master of science in horticulture...shoot tip and nodal segment explants 40 10 effect of different...
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IN VITRO STUDIES IN ACID LIME (Citrusaurantifolia Swingle)
cv. KAGZI LIME
By
Raj Kumar Verma[2004A73M]
Thesis submitted to the Chaudhary Charan Singh HaryanaAgricultural University, Hisar in the partial fulfillment of the
requirements for the degree of
Master of Science in
Horticulture
COLLEGE OF AGRICULTURE CCS HARYANA AGRICULTURAL UNIVERSITY
HISAR-125004
2006
DedicatedTo
Farming Community
CERTIFICATE – I
This is to certify that this thesis entitled, “In vitro
studies in acid lime (Citrus aurantifolia Swingle) cv. Kagzi
Lime”, submitted for the degree of Master of Science in the
subject of Horticulture of the Chaudhary Charan Singh Haryana
Agricultural University, Hisar, is a bonafide research work carried
out by Mr. Raj Kumar Verma under my supervision and that no
part of this thesis has been submitted for any other degree.
The assistance and help received during the course of
investigation have been fully acknowledged.
[Dr. Sultan Singh]Associate Professor
Department of Horticulture CCS Haryana Agricultural University
Hisar – 125 004
CERTIFICATE – II
This is to certify that this thesis entitled, “In vitro
studies in acid lime (Citrus aurantifolia Swingle) cv. Kagzi
Lime”, submitted by Mr. Raj Kumar Verma to the Chaudhary
Charan Singh Haryana Agricultural University, in partial
fulfilment of the requirements for the degree of Master of Science
in the subject of Horticulture, has been approved by the
Student’s Advisory Committee after an oral examination on the
same.
MAJOR ADVISOR
HEAD OF THE DEPARTMENT
DEAN, POSTGRADUATE STUDIES
Acknowledgement
Gratitudes cannot be seen or expressed, it can only be
felt deep in heart and is beyond description. Although, thanks
are poor expressions of dept of gratitude one feel, yet there is
no better way to express it.
It gives me immense pleasure to gratefully thank the
Almighty Godwhose grace has brought me upto here. I avail
this opportunity to express my immense sense of gratitude to
modest and generous personality Dr. Sultan Singh, Major
Advisor, for his prudent admonition, facile guidance, keen
complicity, arduous din’t and constant inspiration during the
course of this investigation.
It is my proud privilege to express my deepest sense of
gratitude and indeptness to Dr. S.K. Sehrawat, Associate
Professor of Horticulture, CCS Haryana Agricultural University,
my co-major advisor, who conceived, detailed and shaped the
problem and provided intellectual stimulation, continuing
exhortation and sagacious guidance throughout the present
study, without which it could have been a chimera.
I am also thankful to other members of my advisory
committee Dr. J.K. Sandooja, Senior Veg. Physiologist (Plant
Physiology), Dr. Balwan Singh, Scientist (Soil Science) and Dr.
(Mrs.) Urmil Verma, Professor (Statistics) for rendering useful
tips and advice whenever asked for. I am thankful to Dr. V.S.
Beniwal, Former Director (Technical) Centre for Research and
Application in Plant Tissue Culture (CRAPTC), new campus
CCS HAU, Hisar for suggesting me to work on in vitro
multiplication and providing the facilities to carry out research
in his Laboratory in CRAPTC. I record my gratitude and
regards to Dr. M.S. Joon, Professor and Head, Department of
Horticulture for providing necessary facilities.
My sincere appreciation goes to my dear friends and
seniors Ashok Patel, Sarvesh, Anupam, Kaushal, Anuja,
Keerti, Virendra, Ravindra, Deepak, Pawan, Vijay, Ankush and
my other friends and class mates for their precious assistance,
constituent encouragement and nice companionship.
Financial assistance received in the form of J.R.F.
by I.C.A.R., new Delhi is also duly acknowledged.
Date:
Place: (Rajkumar Verma)
CONTENTS
CHAPTER DESCRIPTION PAGE(S)
I INTRODUCTION 1-3
II REVIEW OF LITERATURE 4-14
III MATERIAL AND METHODS 15-32
IV EXPERIMENTAL RESULTS 33-64
V DISCUSSION 65-73
VI SUMMARY AND CONCLUSION 74-76
LITERATURE CITED i-xi
LIST OF TABLES
TableNo.
Description Page(s)
1 Chemical composition of MS (Murashige and Skoog,1962) medium
18
2 MS basal medium treatments used for in vitro seedgermination and axenic seedling production
23
3 MS basal medium treatments used for directregeneration
24
4 Effect of modified MS medium on root differentiation 26
5 Effect of different potting mixtures on hardening andplant growth
28
6 Effect of different durations of sterilization with HgCl2(0.1%) on percent contamination of inoculated acidlime seeds
34
7 Effect of different levels/concentrations of BAP andGA3 alone and in combination on germination of acidlime seeds
35
8 Effect of different levels/concentrations of BAP andGA3 alone and in combination on seedling length at 15and 30 days after germination
37
9 Effect of different levels/concentrations of BAP andNAA on shoot initiation and regeneration from in vitroshoot tip and nodal segment explants
40
10 Effect of different levels/concentrations of BAP andNAA on percent regeneration from in vitro shoot tip andnodal segment explants
42
11 Effect of different levels/concentrations of BAP andNAA on number of shoots/explant and shoot lengthfrom in vitro shoot tip and nodal segment explants
44
12 Effect of different levels/concentrations of BAP andNAA on shoot initiation and regeneration from in vivoshoot tip and nodal segment explants
47
13 Effect of different levels/concentrations of BAP andNAA on percent regeneration from in vivo shoot tip andnodal segment explants
49
14 Effect of different levels/concentrations of BAP andNAA on number of shoots/explant and shoot lengthfrom in vivo shoot tip and nodal segment explants
51
15 Effect of different levels/concentrations of NAA and IBAon root initiation and rooting
54
16 Effect of different levels/concentrations of NAA and IBAon number of roots/plantlet and root length (cm)
56
17 Effect of different levels/concentrations of NAA and IBAon percent rooting and percent survival of rootedplantlet
58
18 Effect of different combinations of potting mixtures onplant height and number of leaves/plant at 15 and 30days after potting
60
19 Effect of different combinations of potting mixtures onpercent survival of plantlets under green house andfield condition
61
20 Effect of different cocultivation and selection conditionson Agrobacterium mediated transformation frequenciesin the acid lime internodal stem segments
64
LIST OF PLATES
PlateNo.
Description
1 In vitro grown seedlings
2 Direct shoot regeneration from in vitro shoot tip
3 Direct shoot regeneration from in vitro nodal segment
4 Direct shoot regeneration from in vivo shoot tip
5 Direct shoot regeneration from in vivo nodal segment
6 Rooted plantlets of acid lime
7 Hardening of in vitro raised plants in green house
8 Hardened plants transplanted in various potting mixture
LIST OF ABBREVIATIONS
AC : Activated charcoalBA : Benzyl adenineBAP : Benzyl amino-purineC.D. : Critical difference cm : Centimeter(s)Cv. : Cultivar d : Daye.g : Exampli gratia (Example)et al. : Et allia (and others)etc. : Etcetra, etcetrag : gram(s)GA3 : Gibberellic acidh : HourIAA : Indole acetic acid IBA : Indole butyric acidKin : KinetinLAF : Laminar air flowME : Malt extractmg : Milligram(s)ml : MillilitresMS : Murashige and Skoog medium (1962)MT : Murashige and Tucker mediumNAA : Naphthalene actic acidNo. : NumberPPM : Parts per millionS.E. : Standard error of meansSTG : Shoot tip grafting uv : Ultra violet v/v : Volume for volumevar. : Varietyviz. : Namely% : Per centM : MicromoleºC : Degree Celsius2,4,5-T : 2,4,5-trichlorophenoxy acetic acid2,4-D : 2,4-dichlorophenoxy acetic acidGUS : -GlucuronidaseNOS : Napoline synthase
NPT : Neomycin phosphotransferase
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CHAPTER-1
Introduction
Citrus is the third most important fruit crop in India
after mango and banana. The genus Citrus includes more than
150 species. Commercially in India mandarins (C. reticulata
Blanco), sweet orange (C. sinensis Osbeck) and acid lime (C.
aurantifolia Swingle) are the most important of all Citrus species
sharing 41, 23 and 23 per cent of production respectively
(Anonymous, 2004).
