4o 117.4p 199.6BC Bhakhar-2002 222.4h 218.6m 203.0o 135.4ub 183.9G Mean 231.6A 219.0B 204.0C 133.7D
Mean sharing the same letter do not differ significantly at P = 5% LSD V= 2.98 S= 1.89 SxV= 1.42
Analysis of variance of K+ concentration of wheat somaclones
SOV DOF SS MS F Replication 4 528.180 132.045 5.7337 Salinity 3 287074.475 95691.658 4155.1566* Varieties 9 6000.605 666.734 28.9512* Salinity x Variety 27 7747.775 286.953 12.4602* Error 156 3592.620 23.030 Total 199 304944.155 K+
: Na+ ratio in wheat somaclones.
K+: Na+ ratio (Table.12) assessed from leaves of wheat somaclones was quite
high under saline conditions it decreased drastically as the salinity level enhanced.
Comparison of treatments means indicated a significant decreased K+: Na+ ratio as
the salinity increased. Maximum K+: Na+ ratio 110.7 was under controlled (0mM
NaCl) while lowest (2.623) K+: Na+ ratio was observed at salinity level 150mM NaCl
and 100mM NaCl level, it was 32.21 and 10.85 respectively.
87
Comparison of means among the genotypes was also significant. Genotype
inqlab-91 had the higher K+: Na+ ratio in comparison with other genotypes. It was
followed by Ufaq- 2002 and LU- 26S at par with almost having the same K+: Na+
ratio while AS-2000 was at lowest with K+: Na+ ratio 23.92.
Interaction between salinity and genotypes was also found significantly.Ufaq-
2002 had maximum k+: Na+ ratio under control (having no salt). Genotype Inqulab-
91 showed maximum K+: Na+ ratio at all the rest salt levels with the value of 62.194,
19.696 and 3.486 respectively.
Table.12: K+/Na+ ratio of wheat somaclonal as affected by salinity Name of Varity 0 mMNaCl 50
mMNaCl 100 mMNaCl 150 mMNaCl Mean
Ufaq-2002 151.618b 28.5l 10.47r 2.5u 48.27B Punjab-94 82.778e 21.6o84 7.302t 2.55u4 34.58D LU-26S 156.038a 26.13mn6 8.542st 1.986u 48.19B Punjab-76 107.344e 43.21k 14.412q 3.06u 42.01C Pasban-90 99.97f 42.214k 14.308q 2.898u 39.95C Inqulab-91 124.9c 62.194j 19.696p 3.486u 5217H Uqab-2000 94.924g 27.558lm 9.024rs 2.93u 3361D Chenab-70 78.622h 24.872n 8.098st 2.636u 28.56E AS-2000 63.012i 22.438o 6.99t 2.078u 23.92F Bhakhar-2002 122.38d 24.894n 9.63rs 2.096u 39.75C Mean 110.7A 32.21B 10.85C 2.623D Mean sharing the same letter do not differ significantly at P = 5% LSD= V = 2.62 V= 1.66 S x V = 1.66 , Analysis of variance of K+/Na+ ratio of wheat somaclones SOV DOF SS MS F Replication 4 66.065 16.516 0.9326 Salinity 3 365249.909 121749.970 6874.361*7 Varieties 9 14782.469 1642.497 92.7402* Salinity x Variety 27 31241.579 1157.096 65.3330* Error 156 2762.874 17.711 Total 199 414162.897
88
Physiological parameters
The present research work was undertaken to understand the mechanism of
salinity resistance in wheat somaclones and to rank them on the basis of their
salinity tolerance level. The following physiological parameters were studied.
Net photosynthesis rate (Pn)
The data presented in Table.13 shows that different levels of applied salinity
significantly reduced the net photosynthesis rate of all the wheat somaclones. The
maximum reduction was observed at higher salinity level, i.e., 150 mm NaCl. The
cultivars did differ significantly from each other. The maximum net photosynthesis
rate was observed in somaclones of Ufaq-2002 and Perwaz-94 and less
photosynthesis rate was shown by Chenab-70 somaclones. A significant interaction
( v x t ) was also observed under stress conditions.
Stomatal conductance
Data shows that different levels of imposed salinity reduced the stomatal
conductance of all the wheat cultivars but difference among them was non-
significant. The maximum reduction was observed at highest level of salinity. The
somaclones differed non-significantly from each other in stomatal conductance.
However, higher value of stomatal conductance was observed in cv.Inqulab-91. The
interaction ( v x t ) was also non-significant. The maximum stomatal conductance
was observed in somaclones of Uqab-2000 at 0, 50 and 100 mM NaCl salinity level
and it was high in Inqulab-91( somaclones) at higher salinity level ( Table.14).
89
Table.13: Effect of Salinity on Net Photo Synthesis(Pn) in 10 wheat somaclones Name of Varity 0 mMNaCl 50
mMNaCl 100 mMNaCl 150 mMNaCl Mean
Ufaq-2002 10.54c 9.72ef 8.2k 6.04q 8.625A Parwaz-94 10.68c 9.98d 8.3jk 5.26s 8.605A LU-26S 10.16d 8.66i 6.62p 4.76t 7.550C Punjab-76 9.6f 7.6lm 5.1s 3.42w 6.430E Pasban-90 9.86ef 7.42mn 5.74r 5.16s 7.015D Inqulab-91 11.42a 9.8e 8.4j 4.72t 8.585A Uqab-2000 11.04b 9.22g 7.3n 4.6t 8.085B Chenab-70 10.52c 8.88h 6.94o 3.96v 7.575C AS-2000 8.38j 6.58p 4.68t 3.56w 5.785F Bhakhar-2002 9.22g 7.72l 6.1q 4.16u 6.805D Mean 10.13A 8.586B 6.73C 4.566D Mean sharing the same letter do not differ significantly at P = 5% LSD V = 0.29 S = 0.18 SxV= 0.18 Analysis of variance of Net Photosynthesis of wheat somaclones SOV DOF SS MS F Replication 4 29.823 7.456 33.1920 Salinity 3 865.310 288.437 1284.0923** Varieties 9 176.365 19.596 87.2399** Salinity x Variety 27 37.714 1.397 6.2184* Error 156 35.041 0.225 Total 199
90
Table.14: Stomatal Conductance (gs) in10 wheat somaclones as affected by salinity Name of Varity 0 mMNaCl 50
mMNaCl 100 mMNaCl 150 mMNaCl Mean
Ufaq-2002 0.312 0.232 0.196 0.132 0.218 Parwaz-94 0.328 0.25 0.20 0.104 0.2205 LU-26S 0.328 0.278 0.204 0.14 0.2375 Punjab-76 0.234 0.182 0.122 0.086 0.156 Pasban-90 0.322 0.23 0.182 0.16 0.2235 Inqulab-91 0.372 0.322 0.27 0.204 0.292 Uqab-2000 0.388 0.292 0.234 0.142 0.264 Chenab-70 0.318 0.266 0.208 0.146 0.2346 AS-2000 0.184 0.136 0.098 0.076 0.1235 Bhakhar-2002 0.234 0.194 0.154 0.10 0.7505 Mean 0.302 0.2382 0.1868 0.129 Analysis of variance of stomatal conductance of wheat somaclones SOV DOF SS MS F Replication 4 0.0320 0.008 31.2191 Salinity 3 0.839 0.280 1102.2707NS Varieties 9 0.412 0.046 180.3509NS Salinity x Variety 27 0.053 0.002 7.7493NS Error 156 0.40 0.000 Total 199 1.375
91
Response of somaclones derived from wheat cultivars to salinity
Wheat breeding by tradition means has been in practice for centuries, but no
success has been made for a breakthrough to cope with worlds demand. The varieties
thus developed do not last long, long term objectives can not be achieved unless more
genetic variability is generated. In vitro technology complements the conventional
means of wheat breeding in generating diversity when both are combined with each
other. So biotechnological approaches are being evaluated to induce genetic variability.