Acid lime (C. aurantifolia Swingle), a member of
Rutaceae family, is native of India. The acid lime is extensively
grown in almost all parts of tropical and subtropical regions. It is
highly polyembryonic distinct species of great commercial
importance. The flowering and fruiting takes place throughout
year. It is susceptible to tristeza and citrus canker.
The acid lime occupies 1.61 lakh hectares with the
production and productivity of 14.13 lakh tonnes and 8.8
tonnes/ha., respectively (Anonymous 2004). It is rich source of
vitamin C and used to prepare excellent pickle, RTS and
marmalades.
-2-
Acid lime is commercially propagated by seed and
air-layering but the rate of multiplication is slow by following
these two methods of propagation. The limitations in propagation
through nucellar seedling plants are excessive thorniness and
slowness in coming to bearing. Nucellar seedlings may require 6-8
years before coming to bearing. In these aspects, they resemble
with the sexual seedlings. Air layering is generally followed in
humid and mild temperature conditions and is not satisfactory in
north-western parts of the country. These limitations can be
easily overcome by in vitro propagation which offers the advantage
of rapid multiplication of clones of elite genotypes in terms of time
and space with no limitations of growth season.
In vitro propagation is also useful in seedless citrus
which bear fruits but never produce seeds (Grosser et al., 1993
and Bowman 1994). Non availability of seeds in some species and
low percentage of polyembryony in other species necessitates the
application of the technique of in vitro micro propagation (Kitto
and Young 1981., Barlas and Skene, 1982, Edriss and Burger,
1984; Moore, 1986).
A many fold increase in the rate of multiplication over
conventional method has been achieved very recently by way of
tissue culture. A few scientists have tried to obtain differentiation
of shoot-buds and plantlets from in vitro cultured explants of
different citrus species (Grinblat, 1972, Fitchet, 1990, Grosser et
-3-
al., 1993; Baruah et al., 1996, Thirumalai and Thamburaj, 1996;
Perez et al., 1997).
For citrus improvement using tissue culture
technique, it is essential to develop methodology for high
frequency of plant regeneration from organ culture. Also, the
planting material for commercial production should be virus and
pathogen free using recommended technique of tissue culture.
Large number of viruses are transmitted through seeds, so the
only way to get the true-to-type, pathogen and virus free plants on
large scale is to use the in vitro technique. Efforts were also made
on Agrobacterium-mediated transformation in acid lime so that a
protocol may be standardized for gene transfer at later stage.
Keeping in view the above mentioned points regarding
difficulties in propagation, lack of planting material acting as
bottleneck in the increase in area under acid lime crop, the
present investigation entitled “In vitro studies in acid lime” (Citrus
aurantifolia Swingle) var. Kagzi Lime was proposed with following
objectives
1. To standardize the seed sterilization duration and in vitro
germination media.
2. To standardize the protocol for in vitro multiplication of acid
lime.
-4-
CHAPTER-2
Review of Literature
Micro propagation is a potential bio-technical tool that
has become a commercially viable method of in vitro clonal
propagation of a wide range of herbaceous and woody plants.
Studies on in vitro culture of citrus tissues and organs are being
conducted since last three decades. Most investigations were
restricted to nucellus tissues and embryos, but some research
has been on different explants and on nutrient media for
elimination of viruses and other pathogens. The available
literature on these aspects has been reviewed here in following
paragraphs.
2.1 In vitro germination of acid lime seeds
Ashari et al (1988) gave three treatments of
sterilization duration with 0.1% HgCl2 (5,10 and 15 minutes) on
five polyembryonic citrus root stock seeds before inoculation and
observed that there was minimum contamination with 10 minutes
-5-
sterilization (12.5%) and maximum with 5 minutes sterilization
(30.6%).
Singh et al. (1994) observed six citrus species with
regard to seed germination and reported that in vitro condition
helped in early germination of rough lemon (142 days) followed by
sour orange (15.1 days) and Rangpur lime (15.3 days).
Germination frequency was maximum for rough lemon (98.2%)
followed by sour orange (94.6%) and Rangpur lime (93.7%).
2.2 Shoot regeneration and rooting
Bouzid (1975) cultured about 1cm long stem segments
of sweet orange, sour orange, mandarin and lemon tree in several
media and found that development of shoots were affected by
medium, age of cutting, part of the stem from which it was taken
and the time of a year. Cutting from mature trees rooted with
difficulty but those from seedling rooted easily.
Navarro et al. (1979) reported that Navel orange plants
obtained by culturing ovules from 2, 4, 6, 8 and 10 week old
fruits were free from exocortis, concave gum and other viruses.
Such plants were uniform in growth and were morphologically
normal. Stem segments of different citrus species were cultured
in different media supplemented with micro nutrients and growth
regulators and significant success was reported by various
workers (Kitto and Young, 1981).
-6-
Sauton et al. (1982) observed multiple shoot
production in orange, lemon and citrange explants taken from
juvenile and mature trees on MS medium supplemented with BA
at 10M. A hybrid (Carrizo citrange) had the highest regenerative
capacity. NAA at 10M induced the best rooting (Barlass and
Skene 1982). The regeneration potential of epicotyl, cotyledon,
hypocotyl and root tissue of Valencia orange was determined on
different modifications of Murashige-Tuckers basal medium.
Epicotyl and hypocotyls segments had the greatest potential for
bud formation on a medium containing BA 1.0 mg/l and NAA
0.01 mg/l or on a medium devoid of hormones. Epicotyl segments
and cotyledons formed roots on a medium containing BA 0.02
mg/l and NAA 1.0 mg/l. Root segments formed buds on medium
containing BA 1.0 mg/l and NAA 0.01 mg/l.
Singh and Sandhu (1985) studied effect of IBA and
NAA on callusing, rooting and sprouting of cuttings of C. jambhiri
Lush and reported that the highest rooting percentage with 50
ppm NAA followed by 50 ppm IBA.
Moore (1986) observed shoot initiation of citrus
rootstock by culturing internodal seedling stem section in media
containing varying concentrations of BA and NAA.
Similarly, Grosser and Chandler (1986) reported
successful multiplication in young Swingle Citrumelo seedlings
using 0.5 cm and 1.0 cm nodal and internodal segments as
-7-
explants cultured on basal medium containing different levels of
coumarin growth regulatory substance.
Starrantio and Caruso (1988) obtained better
percentage of multiplication in Troyer and Carrizo citrange and
the citrumelo CPB 4475 with 1mg/l of BAP and 0.5 mg/l of IBA.
The best rooting was obtained on the basal MS medium
supplemented only with NAA 1 mg/l.
Duran et al. (1989) studied morphogenesis and callus
culture of sweet orange, citron, and lime. The optimum
concentrations of NAA to induce root formation on stem segment
were 10 mg/l for sweet orange and lime and 3 mg/l for citron.
The optimum BA concentration for shoot and bud proliferation
was 3 mg/l for sweet orange and citron and 1 mg/l for lime.
Fitchet (1990) studied clonal propagation of Queen
and Smooth Cayenne pineapples using lateral buds from the
crown and incubated in MT Medium supplemented with NAA, IBA
and kinetin (2 mg/l each). Multiplication rate was more in liquid
media as compared to semi solid media.
Lukman et al. (1990) reported that shoot apices of
Troyer citrange cultured in liquid MS medium, supplemented with
BA and GA3 produced the most and the longest shoots. Rooting
occurred when the shoots were cultured in half strength MS
medium with no NAA.
-8-
Otoni and Teixeira (1991) studied the effect of size of
explants used for in vitro culture in MS medium and observed that
explant size affected the number of auxiliary shoot development
from them. This number was greatest with 0.5 cm nodal
segments.
Can et al. (1992) observed that shoot initiation was
best in medium containing BA 2.0 mg/l with or without GA3 4.0
mg/l. The rooting of these shoots was optimal in medium
containing IBA 1.0 mg and NAA 1.0 mg/l.
Huang and Xiang (1992) reported that shoots
multiplied in MT medium supplemented with BA 0.25-1.0 mg/l.
Multiplication rate was highest with BA 0.5 mg/l and shoot
proliferation rate was 3.2-4.2 in Jianshui Seedless tangerine.
Lin-B et al. (1992) studied the culturing of lateral buds
of several cultivars of citrus on different combinations of BA, IAA
and GA3 concentration. For proliferation best combination was
BA 0.1 mg, IAA 0.05 mg and GA3 0.1 mg/l. The highest rooting
percentage (70%) was observed on half strength MS medium
supplemented with NAA 0.2 mg/l, agar 0.3% and 15% sucrose.
Omura and Hidaka (1992) have investigated the
initiation, sub culture, rooting and acclimatization of shoot tip
explants of Satsuma cv. Aoshima Unshice and tangor cv. Kiyomi
and reported that MS medium containing 50 M GA3, IM BA and
0.1 M NAA was suitable for best multiplication and growth.
-9-
Multiplication was obtained with media containing 5-10 M GA3+
1M NAA and 10 M IBA resulted in good rooting.
Raman et al. (1992) observed that per cent callus
induction was more when the stem segments from 14-21 days old
axenic seedling of C. limon and C. jambhiri were cultured on MS
media supplemented with NAA 10 mg/l + kinetin 0.2 mg/l. The
highest percentage of shoot regeneration was obtained on ½ MS
medium supplemented with BA 5.0 mg/l alone or in combination
with GA3 3.0 mg/l. Regenerated shoots rooted most easily on ½
strength MS medium + NAA 1.0 mg/l + 2% sucrose.
Mas et al. (1994) obtained good multiplication rate
(5:1) by culturing stem segment of citremon 1452 on MS medium
supplemented with BA 0.5 mg and NAA 0.5 mg/l.
Nagao et al. (1994) studied regeneration of Poncirus
trifoliata in vitro culture of apical buds cultured on MS medium
supplemnted with BA 1.0 mg/l and NAA 1.0 mg/l and varying
sucrose and inorganic nitrogen levels. The best results were
obtained on medium supplemented with sucrose 30-45 g/l and
double dose of inorganic nitrogen.
Singh et al. (1994) obtained multiple shoots from shoot
tips from mature plants of mandarin cv. Khasi and Assam lemon
when cultured on MS medium supplemnted with BA 1.0, Kinetin
0.5 and NAA 0.5 mg/l. Baruah et al. (1995) observed better shoot
-10-
proliferation on MS medium supplemented with BA when shoot
explants of axenic seedling of pummelos were cultured.
Das et al. (1995) obtained shoot bud formation from
nodal segment. The best growth regulator combination was NAA
0.1 mg/l+ BA 0.5 mg/l for orange cv. Mosambi. Rey et al. (1995)
reported that plant regeneration of citrus species via in vitro
somatic embryogenesis from nucellar explants is reviewed and a
protocol developed from orange cv. Valencia late and grape fruit
cv. Ruby.
According to Morsy and Miliet (1996), the nodal
segment taken from different stages of the rhythmic growth from
different positions of sour orange had significant influence on
growth.
Rahaman et al. 1996) obtained in vitro cotyledonary
embryoids from the ovules culturing on MS medium
supplemented with either Kinetin or BA. Thirumalai and
Thamburay (1996) observed that callus induced from inter nodal
segments of sweet orange cv. Sathgudi and acid lime cv. PKM-I on
MS medium containing 0.25 mg /l BA and NAA at 9-10 mg/l.
Formation of shoot buds took place NAA 0.1 mg/l and BA 0.25
mg/l.
Similarly, Desai et al. (1996) reported that maximum
production of shoot were obtained on MS medium supplemented
with BA 0.25 mg/l + ME 200 mg/l and maximum roots were
-11-
occurred in half strength MS media with NAA 0.1 mg/l in acid
lime cv. Kagzi Lime.
Harada and Murai (1996) reported that in P. trifoliate
induction of regeneration of shoots occurred on MS solid medium
supplemented with BA 44.4 M, 3% sucrose and 0.8% agar.
Regenerated shoots rooted on half strength MS basal medium
with or without IBA 0.5 mg/l.
Belarmino and Posas (1997) reported that rapid shoot
proliferation were occurred by using shoot tip and single-node
stem segment on MS medium containing BA 0.5 mg/l, IBA 0.5
mg/l and adenine 40 mg/l in pummelo.
Normah et al. (1997) reported that maximum number
of shoot was achieved by using hypocotyl explants cultured on MS
medium supplemented with 2.2-11.1 M BA in wild C. halimii.
Perez et al. (1997) studied in vitro regeneration by
organogenesis starting from inter nodal stem segments from
seedling of Maxican lime and mandarin on MS medium with
vitamins from B5 medium, Sucrose 5%, BA 33.3 M and NAA 5.4
M and incubated at 25±2ºC for 21 days in darkenss followed by
29 days on 15-H/8-h light /dark cycle. They obtained an average
7.8 well differentiated shoots per explant in Mexican lime and 5.1
in Mandarin.
According to Mohanty et al. (1998) shoot meristems of
C. sinensis cv. Musambi cultured in MS medium containing NAA
-12-
developed callus and regenerated shoots to the tune of 30 and 45
percent, respectively. The nodal segments formed shoots best in
MS medium containing BA and IAA, root development only
occurred in media containing kinetin + NAA or NAA + IBA.
Ramsundar et al. (1998) reported rapid regeneration
from leaf derived callus of acid lime. They reported that MT
medium was better than MS for callus induction. Shoot
differentiation was best with 2,4-D 2.0 mg./l. The highest number
of shoots/ explant was achieved with BA 5.0 mg/l. Maximum
response to elongations was achieved with GA3 5.0 mg/l and best
rooting occurred with NAA 2.0 mg/l.
Kamble et al (2005) carried out micro propagation of
acid lime (C. aurantifolia S.) var Sai-Sharbati using shoot tip and
nodal segment explants obtained from axenically grown seedlings
of 21 to 30 days age. Direct regeneration of shoots and roots
without intervention of callus was obtained from both the
explants on MS basal media. In both the explants, 100 Per cent
shoot proliferation was induced on MS + BA 0.25 mg/l-1 + malt
extract 200 mg1-1 media. The shoot proliferation rate was more
(5.0) in nodal segment as compared to the shoot tip explant (4.3).
Half strength MS medium supplemented with IBA 1.0 mgl-1 was
best for root initiation within 15.66 days. A higher kinetin/IAA
ratio favoured stem elongation more than root formation whereas
-13-
a lower kinetin/IAA ratio favoured root formation and inhibited
stem elongation. The beneficial effect of cytokinin in shoot
regeneration has also been reported in C. grandis (Chaturvedi and
Mitra, 1974), C. sinensis, C. paradisi C. aurantifolia (Bhansali and
Arya 1978) and C. limettoides (Bhansali and Arya, 1979).
2.3 Hardening
Kamble et al. (1995b) reported that soil rite mix was
found best suitable potting mixture for survival and growth of
rooted plantlets under green house condition as well as field
condition.
Shah et al. (1999) rerpoted that the potting mixture
containing soil, sand and FYM (1:1:iv/v) was found to be the most
suitable followed by soilrite mix for best survival and growth. All
the other potting mixtures were resulted in high mortality and
poor growth of the plantlets. These results were encouraging as
soil, sand and FYM were cheaper and easily available sources as
compared to vermiculite, sphagnum mass or soil rite mix.
2.4 Agrobacterium-mediated transformation in acid
lime (Citrus aurantifolia Swingle) var. Kagzi Lime
The Agrobacterium-mediated transformation method is
the most efficient method for producing transgenic citrus plants
(Pena et al., 1995).