In vitro cultures exhibit somaclonal variation and regenerated plants show heritable
variation (Ahloowalia,1982).the production profile of wheat from salt effected areas is
extremely poor. The yield in these areas can be increased through development of salt
tolerant wheat.
R0 regenerated plants of 10 wheat somaclones along with their parents were
tested in pots having artificially developed four salinity levels with NaCl salts .i.e. 0, 4. 8,
and 12dSm-1 ECe. Salt level were prepared as per method given in Hand Book No. 60,
United States Department of Agriculture(Anonymous,1958). A normal non- saline, non-
sodic sandy loamy soil was taken from the upper 15 cm layer of a field located in the
research area of Ayub Agricultural Research Institute, Faisalabad. The soil was first air
dried, ground and passed through 2 mm sieve and was mixed thoroughly, a
representative sample from the soil was taken for physical and chemical analyses. With
out any provision of leaching, 10kg of soil were filled in each of the 400 glazed pots the
soil was artificially salinized to different salinity level using NaCl. Calculated amounts of
92
salt) was mixed thoroughly with the soil of each pot separately, followed by
equilibrization for 15 days by giving alternate wetting and drying cycles.
10 healthy R0 seed each of both somaclones and their parents were dry-sown in
each part on 15th November 2008 and crop was harvested on 25th April,2009. During
the studies data regarding plant height, number of tillers, number of spikes, number of
spikelets , 100 grain weight and yield / plant were also recorded.
The major effects of somaclones to its parent , salinity and their interaction was
significant in almost all the parameters. It indicated that both somaclones and parents
were differential in their responses to salinity. Therefore, in discussing this results,
emphasis was given to the relative performance of various genotypes and wheat
somaclones under different stress conditions.
Plant Height:
The data presented in Table.15. represents the plant height of wheat genotypes
and somaclones at different salinity levels. The data reflects that plants attained more
height at lower salinity levels as compare to higher ones and the differences at different
salinity levels were significant. The mean plant height was 89.48, 75.82, 59.38 and
55.76 cm for 0, 4, 8 and 12 dSm-1 ECe levels. It was also noted that height of
somaclones was almost less affected than their parents. The findings are in line with the
results of different researchers working on wheat and barley (Poustini and Salmasi,
1997; Barkat and Haris, 1998; Lutts et al., 1999 and Ahmad et al., 2002.).
93
According to the data regarding plant height of individual genotypes at all the
salinity levels the maximum plant height (80cm) was attained by Ufaq.2002(S) and
minimum (64.95cm) by Chenab-70(P). The plant height of all the 20 genotypes was in
order as under:
Ufaq.2002(S) > Ufaq-2002(P) >Punjab-96(S) ) > Punjab-76(S) >LU-26S(S) > Inqulab-
91(S) >Bhakhar-2002(S) >AS-2002(S) > Chenab-70(S) > Pasban-90(S) > Uqab-
2000(S) > Punjab-76(P) > Punjab-96(P) > AS-2002(P) >LU-26S(P) > Uqab-2000(P) >
Inqulab-91(P) > Bhakhar-2000(P) > Pasban-90(P). > Chenab-70(P).
94
Table.15. Effect of Salinity on Plant Height in 10 wheat somaclones alongwith parents Name of Varity
0 dSm-1 4 dSm-1 8 dSm-1 12 dSm-1 Mean
Ufaq-2002 S 113.00a 80.80hi 70.40nopqr 55.80tuvwx 80.00A Ufaq-2002 P 96.20c 85.80fg 66.40r 56.20tuvwx 76.15B Punjab-96 S 101.80b 83.80fgh 60.40stu 55.00wx 75.25BC Punjab-96 P 83.40fgh 75.60jklm 57.20 stuvwx 56.00tuvwx 74.95BC LU-26S S 95.60c 81.80ghi 60.00stuv 56.40stuvwx 73.40CD LU-26S P 80.20hij 74.60klmn 59.00stuvwx 55.80tuvwx 71.35DE Punjab-76 S 101.80b 80.40hij 60.60st 57.00stuvwx 71.10DE Punjab-76 P 84.80fgh 77.40ijk 55.80tuvwx 55.00wx 69.60EF Pasban-90 S 81.00ghi 77.40ijk 59.60stuvw 55.80tuvwx 69.40EFG Pasban-90 P 79.60hij 73.00klmnopq 59.80stuvw 55.00wx 68.45FGH Inqulab-91S 91.80Cde 77.00ijkl 61.20s 55.40vwx 68.20FGH Inqulab-91P 84.40fgh 68.20qr 59.60stuvw 55.60uvwx 68.15FGH Uqab-2000 S 88.20def 74.00klmno 55.60uvwx 55.00wx 68.05FGH Uqab-2000 P 84.40fgh 71.80mnopq 58.00stuvwx 55.40vwx 67.70FGH Chenab-70 S 92.60cd 72.40lmnopq 57.60stuvwx 55.00wx 67.40FGHI Chenab-70 P 81.60ghi 68.60pqr 54.60x 55.00wx 67.40FGHI AS-2002 S 94.20c 69.60opqr 59.00stuvwx 55.60uvwx 67.40FGHI AS-2002 P 87.40ef 73.40klmnop 55.00wx 55.00wx 66.90HI Bhakhar-2000 S
87.20efF 80.20hij 59.00stuvwx 58.00stuvwx 66.85HI
Bhakhar-2000 P
80.80hi 70.80mnopqr 58.80stuvwx 57.20stuvwx 64.95I
Mean 89.48A 75.82B 59.38C 55.76D Mean sharing the same letter do not differ significantly at P = 5% LSD= V = 2.491 S = 1.114 SxV= 4.983 Analysis of variance of plant height of wheat somaclones SOV DOF SS MS F Replication 4 679.585 169.896 105959 Salinity 3 72885.640 24295.213 1515.2097* Varieties 19 5815.460 306.077 19.0890* Salinity x Variety 57 5457.660 95.748 5.9715* Error 316 5066.815 16.034 Total 399
95
The interaction among the varieties showed that maximum plant height
(113cm) was recorded in genotypes Ufaq-2002(S) followed by Punjab-96(S) with
101.80cm height at 0 dSm-1. The accessions Ufaq-2002(P) and Punjab-96(S)
showed best performance at 4 dSm-1 with the values of 85.80 and 83.80cm
respectively. Ufaq-2002(S) was again at the top with 70.40cm height at salinity level
8 dSm-1 while Ufaq-2002(P) maximum height of 56.20cm at salinity level 12 dSm-1 .