Bond and Roose (1998) obtained transgenic
Washington Navel orange (Citrus sinensis Osbeck) using
-14-
Agrobacterium mediated transformation of internodal segment
tissues.
Yaang et al. (2000) successfully recovered genetically
modified plants of grape fruit (Citrus paradise Mact) by
Agrobacterium mediated gene transfer using internodal segments
as explants.
According to Almeida et al. (2002) recovery of
transformed plants from mature tissues in citrus has been
reported only from internodal segments of Citrus sinensis cv.
Pineapple.
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CHAPTER-3
Materials and Methods
The present investigation entitled, “In vitro studies in
acid lime (Citrus aurantifolia Swingle) var. Kagzi Lime” was
conducted during the year 2004-2006 in the Department of
Horticulture, CCS HAU, Hisar in collaboration with the Centre for
Research and Application in Plant Tissue Culture (CRAPTC) at
CCS HAU, Hisar.
3.1 Materials
3.1.1Sources of explants
For micro propagation experiments, the required seeds
and explants were collected from mature tree growing at
experimental orchard of Department of Horticulture, CCS HAU,
Hisar and some explants were obtained from in vitro grown
seedlings of acid lime cv. Kagzi Lime at CRAPTC, Hisar.
3.1.2Chemicals
Care was taken to use chemicals of higher purity
throughout the course of investigation. The chemicals viz., growth
hormones, vitamins, myo-inositol, glycine, chelating agents
-16-
(EDTA) sodium salts) etc were procured from Sigma (USA) and
Hi-Media company (India). However, the other chemicals like
sucrose, agar-agar were procured from SRL, BDH and Hi-Media
companies.
3.1.3Culture vessels and instruments
Borosilicate glasswares of Borosil brand were used for
the experiments. For nutritional studies of different cultures, the
wide mouth bottles with autoclavable plastic caps were used.
Similarly, the test tubes of 150 mm x 25 mm were also used. For
stock solution preparation, media preparation and other work,
Erlenmeyer flasks and beakers were used. Micro-pipettes of
Himedia were also used. The instruments such as stereoscopic
microscope, forceps, scalpels, needles, spatulas and sterile blade
were used for aseptic manipulations.
3.1.4Washing of glasswares
Glasswares were first washed in boiled soda water for
two hours, thereafter, rinsed with tap water and dipped overnight
in dilute nitric acid solution. On the following day, these were
thoroughly washed in tap water and followed by distilled water.
Washings before drying on draining racks.
3.1.5Sterilization of culture vessels and instruments
Cotton plugged test tubes, flasks and culture bottles
along with aluminium wrapped, pipettes, petri dishes, etc. were
-17-
autoclaved at 15 psi pressure and 121ºC for 15-20 minutes.
These were, then, dried in hot air oven at 80-100ºC for 2-4 hours.
The instruments used for aseptic manipulations such
as forceps, scalpels, needles, spatulas and blades were sterilized
by dipping in 95 Per cent ethanol followed by flaming and cooling.
3.2 MEDIA
3.2.1Composition of media
In the present study, MS (Murashige and Skoog, 1962)
basal medium was used. The chemical composition of media is
given in Table 1.
3.2.2Preparation of stock solution
Separate stock solutions of major nutrients, minor
nutrients, potassium iodide, iron and various vitamins were
prepared by dissolving each chemical separately in small quantity
of double distilled water and making up the required volume with
double distilled water. The stock solutions of growth regulators
were prepared by dissolving them in small quantity of appropriate
solvents, heating gently and then making up the volume with
double distilled water. Auxins were dissolved in absolute ethyl
alcohol, while the cytokinins were dissolved in 1 N NaOH.
-18-
Table 1: Chemical composition of MS (Murashige andSkoog, 1962) medium
Stock Constituents Concentration instock solution
g/litre
Volume of stockin final volume
mg/litre (for 1 liter)
Finalconcentration in
medium(mg/litre)
A NH4NO3 82.50 20 ml 1650.00
B KNO3 95.00 20 ml 1900.00
C H3BO3 1.24 6.20
KH2PO4 34.00 170.00
KI 0.166 0.83
Na2MoO4.2H2O 0.050 5 ml 0.25
CoCl2.6H2O 0.005 0.025
D CaCl22H2O 88.00 5 ml 440.00
E MgSO4.7 H2O 74.00 370.00
MnSO4.4 H2O 4.46 5 ml 22.30
ZnSO4.7 H2O 1.72 8.60
CuSO4.5 H2O 0.005 0.025
F Na2EDTA 7.40 5 ml 37.55
FeSO4.7 H2O 5.57 27.85
G Thiamine HCl 0.02 0.10
Nicotinic acid 0.10 5 ml 0.50
Pyridoxine HCl 0.10 0.50
Glycine 0.40 2.0
Myo-inositol - 100 mg 100.0
Sucrose - 30 g 30g
Agar - 08.0 g 08.0 g
-19-
3.2.3Preparation of media
Specific quantities of stock solutions of major and
minor nutrients, organic constituents and growth regulators were
pipetted out in a beaker. Sucrose and myo-inositol were added
and dissolved. The volume was made upto 1 litre using double
distilled water and the pH of the medium was adjusted to 5.7-5.8
using 0.1 N NaOH or 0.1 N HCl. The medium was then boiled and
agar 0.8 per cent was added in the boiling medium and dissolved
to homogenize the medium. It was poured hot at the rate of 40-50
ml per sterilized bottle and 10-15 ml per sterilized culture tubes
(150 mm x 25 mm).
The bottles were plugged with autoclavable screw caps
and culture tubes were plugged with non-absorbent cotton and
autoclaved at 15 lbs (1.06 kg cm-2) pressure a temperature of
121ºC for 20 minutes (Bhojwani and Razdan, 1983). After
sterilization the medium was cooled to room temperature and
stored in cool dry inoculation room until used.
3.2.4Transfer area and aseptic manipulations
All the aseptic manipulations such as surface
sterilization of explants, preparation and inoculation of explants
and subsequent subculturing were carried out under aseptic
conditions in the hood of a clean laminar airflow chamber. The
working table of the laminar air flow chamber was first surface
sterilized with absolute alcohol and then by switching on the U.V.
-20-
light for 20-30 minutes before work started. The Petri dishes as
well as instruments used for inoculation were earlier steam
sterilized in an autoclave at 15 lbs pressure and 121ºC for 20
minutes were flame sterilized before each inoculation. Hands were
also swabbed in 70 per cent alcohol before inoculation.
3.2.5Culture room
The inoculated cultures were incubated at 25±2ºC in
an air-conditioned culture room with a light intensity of
2000-3000 Lux by cool while fluorescent tubes. Photoperiod was
maintained 16 hours daily.
3.3 METHODS
3.3.1 In vitro production of aseptic seedlings
Aseptic seedlings of Kagzi Lime were produced by
using the following methods.
1. Freshly extracted healthy seeds were taken and their
seed coats were removed.
2. De-coated seeds were surface sterilized in a 0.1 per cent
HgCl2 solution for 10 minutes.
3. Then seeds were removed from HgCl2 solution and were
given 3-4 washings of sterilized distilled water in a
laminar air flow cabinet.
4. Seeds were plated in the MS basal salt solid medium in
culture bottles.
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5. Incubate it at 25±2ºC temperature and 16/8 hours
light/dark cycles of photoperiod.
6. In due course of time seeds were germinated and aseptic
seedlings produced were used as explant in further
study.
3.3.2Collection of explants
The different explants used in micropropagation study
were nodal segments and shoot tips. These explants were excised
from the axenic seedlings of 10-30 days old and from in vivo
grown mature tree.
3.3.3Excision and sterilization of explants
After removing the axenic seedlings from culture
bottles, they were washed with sterile distilled water. The explants
of optimum size were surface sterilized in 0.1 per cent HgCl2
solution for 2-3 minutes for avoiding bacterial and fungal growth
followed by 2-3 washings with sterile distilled water.
The explants whether taken from in vitro grown
seedling or in vivo grown mature tree were cut aseptically in
Laminar Air Flow cabinet to get explants of appropriate size and
shape. The shoot tips of 0.5-1.0 cm having 2-3 leaf primordial,
nodal segments of 1.0-1.5 cm with at least one axillary bud.