The minimum values were shown by Pasban-90(S), Inqulab-91(P), Chenab-70(P) at
0, 4, and 8 dSm-1 salinity levels and Punjab-96(S) along with five varieties were at
the bottom at 12 dSm-1 salt level . The remaining accessions fell in the these
extremes. The Ufaq-2002(S) and Ufaq-2002(P) stood almost at the top at all the
salinity levels which cab be used as salt tolerant wheat lines for salt affected soils.
No. of Tillers Per Plant
Data Table.16. depicts that all the genotypes and somaclones performed well
and produced more tillers per plant at lowest salinity level. Mean values recorded for
this parameter were 8.10, 4.88, 4.13 and 3.18 at 0, 4, 8 and 12dSm-1 respectively.
The differences in no. of tillers at different salinity level were statistically significant.
These results are in line with the finding of Poustini (1995), Barakat and Haris
(1998), khan et al.(2004) and Yadav et al. (2004).
Maximum tillers were produced by genotype Punjab-96(P) reflecting
maximum tolerance followed by Inqlab-91(P) whereas maximum sensitivity was
96
shown by Uqab-2000(S). When this somaclones were compared with their parent,
they were in the following order in producing the No. of tillers per plant.
Punjab-96(P) > Inqlab-91(P) > Pasban-90(P) > Inqlab-91(S) > Punjab-70(P) >
Bhakhar-2000(P) > Pasban-90(S) > Bhakhar-2000(S) > Ufaq-2002(P) > LU-26S (P)
> Punjab-96(S) >AS-2002(P) > LU26-S(S) > Uqab-2002(S) > Chenab-70(p) > Uqab-
2000(P) > Punjab-76(S) > AS-2000(S) ) > Ufaq-2002(S) > Chenab-70(S).
Interaction between the genotypes (P & S) and salt levels was found
significantly variable among themselves.
Table.16. Salinity effect on No. of Tillers in Wheat Somaclones
Name of Varity 0 dSm-1 4 dSm-1 8 dSm-1 12 dSm-1 Mean Ufaq-2002 S 8.6bcd 5.0klmn 3.2tuvw 2.6wxyz 4.850DE Ufaq-2002 P 8.8bc 5.4kl 3.6rstu 2.8vwxy 5.150CD Punjab-96 S 8.2cde 4.4nopq 4.2opqr 3.8qrst 5.150CD Punjab-96 P 8.8bc 7.2ghi 5.4kl 3.8qrst 6.3A LU-26S S 7.4fgh 4.6mnop 4.6mnop 3.6rstu 5.050DE LU-26S P 7.8efg 4.2opqr 4.6mnop 4.0pqrs 5.150CD Punjab-76 S 6.6ij 5.0lkmno 3.6rstu 2.2yz 4.350F Punjab-76 P 8.6bcd 5.6k 4.4nopq 3.2tuvw 5.45BC Pasban-90 S 8.2cde 5klmn 4.6mnop 4.0pqrs 5.450BC Pasban-90 P 8.8bc 5.4kl 4.8lmno 3.8qrst 5.700B Inqulab-91S 9.2b 5.2klm 4.2opqr 3.8qrst 5.600B Inqulab-91P 11a 4.8lmno 5.2klm 3.8qrst 6.200A Uqab-2000 S 6.8hij 3.4stuv 3.0uvwx 2.4xyz 3.900G Uqab-2000 P 8.6bcd 4.2opqr 3.4stuv 2.8vwxy 4.750E Chenab-70 S 6.8hij 3.2tuvw 3.2tuvw 2.0z 3.800G Chenab-70 P 8.2cde 4.2opqr 3.6rstu 3.0uvwx 4.750E AS-2002 S 6.4j 4.4nopq 3.6rstu 2.0z 4.1000FG AS-2002 P 8.2cde 5.6k 4.0pqrs 2.6wxyz 5.100CDE Bhakhar-2000 S 7.0hij 5.6k 4.6mnop 3.6rstu 5.200CD Bhakhar-2000 P 8.0def 5.2klm 4.8lmno 3.8qrst 5.450BC Mean 8.100A 4.880B 4.130C 3.180D
Mean sharing the same letter do not differ significantly at P = 5% LSD V=0.36 S=0.16 SxV= 0.72
97
Analysis of variance of No. of tillers of wheat somaclones SOV DOF SS MS F Replication 4 2.285 0.571 1.7076 Salinity 3 1367.268 455.756 1362.3312* Varieties 19 172.647 9.087 27.1617* Salinity x Variety
57 104.983 1.842 5.5054*
Error 316 105.715 0.335 Total 399
No. of Spikes
Data given in Table.17. reveal that all the mean genotypes and their
somaclones showed better performance and produced more no. of spikes at lower
salinity levels as compared to higher ones. No. of spikes were decreased as the
salinity increased. These were 8.20, 4.80, 4.08 and 3.17 at EC 0, 4, 8 and 12 dSm-1
respectively. These differences among the No. of spikes at different salinity levels
were statistically significant. The result indorsed the result of Grieve and Francoise,
1992, Khan et. al., 1992 and Poustini, 1995. Maximum No. of spikes among the
genotypes were produced by Punjab-96(P) and was at par with the Inqlab-91(P)
followed by Bhakhar-2002(S) and Pasban-90(P) whereas minimum no. of spikes
were produced by Chenab-70 (S) and Uqab-2000(S). Other genotypes were in
between these two peaks.
The interaction between salinity and genotypes/somaclones showed
that at EC 4d Sm-1 genotype Punjab-96(P) and Bhakhar-2000(S) were most tolerant
and Chenab-70(S) and Uqab-2000(S) were more salt sensitive. At EC 8 dSm-1 the
effect was similar but at EC12 dSm-1 , variety Punjab-96(P) along with LU-26S(P),
Pasban-90(P&S), Inqulab-91(P&S) and Bhakhar-2000(P&S) produced more no. of
LSD = 0.22
98
spikes which showed their improved tolerance at this level of salinity. The interaction
among the salinity x genotypes/somaclones were statasticaly significant (Fig.XIX).
Table.17. Effect of Salinity on No. of Spikes in 10 Wheat Somaclones alongwith parents
Name of Varity 0 dSm-1 4 dSm-1 8 dSm-1 12 dSm-1 Mean Ufaq-2002 S 8.2cde 4.8lmno 3.2tuvw 2.6wxyz 4.70H Ufaq-2002 P 8.8bc 5.4jkl 3.6rstu 2.8vwxy 5.15F Punjab-96 S 8.0def 4.4nopq 4.0pqrs 3.6rstu 5.00FGH Punjab-96 P 8.6bcd 7.2gh 5.2klm 3.8qrst 6.20A LU-26S S 7.4fg 4.6mnop 4.6mnop 3.6rstu 5.05FG LU-26S P 7.8efg 4.2opqr 4.6mnop 3.8qrst 5.10F Punjab-76 S 6.0ij 5.0klmn 3.6rstu 2.2yz 4.20I Punjab-76 P 8.2cde 5.2klm 4.4nopq 3.2tuvw 5.25EF Pasban-90 S 7.8efg 5.0klmn 4.6mnop 3.8qrst 5.30DEF Pasban-90 P 8.6bcd 5.2klm 4.8lmno 3.8qrst 5.60CDBCD Inqulab-91S 9.0b 5.2klm 4.2opqr 3.8qrst 5.55CDE Inqulab-91P 10.4a 4.8lmno 5.0klmn 3.8qrst 6.00AB Uqab-2000 S 6.6hi 3.4stuv 3.0uvwx 2.4xyz 3.85J Uqab-2000 P 8.6bcd 4.0pqrs 3.4stuv 2.8vwxy 4.70H Chenab-70 S 6.6hi 3.2tuvw 3.0uvwx 2.0z 3.70J Chenab-70 P 8.2cde 4.2opqr 3.4stuv 3.2tuvw 4.75GH AS-2002 S 9.0b 4.2opqr 3.6rstu 2.0z 4.70H AS-2002 P 8.8bc 5.2klm 4.0pqrs 2.6wxyz 5.15F Bhakhar-2000 S 8.8b 5.6jk 4.6mnop 3.8qrst 5.70BC Bhakhar-2000 P 8.6bcd 5.2klm 4.8lmno 3.8qrst 5.60CD Mean 8.20A 4.80B 4.08C 3.17D
Mean sharing the same letter do not differ significantly at P = 5% LSD V= 0.322 S= 0.144 SxV= 0.644 Analysis of variance of No. of spikes of wheat somaclones
SOV DOF SS MS F Replication 4 3.200 0.800 2.9811 Salinity 3 1445.968 481.989 1796.0917** Varieties 19 161.388 8.494 31.6525* Salinity x Variety
57 92.082 1.615 6.0200*
Error 316 84.800 0.268 Total 399
99
No. of Spikelets
Data on No. of spikelets are presented in Table.18. It narrates that all the
genotypes/somaclones produced more spikelets per ear at controlled level where no
salt was added as compared to spikelets at higher salinity level. i.e. 12 dSm-1. the
average No. of spikelets per ear was13.17, 12.31, 10.15 and 6.05 in case of 0, 4, 8
and 12dSm-1 respectively. These results are in agreement with the results of Grieve
et al.(1994); Khan et al.(1992); Barakat and Haris (1998) and Yadav et al. (2004) .
Whereas the performance of individual accessions is concerned maximum number
of spikelets per ear (11.95) was produced by Pasban-90(P) followed by Bhakhar-
2000(P) and Inqlab-91(P) with the value of 11.85 spikelets/ear by each genotype
and Inqulab-91(S) with value of 11.80 spikelets per ear. The minimum values were
observed in Chenab-70(S) and Uqab-2000(S) with the value of 9.30 and 10.05
spikelets per ear. All the remaining values fell in between these lower and higher
performance levels.
The interaction of accession with salinity levels showed that at EC 4dSm-1
Bhakhar-2000(P) and Inqulab-91(S) and LU-26S (P) were more tolerant than others
while Uqab 2000(S) and Chenab-70 (S) were salt sensitive. At EC 8dSm-1 Bhakhar-
2002(S) and Bhakhar-2000(P) showed best performance. At higher salinity level of
EC 12 dSm-1 , the accessions LU-26S (P) and Pasban-90(P) were at the top while
Punjab-96(S) was at lower side in producing the No. of spikelets per ear.