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3.3.4Inoculation of explants
The surface sterilized and aseptically excised explants
were finally placed on media by working on LAF cabinet. The
bottles containing medium prepared as per different treatments,
were unplugged by holding them over spirit lamp and inoculations
were performed by placing explants on the surface of the medium
with the help of flame sterilized long forceps and again plugged
with screw cap of the bottles. During inoculation the explants
were properly positioned on the media and were gently pressed
with forceps to secure their firm contact with the media.
3.3.5Incubation of culture
The culture bottles/tubes after inoculation were kept
in culture room at 25±2ºC temperature under complete dark for
germination and also for callus induction. The explants incubated
for shoot induction/proliferation were cultured by maintaining
25±2ºC temperature and photoperiod (2000-3000 lux) of 16 hours
light and 8 hours dark in culture room.
3.4 EXPERIMENTAL DETAILS
3.4.1 In vitro Seedlings
Axenic seedlings of acid lime var. Kagzi Lime were
produced by using MS basal medium as shown in table 2.
-23-
Table 2: MS basal medium treatments used for invitro seed germination and axenic seedlingproduction
Sr. No. Treatments
1 (Control) MS Basal Media
2 MS + BAP 0.5 Mg/l
3 MS + GA3 0.5 mg/l
4 MS + BAP 0.5 mg/ l + GA3 0.5 mg/l
5 MS + GA3 1.0 mg/ l
6 MS+ BAP 1.0 mg/ l
7 MS + BAP 1.0 mg/l + GA3 1.0 mg/ l
The observation on days required for initation and
completion of germination, per cent germination, and seedling
length at 15 and 30 days after germination were recorded.
3.4.2Micro propagation
For micro propagation studies, shoot tip and nodal
segments from Kagzi Lime axenic seedlings and in vivo grown
mature tree were used as explants. The shoot multiplication was
achieved using MS basal medium as shown in table 3.
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-25-
Table 3: MS basal medium treatments used fordirect regeneration
Sr. No. Treatments
1 MS (Control)
2 MS + BAP 0.25 mg/l
3 MS + BAP 0.25 mg/l+ NAA 0.25 mg/l
4 MS + BAP 0.25 mg/l+ NAA 0.50 mg/l
5 MS + BAP 0.50 mg/l
6 MS + BAP 0.50 mg/l+ NAA 0.25 mg/l
7 MS + BAP 0.50 mg/l+ NAA 0.50 mg/l
8 MS + BAP 0.75 mg/l
9 MS + BAP 0.75 mg/l+ NAA 0.25 mg/l
10 MS + BAP 0.75 mg/l+ NAA 0.50 mg/l
11 MS + BAP 0.10 mg/l
12 MS + BAP 0.10 mg/l + NAA 0.25 mg/l
13 MS + BAP 0.10 mg/l + NAA 0.50 mg/l
Following observations were recorded periodically after
inoculation for shoot regeneration in micro propagation.
3.4.3Observations recorded
3.4.3.1 Number of days for shoot initiation: The number of
days taken for shoot initiation were counted numerically from the
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date of inoculation for regeneration separately for each treatment
and represented as number of days required for shoot initiation.
3.4.3.2 Number of days for completion of regeneration: The
number of days taken for complete regeneration were counted
numerically from the date of inoculation for regeneration till the
regeneration completed for each treatment separately and
represented as number of days required for completion of
regeneration.
3.4.3.3 Per cent shoot regeneration: The number of
cultures which responded to shoot regeneration were counted in
each treatment medium excluding the contaminated of cultures
inoculated and multiplied with 100 were calculated and
represented as per cent shoot regeneration.
No of culture regenerated Per cent shoot regeneration = ––––––––––––––––––––––––––×100
Total no of cultures inoculated
3.4.3.4 Number of shoots/explant: The number of shoots
developed from each explants were numerically counted
separately in each treatment after complete regeneration. The
number of shoots/explant from different media were recorded.
3.4.3.5 Shoot length (cm): The length in centimeter of each
shootlet was measured under aseptic condition before
transferring them to media for root differentiation.
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3.4.4Root differentiation: Shoot above 1.5-2.5 cm height or
minimum four leaves, were transferred for root induction. The
root organogenesis was obtained by using full and ½ strength MS
medium supplemented with different concentrations of auxin
(NAA or IBA) as shown in table 4.
Table 4: Effect of modified MS medium on rootdifferentiation
Sr. No Treatments
1. MS + NAA 0.25 mg/l
2. MS + NAA 0.50 mg/l
3. MS + NAA 1.0 mg/l
4. MS + IBA 0.25 mg/l
5. MS + IBA 0.50 mg/l
6. MS + IBA 1. mg/l
7. ½ MS + NAA 0.25 mg/l
8. ½ MS + NAA 0.50 mg/l
9. ½ MS + NAA 1.0 mg/l
10. ½ MS + IBA 0.25 mg/l
11. ½ MS + IBA 0.50 mg/l
12. ½ MS + IBA 1.0 mg/l
-28-
The following observation were recorded periodically
after inoculation of shootlets for rooting
3.4.4.1 Number of days for initiation of roots
The number of days taken for root initiation were
counted numerically from the date of inoculation for rooting
separately for each treatment and represented as number of days
for root initiation.
3.4.4.2 Number of days for completion of rooting
The number of days required for completion of rooting
were counted numerically from date of inoculation for rooting till
the rooting completed for each treatment separately and
represented as number of days required for completion of rooting.
3.4.4.3 Number of roots/plantlet
The number of roots differentiated from each plantlet
were numerically counted separately in each treatment after
complete differentiation of rooting and the number of
roots/plantlet from different media were recorded.
3.4.4.4 Root length (cm)
The length of each root in centimeter was measured
during transferring them to potting mixture for hardening.
3.4.4.5 Per cent rooting
The number of shoots or plantlets which responded to
the media for root initiation were counted in each treatment
excluding the contaminated shoots and the ratio of this to that of
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the total number of shoots inoculated for rooting were calculated
and multiplied with 100 and represented as per cent rooting.
3.4.4.6 Per cent survival of rooted plantlets
The number of surviving rooted plantlets and dead
plantlets were counted numerically in each treatment and the
ratio of these was calculated and multiplied with 100 represented
as per cent survival of rooted plantlets.
3.4.5Hardening
The well developed plantlets with root and shoot were
transferred in different potting mixtures and handened in green
house and in field condition. For the hardening of plantlets
following potting mixtures were used as shown in table 5.
Table 5: Effect of different potting mixtures on hardeningand plant growth
Sr. No. Treatments
T1 Soil : sand : 1:1v/v
T2 Soil : sand : FYM 1:1:1 v/v
T3 Soil : sand : FYM 2:1:1 v/v
T4 Soil : sand : vermicompost 1:1:1 v/v
T5 Soil : sand : vermicompost 2:1:1 v/v
T6 Soil : sand : vermiculite 1:1:1 v/v
T7 Soil : sand : vermiculite 2:1:1 v/v
T8 Soil : vermiculite 1:1 v/v
T9 Soil : vermiculite 1:2 v/v
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T10 Soil site mix
The following observations were recorded periodically,
after transferring in green house
3.4.5.1 Height of plant (cm)
The height in centimeters of each plantlets was
measured in each potting mixture treatment at 15 and 30 days
after potting as to represent height of plant.
3.4.5.2 Number of leaves per plant
Number of leaves/plant was counted numerically in
each potting mixture treatment at 15 and 30 days after potting.
3.4.5.3 Per cent survival of plantlets under green house
condition this was calculated by:
Number of plantlets survived = –––––––––––––––––––––––––––––––––––––––––––––––––– x 100 Number of plantlets transferred in the plastic bags
3.4.5.4 Per cent survival of plantlets in open condition-
this was calculated by:
Number of plantlets survived = –––––––––––––––––––––––––––––––––––––––––––––––––––x 100 Number of plantlets transferred in the open condition
3.5 Statistical analysis
The experiment on In vitro studies on Citrus
aurantifolia Swingle was carried out in Completely Randomized
Design (CRD) following the procedures given by Panse and
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Sukhatme (1985). Ten culture bottles having two explants of each
treatment was used for recording different observations.