100
Table.18. Salinity effect on No. of Spikelets in 10 wheat somaclones alongwith parents
Name of Varity 0 dSm-1 4 dSm-1 8 dSm-1 12 dSm-1 Mean Ufaq-2002 S 14.00bcde 12.6ghijkl 9.0yz 8.8z 11.10CD Ufaq-2002 P 14.00bcde 13.4defgh 10.6qrstuv 9.0z 11.75A Punjab-96 S 13.00efghij 12.8fghijk 10.4rstuvw 8.4z 11.15BCD Punjab-96 P 14.00bcde 13.6cdefg 9.4wxyz 9.0yz 11.50ABC LU-26S S 13.2defghi 12.4hijklm 11.2nopqrs 10.0tuvwxy 11.70AB LU-26S P 12.8fghijk 13.8bcdef 10.6qrstuv 9.0yz 11.55ABC Punjab-76 S 11.6lmnopq 11.6lmnopq 8.8z 8.8yz 10.20EF Punjab-76 P 13.0efghij 13.2defghi 9.4wxyz 9.0yz 11.15BCD Pasban-90 S 13.4defgh 12.0jklmno 10.2stuvwx 8.6yz 11.05CD Pasban-90 P 14.2bcd 12.8fghijk 10.8pqrstu 10.0tuvwxy 11.95A Inqulab-91S 14.6abc 13.8bcdef 9.6vwxyz 9.2y 11.80A Inqulab-91P 14.6abc 13.0efghij 11.0opqrst 8.8z 11.85A Uqab-2000 S 12.6ghijkl 9.8uvwxyz 9.0yz 8.8z 10.05F Uqab-2000 P 11.2nopqrs 12.4hijklm 10.4rstuvw 8.8z 10.70DE Chenab-70 S 9.2xyz 9.8uvwxyz 9.2xyz 9.0yz 9.30G Chenab-70 P 13.4defgh 11.2nopqrs 9.6vwxyz 8.8z 10.75DE AS-2002 S 11.4mnopqr 11.0opqrst 10.4rstuvw 9.6vwxyz 10.60DEF AS-2002 P 15.4a 11.8klmnop 10.2stuvwx 9.0yz 11.60ABC Bhakhar-2000 S 13.0efghij 13.0efghij 11.6lmnopq 9.6vwxyz 11.80A Bhakhar-2000 P 14.8ab 12.2ijklmn 11.6lmnopq 8.8z 11.85A Mean 13.17A 12.31B 10.15C 6.05D
Mean sharing the same letter do not differ significantly at P = 5% LSD V= 0.57 S= 0.25 SxV= 1.14 Analysis of variance of No. of spikelets per spike of wheat somaclones
SOV DOF SS MS F
Replication 4 94.115 23.529 27.8797 Salinity 3 1083.440 361.147 427.9294** Varieties 19 196.340 10.334 12.2446* Salinity x Variety 57 225.860 3.962 4.6952* Error 316 266.685 0.844 Total 399
101
100 Grain weight.
Data given in Table.19. indicate that salinity significantly decreased the grain
yield in most of the wheat genotypes and in somaclones as well. All the accessions
performed well and produced more weight per 100 grains at lower salinity levels as
compare to higher ones. The weight per 100 grains was 3.375, 3.099, 2.841 and
2.790 grams at 0 , 4 , 8 , and 12 dSm-1 respectively. These differences in weight per
100 grains at different salinity levels were statistically significant. The results confirmed
the findings of Poustini, 1995 and Ozgen et al. 1996.
Regardless of salinity levels Bhakhar-2000 (P) with 3.301 grams proved itself the
best followed by AS-2002 (P) and Bhakhar-2000 (S) with weight of 3.253 and 3.210
grams per 100 grains. The minimum weight per 100 grains was observed in Chenab-
70(S) i.e. 2.861 grams followed by Punjab-76(S) 2.869. The rest genotypes /
somaclones performed in between these limits. Inqulab-91 (P) was most tolerant at EC
4dSm-1 followed by Inqulab-91 (S) and Bhakhar-2000 (S) while Chenab-70 (S) was
least tolerant. At EC 8dSm-1 LU-26S(P) and Bhakhar-2000 (S) noted as the best
tolerant and Chenab-76 (S) improved its tolerance while performance of Uqab-2002(S)
was most sensitive at this level of salinity. At EC 12 dSm-1 Inqulab-91 (P) showed more
tolerance while Punjab-76 (P) yielded the least weight of 100 grams).
102
Table.19. Effect of Salinty on 100 Grain weight (g) of 10 Wheat Somaclones alongwith parents
Name of Varity
0 dSm-1 4 dSm-1 8 dSm-1 12 dSm-1 Mean
Ufaq-2002 S 3.168ghijklmn 3.114ijklmnop 2.798vwx 2.772x 2.963GHI Ufaq-2002 P 3.320defgh 3.110jklmnop 2.930pqrstuvwx 2.770x 3.033FG Punjab-96 S 2.810vwx 3.030nopqrs 2.922pqrstuvwx 2.788vwx 2.888IJ Punjab-96 P 2.780vwx 3.094lmnopq 2.824uvwx 2.776wx 2.868IJ LU-26S S 3.426cde 3.086lmnopqr 2.814vwx 2.782vwx 3.027FGH LU-26S P 3.288efghijk 3.228fghijklm 2.956opqrstuvwx 2.796vwx 3.067DEF Punjab-76 S 2.906qrstuvwx 2.968opqrstuvw 2.816vwx 2.788vwx 2.869IJ Punjab-76 P 3.498cd 3.100klmnop 2.784vwx 2.770x 3.038EFG Pasban-90 S 3.040mnopqrs 3.060mnopqr 2.862stuvwx 2.772x 2.934HIJ Pasban-90 P 3.332defg 3.258efghijkl 2.898rstuvwx 2.824uvwx 3.078DEF Inqulab-91S 3.554c 3.400cdef 2.788vwx 2.786vwx 3.132CDE Inqulab-91P 3.300efghij 3.430cde 2.930pqrstuvwx 2.972opqrstuv 3.158BCD Uqab-2000 S 3.398cdef 2.850stuvwx 2.766x 2.772x 2.947GHIJUqab-2000 P 3.446cde 3.132hijklmno 2.770x 2.774x 3.030FG Chenab-70 S 3.100klmnop 2.778wx 2.776wx 2.788vwx 2.861J Chenab-70 P 3.128hijklmno 2.772x 2.810vwx 2.772x 2.871IJ AS-2002 S 3.370cdef 3.018nopqrst 2.822vwx 2.778wx 2.997FGH AS-2002 P 4.450a 3.016nopqrstu 2.776wx 2.772x 3.253AB Bhakhar-2000 S
3.810b 3.306defghi 2.952opqrstuvwx
2.770x 3.210ABC
Bhakhar-2000 P
4.374a 3.226fghijklm 2.832tuvwx 2.772x 3.301A
Mean 3.375A 3.099B 2.841C 2.790D Mean sharing the same letter do not differ significantly at P = 5% LSD V= 0.96 S= 0.04 SxV= 0.19 Annexure-22: Analysis of variance of 100 grain weight of wheat somaclones
SOV DOF SS MS F Replication 4 1.451 0.363 14.8082 Salinity 3 21.698 7.233 295.3331** Varieties 19 6.681 0.352 14.3578* Salinity x Variety 57 14.982 0.263 10.7327* Error 316 7.739 0.024 Total 399 52.551
103
Yield per plant
Data given in Table.20. told that all the genotypes / somaclones responded
differently at all the salinity levels. Increase in salinity level , significantly decreased the
yield per plant. All the accessions exhibited more yield per plant at control level i.e. 0
dSm-1 compared to salinity levels of 4, 8, and 12 dSm-1. Salinity levels reduced the
yield/ plant significantly. The yield per plant was 7.138, 3.832, 2.857 and 2.791 grams
per plant at EC 0, 4, 8 and 12 dSm-1 respectively. Similar results were reported by
Barkat and Haris, 1998. As a whole cultivar AS-2002(P) produced better yield
(5.139g/plant ) followed by Bhakhar-2000(P) , Inqulab-91 (P) and Inqulab-91 (S) i.e.
5.107, 5.068 and 5.002 g/plant.
Interaction salinity x genotype showed that Punjab-96 (P) was tolerant at EC 4d
Sm-1 followed by Inqulab-91(S) , Bhakhar-2000 (S) and Ufaq-2002(P) while Chenab-70
(S) and Chenab-70 (P) was less tolerant followed by Uqab-2000 (S). At the salinity
level of 8 dSm-1 Inqulab-91(P) and Bhakhar-2000 (S) performed better with the yield of
3.206 and 3.154 grams per plant while minimum yield (2.524g/plant) was obtained in
Ufaq-2002(S) at the same salinity level. At EC 12 dSm-1 Uqab-2002(P) responded well
later by Punjab-76 (P) and Punjab-96 (P) with the values of 2.912, 2.852 and
2.822g/plant while minimum yield per plant was recorded in LU-26S(S) Inqulab-91(P)
with the average yield of 2.748and 2.762 grams per plant.
104
Table.20. Yield/Plant (g) of 10 Wheat Somaclones alongwith parents as affected by salinity
Name of Varity
0 dSm-1 4 dSm-1 8 dSm-1 12 dSm-1 Mean
Ufaq-2002 S 7.38def 3.764opqrst 2.524w 2.768vw 4.109EFGHUfaq-2002 P 8.154d 4.500mno 2.786vw 2.788vw 4.557CDE Punjab-96 S 5.498jkl 3.426qrstuvw 2.764vw 2.804uvw 3.623IJKL Punjab-96 P 6.638efgh 6.054ghij 2.762vw 2.822uvw 4.569CDE LU-26S S 7.122ef 3.474qrstuv 2.914tuvw 2.748vw 4.064FGHI LU-26S P 5.802hijk 3.642opqrstuv 2.862tuvw 2.794uvw 3.775HIJK Punjab-76 S 4.038nopqrs 3.436qrstuvw 2.772vw 2.764vw 3.253LM Punjab-76 P 7.448de 4.230mnopq 2.836uvw 2.852tuvw 4.341DEFGPasban-90 S 6.502fghi 3.660opqrstuv 2.772vw 2.772vw 3.927GHIJ Pasban-90 P 8.218d 4.432mnop 3.038tuvw 2.778vw 4.616BCD Inqulab-91S 9.386c 4.902klmn 2.952tuvw 2.768vw 5.002ABC Inqulab-91P 10.05bc 4.256mnopq 3.206rstuvw 2.762vw 5.068AB Uqab-2000 S 5.630ijkl 2.776vw 2.768vw 2.804uvw 3.494JKL Uqab-2000 P 6.786efg 3.120stuvw 2.780vw 2.912tuvw 3.900GHIJ Chenab-70 S 3.530pqrstuv 2.762vw 2.766vw 2.780vw 2.960M Chenab-70 P 6.892efg 2.762vw 2.804uvw 2.764vw 3.806HIJ AS-2002 S 4.988klm 2.808uvw 2.762vw 2.776vw 3.333KLM AS-2002 P 11.300a 3.710opqrstu 2.774vw 2.770vw 5.139A Bhakhar-2000 S
7.024ef 4.830lmn 3.154stuvw 2.788vw 4.449DEF
Bhakhar-2000 P
10.370b 4.100mnopqr 3.144stuvw 2.808uvw 5.107A
Mean 7.138A 3.832B 2.857C 2.791D Mean sharing the same letter do not differ significantly at P = 5% LSD V= 0.46 S= 0.50 SxV= 0.9
Analysis of variance of yield per plant of wheat somaclones
SOV DOF SS MS F Replication 4 28.071 7.018 12.7714 Salinity 3 1254.752 418.251 761.1618** Varieties 19 162.480 8.552 15.5628* Salinity x Variety 57 301.918 5.297 9.6395* Error 310 173.639 0.549 Total 399 1920.859
105
CHAPTER 5
DISCUSSION
During the last few years tissue culture methods have been emerged that can
be used for the improvements of crop plants. The potential value of cell, tissue and
anther culture technique to use in the improvement of field crops has been described
(green,1977; vasil 1987). The regeneration of whole plant is possible today from
cereal species; such as bread wheat (Redway et al., 1990; Vasil et al.,1990,) maiz
(Duncan et al., 1985) rice (Vamada et al., 1986) and barley (Luhrs & Lorz, 1987)
In the present studies all the wheat genotypes responded differently to callus
formation. Genotypes Uqab-2002, V-o4189, Ufaq-2002 and Inqulab-91 produced
excellent callus induction frequency having the range from 80-90% under 3.0mgL-1
2,4-D. Different concentration of 2,4-D were used to find out the callogenic response
of seed as explant of wheat. The effect of 2,4-D (2-4dichlorophynoxy acetic acid) as
auxin for callus formation and growth has been adopted for wheat, (Abdrabou and
Moustafa,1993). The excellent callus formation was noticed at 3mgL-1 2,4-D after the
culture duration of 5-6 weeks. These results are in accordance with Mohmand
(1993) who found good callus from mature seed embryos of spring and winter
genotypes of wheat and In contrast to Barabanova et al; (1988) they achieved callus
induction at 1.5mgL-1 2,4-D while Bartok and sagi (1990) found callus formation on
6.0-8.0mgL-1 2,4-D which are quite opposite to the present result. Shah et al. (2003)
106
also supported the finding of present studies who achieved best callus at 3,5 mgL-1
2,4-D. Twenty wheat genotypes which produced callus induction frequency in the
range of 70-90 were further evaluated for callus induction potential under different
levels of salt.
Differences among genotype for callus induction potential under salt stress
were quite evident from the results. Almost all the twenty genotypes produce callus
of varying degree with and without salt. Wheat varieties Kohinoor-83 produce more
callus at 50mM NaCl. While LU-26S produce more callus at all four salinity levels i.e.