3.6 Agrobacterium-mediated transformation in acid
lime (Citrus aurantifolia Swingle) var. Kagzi Lime
3.6.1Plant materials and culture media
Green house grown 6-12 month old acid lime seedlings
var. Kagzi Lime were used as a source of tissue for
transformation. Stem pieces (2cm long) were stripped of leaves
and thorns, disinfected for 10 minutes in 2% (vol/vol) sodium
hypochlorite solution and rinsed three times with sterile water.
MSB1 medium consisted of salts of MS medium vitamins of white
medium, 3% sucrose supplemented with 1mg/l BAP. The medium
having pH 5.7 was solidified with 0.8% agar (wt./vol) when
needed (Duran-Vilo et al. 1989). Kanamycin or geneticin,
cefotaxime (600mg/l) and vancomycin (250mg/l) were filter
sterilized and added to the autoclaved medium when needed.
Tomato feeder plates were prepared by pipetting 2ml
of 6 to 7 days old tomato cell suspension (TCS). TCS medium
consisted of Murashige and Skoog (1962) salts, 1mg/l
thiaminehydrochloride, 1mg/l pyridoxine hydrochloride, 1 mg/l
nicotinic acid, 3% sucrose (wt/vol), 2mg/l indole-3-acetic acid,
1mg/l 2-isopentyl adenine, 2mg/l 2, 4-dichlorophenoxy acetic
acid, 0.8% agar (wt/vol), pH 5.7 (Duran-vilo et al. 1995). Tomato
cell suspensions were maintained in TCS liquid medium in a
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shaker at 100 rpm and 25ºC and were subcultured to fresh
medium every 15 days.
3.6.2Bacterial strain and vector
Agrobacterium tumefaciens EHA 105 (Hood et al. 1993)
harboring the binary plasmid pCAMBIA-1301 (Vancanneyt et al.
1991) was used as the vector system for transformation. The uid
A gene driven by the CaMV 35S promoter and terminator
sequences served as reporter gene. The intron in the uid A gene of
PCAMBIA-1301 blocks its expression in A. tumefaciens. The
neomycin phosphotransferase (PTII) gene driven by the napoline
synthase (NOS) promoter and terminator sequences was used as
the selectable marker gene. Bacteria were cultured overnight at
28ºC in LB medium (Sambrook et al. 1989), containing 25mg/l
kanamycin and 25 mg/l halidixic acid. Bacterial cells were
pelleted at 3500 rpm for 10 minutes suspended in liquid MSB1
medium and diluted at 107 cells/ml in the same medium.
3.6.3Transformation and regeneration
Internodal stem segments (1cm long) were cut
transversely from the stem pieces, inoculated with A. tumefaciens
in liquid MSB 1 medium for 15-30min. blotted dry with sterile
filter paper and placed horizontally on solid MSB 1 medium or
tomato feeder plates for a 3-day co cultivation period. After
cocultivation, the explants were blotted dry with sterile filter
paper and transferred to MSB 1 plates containing 500mg/l
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cefotaxime and 250 mg/l vancomycin to control further bacterial
growth. The cultures were maintained in the dark for 15 days at
26ºC and then transferred to 16h photoperiod, 45 Em-2s-1
illumination, 26ºC and 60% relative humidity. To compare
transformation frequencies among different cocultivation and
selection conditions, the explants were - glucuronidase (GUS) –
assayed after 6 weeks in selective medium. Transformation
frequency (%) was evaluated as the number of explants with
transformation frequency (%) was evaluated as the number of
explants with transformation events (blue spots) per total number
of Agrobacterium incubated explants.
In experiment to obtain regenerated shoots of acid
lime, cocultivation was performed only on feeder plates. The
explants were subcultured to fresh MSB 1 medium every weeks.
Regenerated shoots (0.2-0.8 cm long) were harvested from the
stem segments and excised in two pieces. The shoot basal ends
were assayed for GUS activity.
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CHAPTER-4
Experimental Results
The research findings obtained in the present
investigation on “In vitro studies in acid lime (Citrus aurantifolia
Swingle) var. Kagzi Lime” are presented in this chapter.
4.1 In vitro seed germination
4.1.1Sterilization duration
The effect of different durations of sterilization with
HgCl2 (0.1%) on per cent contamination of inoculated acid lime
seeds is presented in Table 6. The three different durations of
sterilization were 5, 10 and 15 minutes. The minimum
contamination was recorded with 10 minutes (12.00%) which was
significantly superior over other two durations of 5 (31.00%) and
15 minutes (40.00%).
4.1.2Number of days for initiation of germination
The effect of different concentrations of BAP and GA3
on seed germination is presented in Table 7. Number of days
taken for initiation of seed germination ranged from
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Table 6: Effect of different durations of sterilizationwith HgCl2 (0.1%) on percent contamination
of inoculated acid lime seeds
Treatments Percent contamination
5 Minutes 31.00±5.09
10 Minutes 12.00±1.22
15 Minutes 40.00±4.47
±SEm
CD at 5% 12.39
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-37-
Table 7: Effect of different levels/concentrations of BAP and GA3 alone and in combination on
germination of acid lime seeds
Treatments Time taken to startgermination (days)
Time taken to completegermination (days)
Percent germination
T1 (Control) 23.00±1.22 34.40±0.24 42.22±1.00
T2 BAP (0.5 mg/l) 15.00±0.70 33.00±0.63 44.98±2.03
T3 GA3 (0.5 mg/l) 8.60±0.24 15.40±0.24 72.16±1.60
T4 BAP (0.5 mg/l) + GA3 (0.5mg/l)
14.20±1.11 24.20±0.66 47.06±1.84
T5 GA3 (1.0 mg/l) 9.40±0.24 17.00±0.31 65.85±1.95
T6 BAP (1.0 mg/l) 15.60±0.24 23.20±0.374 47.86±1.82
T7 BAP (1.0 mg/l) + GA3 (1.0mg/l)
13.40±0.51 23.60±0.40 53.12±1.12
±SEm 2.21 1.28 4.86
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-38-
CD at 5%
-39-
-39-
8.60 to 15.60 days. The seeds inoculated on MS + GA3 0.5 mg/l
induced the earliest germination (8.60 days) which was
significantly at par with MS+GA3 1.0 mg/l (9.40 days). Among the
various different levels of BAP and GA3 maximum days required
for initiation was noted on MS medium + BAP 1.0 mg/l (15.60)
which was at par with MS medium + BAP 0.5 mg/l (15.00 days),
while, in control the time taken to start germination was 23.00
days.
4.1.3Number of days for completion of germination
The number of days required for completion of seed
germination on MS media supplemented with different levels of
BAP and GA3 are depicted in Table 7. Number of days required
for completion of seed germination varied from 15.40 to 33.00
days. The seeds inoculated on MS medium + GA3 0.5 mg/l
completed germination at the earliest (15.40 days) followed by MS
medium + GA3 1.0 mg/l (17.00 days). Among the different MS
media compositions maximum days for completion of seed
germination were recorded on MS medium + BAP 0.5 mg/l (33.00
days), while in control, it was 34.40 days.
4.1.4Per cent germination
-40-
-40-
The effect of different concentrations of GA3 and BAP
on per cent germination are given in Table 8. It was observed that
the maximum germination per cent was recorded
-41-
-41-
Table 8: Effect of different levels/concentrations of BAP and GA3 alone and in combination on
seedling length at 15 and 30 days after germination
Treatments Seedling length at 15 days aftergermination (cm)
Seedling length at 30 days aftergermination (cm)
T1 (Control) 1.82±0.10 2.80±0.03
T2 BAP (0.5 mg/l) 3.26±0.10 3.72±0.11
T3 GA3 (0.5 mg/l) 3.62±0.02 4.68±0.07
T4 BAP (0.5 mg/l) + GA3(0.5 mg/l)
3.82±0.02 5.40±0.06
T5 GA3 (1.0 mg/l) 2.76±0.05 4.20±0.03
T6 BAP (1.0 mg/l) 3.10±0.03 3.54±0.02
T7 BAP (1.0 mg/l) + GA3(1.0 mg/l)
3.42±0.11 4.36±0.06
±SEm 0.22 0.19
-42-
-42-
CD at 5%
-43-
-43-
on MS medium supplemented with GA3 0.5 mg/l (72.16%),
whereas, it was minimum on control (42.22%).
4.1.5Seedling length
The seedling height after 15 and 30 days of
germination on MS medium supplemented with different levels of
BAP and GA3 was recorded (Table 8, Plate - I). Maximum height
was observed on MS medium + GA3 0.5 mg/l + BAP 0.5 mg/l
(3.82 cm) which was significantly at par with MS medium + GA3
0.5 mg/l (3.62 cm). Minimum height was recorded on MS medium
+ GA3 1.0 mg/l (2.76 cm), amongst the various MS media
compositions, whereas, it was 1.82 cm in control.
Maximum height was observed on MS medium + GA3
0.5 + BAP 0.5 mg/l (5.40 cm) which was significantly superior
over other media composition. The minimum height was recorded
on MS medium + BAP 1.0 mg/l (3.54 cm). It was 2.80 cm in
control.
4.2 Micropropagation
The micropropagation in acid lime var. Kagzi Lime was
studied by culturing in vitro and in vivo shoot tip and nodal
segments as explants. The results of various observations
recorded are presented in brief below.
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-44-
a) In vitro explants
4.2.1Number of days for shoot initiation
4.2.1.1 In vitro shoot tip explant
The number of days required for shoot initiation
ranged from 8.40 to 24.80 days (Table 9). The in vitro shoot tips
cultured on MS medium + BAP 0.25 mg/l induced shoot initiation
at the earliest (8.40 days) which was at par with MS + BAP 0.25 +
NAA 0.25 mg/l (8.60 days) and MS medium + BAP 0.25 + NAA 0.5
mg/l (8.80 days). The maximum days required for shoot initiation
were observed on MS + BAP 1.0 + NAA 0.25 mg/l (24.80 days). In
control, the days required for shoot initiation were 26.20.
4.2.1.2 In vitro nodal segments
From Table 9, it was observed that the number of days
required for shoot initiation from in vitro nodal segments ranged
from 10.40 to 28.40 days. The in vitro nodal segments inoculated
on MS medium + BAP 0.25 mg/l induced shoot initiation at the
earliest (10.40 days) which was at par with MS medium + BAP
0.25 + NAA 0.25 mg/l (10.50 days) and MS medium + BAP 0.25 +
NAA 0.5 mg/l (10.40 days). Maximum days taken for shoot
initiation was noted on MS medium + BAP 1.0 mg/l+NAA 0.25
mg/l (25.40 days), which was significantly at par with control
(28.20 days).
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-45-
Table 9: Effect of different levels/concentrations of BAP and NAA on shoot initiation and regenerationfrom in vitro shoot tip and nodal segment explants
Treatments Number of days required forshoot initiation
Number of days required forcompletion of regeneration
Shoot tip Nodal segment Shoot tip Nodal segment T1 (Control) 26.20±0.37 28.20±0.20 41.40±0.24 43.40±0.24
T2 BAP (0.25 mg/l) 8.40±0.24 10.40±0.24 23.40±0.24 25.60±0.24
T3 BAP (0.25 mg/l + NAA (0.25 mg/l) 8.60±0.21 10.80±0.20 23.80±0.20 26.40±0.24
T4 BAP (0.25 mg/l + NAA (0.50 mg/l) 8.80±0.20 10.40±0.24 24.20±0.20 26.60±0.24
T5 BAP (0.50 mg/l) 12.40±0.24 17.40±0.24 26.40±0.24 32.40±0.24
T6 BAP (0.50 mg/l + NAA 0.25 mg/l) 13.40±0.24 17.00±0.00 28.00±0.00 32.00±0.00
T7 BAP (0.50 mg/l + NAA 0.50 mg/l) 12.40±0.24 19.40±0.24 27.60±0.24 34.20±0.20
T8 BAP (0.75 mg/l) 12.80±0.49 18.20±0.20 27.20±0.20 33.60±0.40
T9 BAP (0.75 mg/l + NAA (0.25 mg/l) 17.40±0.24 22.40±0.24 32.40±0.24 37.40±0.24
T10 BAP (0.75 mg/l + NAA 0.50 mg/l) 17.60±0.24 22.80±0.20 32.60±0.24 37.60±0.40
T11 BAP (1.0 mg/l) 24.40±0.24 26.40±0.40 39.60±0.24 41.20±0.20
T12 BAP (1.0 mg/l + NAA 0.25 mg/l) 24.80±0.20 28.40±0.24 38.40±0.24 41.60±0.24
T13 BAP (1.0 mg/l + NAA 0.50 mg/l) 24.40±0.24 26.60±0.24 38.60±0.40 41.80±0.37
-46-
-46-
±SEm
CD at 5% 0.78 0.67 0.68 0.77
-47-
-47-
4.2.2Number of days for completion of regeneration
4.2.2.1 In vitro shoot tip explant
It was noted that the number of days required for
completion of regeneration varied from 23.40 to 41.40 days (Table
9). The in vitro shoot tips cultured on MS medium + BAP 0.25
mg/l completed regeneration at the earliest (23.40 days), which
was at par with MS medium + BAP 0.25 mg/l + NAA 0.25 mg/l
(23.80 days). Maximum days required for completion of
regeneration were recorded on control (41.40 days).
4.2.2.2 In vitro nodal segment
The effect of different levels of BAP and NAA on
number of days required for completion of regeneration are
presented in Table 9. Maximum days were taken for completion of
regeneration on MS medium + BAP 1.0 mg/l + NAA 0.5 mg/l
(41.80 days) which were at par with MS medium + BAP 1.0mg/l
(41.20 days) and MS + BAP 1.0 + NAA 0.25 mg/l (41.60 days) and
was significantly higher than other MS media compositions,
however, in control the regeneration was completed in 43.40 days.
4.2.3Per cent regeneration
4.2.3.1 In vitro shoot tip explant
The effect of different concentrations of BAP and GA3
in MS media on shoot multiplication from in vitro shoot tip is given
in Table 10. It was observed that maximum
-48-
-48-
Table 10: Effect of different levels/concentrations of BAP and NAA on percent regeneration from invitro shoot tip and nodal segment explants
Treatments Percent regeneration Shoot tip Nodal segment
T1 (Control) 29.49±1.53 30.37±1.90
T2 BAP (0.25 mg/l) 90.00±0.00 90.00±0.00
T3 BAP (0.25 mg/l + NAA (0.25 mg/l) 90.00±0.00 90.00±0.00
T4 BAP (0.25 mg/l + NAA (0.50 mg/l) 78.39±5.20 79.43±4.67
T5 BAP (0.50 mg/l) 54.38±1.78 58.31±5.37
T6 BAP (0.50 mg/l + NAA 0.25 mg/l) 53.23±0.51 54.63±1.75
T7 BAP (0.50 mg/l + NAA 0.50 mg/l) 51.81±0.60 53.18±2.97
T8 BAP (0.75 mg/l) 49.24±1.00 51.63±2.08
T9 BAP (0.75 mg/l + NAA (0.25 mg/l) 43.71±1.26 44.29±1.72
T10 BAP (0.75 mg/l + NAA 0.50 mg/l) 42.55±2.39 44.05±1.61
T11 BAP (1.0 mg/l) 40.98±3.28 43.32±3.27
T12 BAP (1.0 mg/l + NAA 0.25 mg/l) 36.83±2.50 40.31±2.35
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-49-
T13 BAP (1.0 mg/l + NAA 0.50 mg/l) 32.49±2.49 35.34±2.85
±SEm
CD at 5% 7.51 7.01
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percent regeneration was recorded on MS medium with BAP 0.25
mg/l (90.00%) which was at par with MS medium + BAP 0.25 +
NAA 0.25 mg/l (90.00%).