50,100 and 150mM NaCl . The data indicate that the callus formation was
dependent on genotype which is according to the finding of Van Sint Jan et al. 1990
in rice, Bomineni and Jauhar,1996; Ozgen et al.1996; Arzani and Mirodjagh,1999 )
in durum wheat and Hess and Carman,1998, in Bread wheat where a high impact of
genotypes on callus induction was found.
Salinity affected the growth rate and relative fresh weight growth as the
salinity increase callus weight and RFWG was also decreased. The relative fresh
weight growth rate after four weeks of culture were 0.1894, 0.1884 and 0.1831 for
Chenab-70, Punjab- 76 and AS-2002 respectively while minimum reduction in
RFWG was recorded in Ufaq-2002. These results are according to the data reported
by Karadimova and Bambova (1993) in durum wheat. They found that higher
concentration of NaCl caused brownish colour and appearent necrosis and reduced
callus growth. Similar results were reported for other genotypes of durum wheat by
Arzani and Mirodjagh in 1999
107
Calli of 10 responsive wheat genotypes under salt stress were subjected to
regeneration and plantlet development on MS medium without having any auxin
(2,4-D). This is also in accordance with those of Varshney et al. (1991), Chen et al.
(1992) and Hussain et. al, (2001) who used immature embryo of Trticum aestivum
as ex-plant and observed plant regeneration on hormone free MS media.
The effect of salts was quite dominant on the development of plant which
decreased as the salinity level increased. Maximum shoots (48) were developed by
LU-26S followed by Pasban-90(40) and Ufaq-2002 with 31 plants. Minimum shoot
development was observed in SA-42 (1). Ozgen et al. (1996) and Poustini and
Salmasi (1997) supported these results who observed that plant regeneration was
apparently influenced by culture medium and found significant reduction in total dry
production
The finding of superior genotypes for salt tolerance together with their high
embryogenic callus induction potential guide us to recommend these varieties as a
good model to observe physiological mechanism involved in in vitro salt tolerance
and in vitro selection for salt tolerance in wheat.
Keeping in view the importance of wheat crop and increasing problem of
salinity, the present studies were under taken to develop wheat somaclones with a
better potential to grow in saline areas where wheat is either grown inefficiently or
not at all. As a finding of these studies , a sufficient callus and plantlets have been
obtained which were used for the salinity evaluation for further parameters. These
research pursuits were carried out in the wire house in pots having artificially
developed saline soils.
108
In saline soils where salts are present in great concentration The growth of
plant is affected adversely through various ways such as osmotic effects, specific ion
effect and nutritional imbalance; all occurring probably simultaneously (Flowers et
al.,1991 ). Initial retarted growth in saline soils is induced by the decreased water
potential of medium due to higher salt presence (Munns et al., 1995). A major effect
of high concentration of Na+ and Cl-1 in the root medium is the suppression of uptake
of essential nutrients such as k+, Ca++ ,Na+ etc. ( Gohrum and wyn jones,1993). In
the present study the slowly increase in NaCl salinity in rooting media reduced the
shoot and root yield, shoot and root length and potassium where as sodium
accumulation was increased. Akhter et al. 2001 and Pervaiz et al. 2002, have also
observed similar adverse effect of salts. The reduction in shoot and root yield of all
the genotypes with the addition of salt may be due to the decrease of physiology
availability of water with increasing in solute suction from saline media or presence
of toxic ions in plants. This data is according to those of Ehret et al. 1990 and
Pervaiz et al. 2002. less reduction in shoot length is because of the toxic effect of the
salts added.
Among gas exchange attributes by applying salinity treatments, there is a
decrease in sub-stomatal conductance. However the significant reduction was there
in the photosynthetic rate and the stomatal conductance (gs). This indicated that an
adverse effect of salinity on the stomatal opening was possible there which tended
to reduce the photosynthetic rate and stomatal conductance in leaves. These results
are in line to those of Sharma (1996) who found that different gas exchange
characteristics declined in leaves under salinity. Similarly Yamamoto et al. (1994)
109
resulted that there was a significant change in gas exchange characteristics
response to salinity and observed a significant reduction in net photosynthesis.
In the present study the photosynthetic rate was significantly decreased due
to salinity stress with increasing concentration of NaCl which is in agreement with
some earlier studies with different crops such as in wheat (Ashraf et al.,2003).
Brassica ( Nazir et al.,2001 ) and sunflower (Ashraf and Rehman,1999). As it has
been earlier studied that this reduction in photosynthetic rate is associated with
stomatal or non- stomatal factors (Debey,2005). In the present study stomatal
conductance was reduced due to salt stress with an accompanied decrease in
photosynthetic rate (A) thus indicating that stomatal limitation was the only factor in
reducing photosynthesis under salt stress.
As for as the mineral contents are concerned, salinity enhanced the sodium
and reduced the potassium contents gradually, different amount of sodium and
potassium concentrations in their root and shoot at all the salinity levels which was
probably due to difference in dry matter yield as well as their individual genetic
behavior.
This tells the suppression effect of sodium on potassium uptake. Epstein
(1977) observed that the basic reason of potassium deficiency in case if salinity was
a competitive inter relationship among sodium and potassium. There is an apparent
relationship in sodium and potassium contents. Although there is proof that cultivar
with high potassium levels can tolerate the presence of high sodium (Caterina and
Guiliani, 2001). The maximum K+ concentration was found in somaclones of Punjab-
76. The relative shoot dry yield produced at high salinity level has been considered
110
tentively to consider as the standard (most rational) critical of relative salt tolerance
of wheat genotypes while the result due to other growth parameters were evaluated
against it. A consistent pattern among the growth parameters and salt tolerance of
genotypes tested was found. Thus the rating of tolerance to salinity based on growth
parameters can not be considered worthwhile. In assessing the plant to salinity
tolerance, both the shoot and root yield at higher salinity level has been used by
different workers. It is found that the considering the relative growth value is
necessary when comparing genotypes that are differed widely in growth habit or, are
grown under various environment conditions (Mass and Hoffman, 1977) more over,
research is required to develop such correlation among genotypes to exploit their
potentials in salt tolerance. In present study an addition of sodium chloride to the
rooting medium caused higher accumulation of sodium ions in leaf and roots which
is commonly seen in salt stress studies (Ashraf and Naz, 1994). However increase
in accumulation of Na+ ions in leaves under saline environments was in all wheat
somaclones, thus indicating the same toxic effect of increase accumulation of Na+
under saline conditions, plant growth is stunted through osmotic effects as well as
specific ion toxicity and nutritional disturbance, it is clear that only accumulation of
Na+ may have a role in increasing the osmotic potential of wheat somaclone..
Estimates of grain yield is another complexity to the salinity response, not just due to
the crops must be grown in uncontrolled environments for long periods of time, but
the conversion of shoot biomass is complex. Highly significant correlations between
the inorganic ions to salt resistance, there were significant negative correlations
between Na+ - contents and yield components of plants. These results are according
111
to the findings of Thalji and Shalaldeh, (2007) and Salam et al. (1999). They also
found the negative correlations between Na+ and yield component in contrast to
Islam et al. (1997) who failed to find such correlations between Na+ and growth
characteristics in barely. There was a positive correlation between K+/Na+ ration and
yield and its components. Positive correlations were also found between K+ and
yield component. These correlations were also obtained for the shoot and root fresh
weight. This supports the conclusions of Thalji and Shalaldeh, (2007) and Salam et
al. (1999) in wheat genotypes that salinity resistance is made up of two factors.
Physiological tolerance or a small relative decrease in growth, and complete
tolerance a high intrinsic growth rate both with and without salinity. The yield
factors, number of grain per plant, 100 grain weight and grain yield per plant were
affected by salts more badly than were plant height. The findings are in good
agreement with the results of Saqib et al. (2004) they found significant decrease in
yield per plant with increasing salinity during experiment of commercial wheat
varieties. Similarly, in hydroponics experiment Salam et al. (1999) also recorded a
decrease in these three yield components when wheat plant were sown under salt
stress. At salinity level 4dSm-1 somaclones of LU-26S, Inqulab-91 and Bhakhar-
2000 performed in plant yield/plant as compare to their parent genotypes and all
other cultivars. At salinity level 8 dSm-1, somaclones of Pasban-90 and LU-26S
were at par with parent genotypes in yield/plant. At 12dSm-1 somaclones of Pasban-
90 and Inqulab-91 were also at par with their parent plants. The rest of the
somaclones produced less yield as compare to their parent plants. These findings
are in accordance with the results of Khan et al. (2004) who observed that
112
somaclones performed better than the source plant. Yadav et al. (2004) also
observed that regenerated plants showed somaclonal variation for spike length,
spikelet number and grain number. Zair et al. (2003) find out that regeneration of
plant from callus initiation on high NaCl levels is a valid method of selection for salt
tolerance.
As the conventional method of variety development takes excessively long
time for the release of a variety due to non availability of desirable variability. This is
found that tissue culture technique can be an important tool in observing the
economy of time and fast selection of desirable variants. Many new traits have
already been identified by this technique. If any character is found to be superior to
mother plant, the somaclones after testing can be released as a new variety.
113
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APPENDICES Annexure-1: List of Wheat Germplasm used to exploit the somaclonal variation
Sr.No. Genotype Sr.No. Genotype
1 Ufaq-2002 26 Pasban-90 2 Mexi Pak 27 Inqalab-91 3 Chenab-70 28 Shahkar-95 4 Barani-70 29 Punjab-96 5 SA-42 30 MH-97 6 Blue Silver 31 Kohistan-97 7 Lyallpur-73 32 Uqab-2000 8 Parwaz-94 33 Chenab-70 9 PARI-73 34 Iqbal-2000
10 Sandal 35 SH-2000 11 SA-75 36 AS-2000 12 LU-26S 37 Sehar-06 13 WL-711 38 Shafaq-06 14 Punjab-81 39 Fareed-2000 15 Pak-81 40 Bhakhar-2000 16 Pothowar 41 GA-2002 17 Punjab-76 42 V-03094 18 Barani-83 43 V-03079 19 Kohinor-83 44 V-04188 20 Fsd-83 45 V-04189 21 Fsd-85 46 V-03138 22 Punjab-85 47 V-04040 23 Chakwal-86 48 V-04067 24 Shalimar-86 49 V-04112 25 Rohtas-90 50 V-04171
Annexure-2: Screening of Wheat Genotypes for Salt Tolerance
Sr.No. Genotype Sr.No. Genotype 1 Ufaq-2002 11 Pasban-90 2 SA-42 12 Inqalab-91 3 Parwaz-94 13 Punjab-96 4 Lu-26S 14 Uqab-2000 5 Pothohar 15 Chenab-2000 6 Punjab-76 16 Iqbal-2000 7 Barani-83 17 AS-2000 8 Kohinoor-83 18 Bhakkar-2000 9 Fsd-83 19 V-03079
10 Chakwal-86 20 V-04189
Annexure-3: Composition of Murashige-Skoog Media for plant tissue and cell culture Macronutrients mg/l mM NH4NO3 1650 20.6 KNO3 1900 18.8 CaCl2
. 2H2O 440 3.0 MgSO4
. 7H2O 370 1.5 KH2PO4 170 1.2 Micronutrients mg/l uM KI 0.83 5 H3BO3 6.2 100 MnSO4
. 4H2O 22.3 100 ZnSO4
. 7H2O 8.6 30 Na2Mo04
. 2H2O 0.25 1.0 CuSo4
.5H2O 0.025 0.1 CoCL2
. 6H2O 0.025 0.1 Fe-Versenate (EDTA) 43.0 100 Vitamins & Hormones mg/l mg/l Inositol 100 100 Nicotinic acid 0.5 1.0 Pyridoxine. HCl 0.5 1.0 Thiamine. HCl 0.1 10.0 IAA 1-30 -- Kinetin 0.04-10 0.1 Sucrose 3000 pH 5.7 --
Annexure-4: Physical and chemical properties of the soil used.
Determination unit value Mechanical analysis Sand % 66.9 Silt % 16.5 Clay % 16.6 Textural class - sandy clay loam Electrical conductivity of dS/m 2.7 Saturation extract (ECe ) - Saturation percentage 32.7 pHs - 7.7 Soluble anions Co3 me/l absent Hco3 “ 6.03 Cl “ 5.0 So4 (By difference) “ 18.0 Soluble cations Ca + Mg “ 13.6 Na “ 13.7 K “ 0.5 .
Annexure-5: Designated, achieved and after crop harvest levels of ECe (dSm-1) ______________________________________________________________________ Soil Designed Achieved After Crop Harvest ECe ECe ECe ______________________________________________________________________ S1 2.7 2.7 2.82 S2 4.0 4.15 4.76 S3 8.0 8.50 8.60 S4 12.0 13.0 12.64 Anneure-6: Salt (mg/100g soil) to develop designed levels of ECe (dS/m) Salt ECe______________________________ ___________________________________________________________________ 0 4 8 12 NaCl - 28.87 101.38 177.90 _____________________________________________________