The minimum per cent regeneration was recorded on
MS medium + BAP 1.0 + NAA 0.5 mg/l (32.49%). In control, the
per cent regeneration was 29.49%.
4.2.3.2 In vitro nodal segment
It can be observed from table 10 that in vitro nodal
segment explants showed maximum per cent regeneration on MS
medium supplemented with BAP 0.25 mg/l (90.00%) and which
was at par with MS medium + BAP 0.25 + NAA 0.25 mg/l
(90.00%). The minimum per cent regeneration was recorded on
MS medium + BAP 1.0 + NAA 0.5 mg/l (35.34%). While, the per
cent regeneration was 30.37% in control.
4.2.4Number of shoots per explant
4.2.4.1 In vitro shoot tip
Data on multiple shoot formation from in vitro shoot
tip explants cultured on MS medium supplemented with different
levels of BAP and NAA is presented in Table 11. In shoot tip
explants, number of shoots regenerated on MS medium
supplemented with BAP 0.25 mg/l (3.40). The minimum number
of shoots were observed on the MS medium + BAP 0.75 mg/l
(1.60).
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Table 11: Effect of different levels/concentrations of BAP and NAA on number of shoots/explant andshoot length from in vitro shoot tip and nodal segment explants
Treatments Number of shoots/explant Shoot length (cm)Shoot tip Nodal segment Shoot tip Nodal segment
T1 (Control) 1.46±0.02 1.74±0.04 1.66±0.04 1.58±0.02
T2 BAP (0.25 mg/l) 3.40±0.24 3.80±0.20 3.48±0.02 4.52±0.08
T3 BAP (0.25 mg/l + NAA (0.25 mg/l) 3.20±0.20 3.60±0.24 3.24±0.04 4.58±0.03
T4 BAP (0.25 mg/l + NAA (0.50 mg/l) 2.86±0.04 2.90±0.03 2.90±0.03 4.56±0.02
T5 BAP (0.50 mg/l) 1.84±0.02 3.00±0.00 3.82±0.09 3.74±0.02
T6 BAP (0.50 mg/l + NAA 0.25 mg/l) 1.66±0.02 2.84±0.04 2.82±0.02 3.6±0.00
T7 BAP (0.50 mg/l + NAA 0.50 mg/l) 1.62±0.02 2.46±0.02 2.62±0.02 2.9±0.00
T8 BAP (0.75 mg/l) 1.50±0.00 2.14±0.05 2.14±0.02 2.21±0.05
T9 BAP (0.75 mg/l + NAA (0.25 mg/l) 1.54±0.02 1.90±0.06 1.86±0.02 3.36±0.04
T10 BAP (0.75 mg/l + NAA 0.50 mg/l) 1.56±0.04 1.98±0.04 1.86±0.02 2.56±0.02
T11 BAP (1.0 mg/l) 1.64±0.04 1.84±0.02 2.02±0.02 2.24±0.04
T12 BAP (1.0 mg/l + NAA 0.25 mg/l) 1.80±0.06 1.94±0.02 2.10±0.04 1.86±0.02
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T13 BAP (1.0 mg/l + NAA 0.50 mg/l) 1.60±0.08 2.08±0.03 1.96±0.04 2.04±0.02
±SEm
CD at 5% 0.10 0.08 0.17 0.10
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4.2.4.2 In vitro nodal segment
The mean number of shoot buds initiated from in vitro
nodal segment explant ranged from 1.84 to 3.80 shoot buds per
explant (Table 11). In vitro nodal segment produced maximum
number of shoots on MS medium + BAP 0.25 mg/l (3.80) followed
by MS+ BAP 0.25 + NAA 0.25 mg/l (3.60). The minimum number
of shoots were observed on MS medium + BAP 1.0mg/l (1.84).
4.2.5Shoot length
4.2.5.1 In vitro shoot tip
Maximum length was observed on the MS medium +
BAP 0.5 mg/l (3.82 cm) which was significantly superior over
other MS media compositions (Table 11, Plate 2). The minimum
length was observed on MS medium + BAP 0.75 + NAA 0.25 mg/l
(1.86 cm). The shoot length was 1.66 cm in control.
4.2.5.2 In vitro nodal segment explant
The average length of shoots regenerated from in vitro
nodal segment explants ranged from 1.86 to 4.58 cm, while, it
was 1.58cm in control (Table 11, Plate 3). Multiple shoots
regenerated from nodal segment explant recorded maximum
length on MS medium + BAP 0.25 + NAA 0.25 mg/l (4.58 cm)
which was at par with MS medium + BAP 0.25 NAA 0.5 mg/l
(4.56cm) and MS medium + BAP 0.25 mg/l (4.52cm). The
minimum shoot length was observed on MS medium + BAP 1.0 +
NAA 0.25 mg/l (1.86 cm).
b) In vivo explants
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4.2.6Number of days for shoot initiation
4.2.6.1 In vivo shoot tip
The number of days required for shoot initiation varied
from 10.60 to 29.00 days (Table 12). MS medium having BAP 0.25
+ NAA 0.25 mg/l induced shoot initiation at the earliest (10.60
day) which was at par with MS medium + BAP 0.25 mg/l (10.80
days) and MS medium + BAP 0.25 + NAA 0.5 mg/l (12.20 days).
The MS medium having BAP 1.0 mg/l took maximum days (29.00)
for shoot initiation, while, it was taken 28.80 days in control.
4.2.6.2 In vivo nodal segment
The average number of days required for shoot
initiation from in vivo nodal segment explants ranged from 12.00
to 29.00 days on different MS media compositions (Table 12). It
was observed that the minimum time taken for shoot initiation
was recorded on MS medium + BAP 0.25 + NAA 0.50 mg/l (12.00
days) which was significantly at par with MS + BAP 0.25 mg/l
(12.60 days) and MS medium + BAP 0.25 + NAA 0.5 mg/l (13.00
days). The maximum time was observed on MS medium + BAP 1.0
mg/l (28.00 days).
4.2.7Number of days for completion of regeneration
4.2.7.1 In vivo shoot tip
The number of days required for completion of
regeneration on MS medium supplemented with different
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Table 12: Effect of different levels/concentrations of BAP and NAA on shoot initiation and regenerationfrom in vivo shoot tip and nodal segment explants
Treatments Number of days required forshoot initiation
Number of days required forcompletion of regeneration
Shoot tip Nodal segment Shoot tip Nodal segment
T1 (Control) 28.80±0.49 31.20±0.80 42.20±1.56 48.00±1.70
T2 BAP (0.25 mg/l) 10.80±0.37 12.60±0.60 29.60±2.44 29.00±0.89
T3 BAP (0.25 mg/l + NAA (0.25 mg/l) 10.60±0.40 13.00±0.77 26.00±0.70 27.00±0.83
T4 BAP (0.25 mg/l + NAA (0.50 mg/l) 12.20±0.37 12.00±0.70 26.20±0.58 27.80±0.80
T5 BAP (0.50 mg/l) 15.20±0.37 19.80±1.15 29.40±1.28 34.60±2.11
T6 BAP (0.50 mg/l + NAA 0.25 mg/l) 16.40±0.51 19.60±0.98 30.00±0.89 33.80±1.35
T7 BAP (0.50 mg/l + NAA 0.50 mg/l) 14.40±0.24 20.00±0.83 30.40±0.67 35.20±1.28
T8 BAP (0.75 mg/l) 16.20±1.24 20.60±0.74 29.40±1.02 34.40±1.50
T9 BAP (0.75 mg/l + NAA (0.25 mg/l) 20.20±0.97 25.00±0.89 36.20±1.35 40.40±1.07
T10 BAP (0.75 mg/l + NAA 0.50 mg/l) 20.20±0.97 25.00±0.83 35.60±1.63 40.60±1.07
T11 BAP (1.0 mg/l) 29.00±1.761 28.00±0.63 35.00±1.78 42.80±0.86
T12 BAP (1.0 mg/l + NAA 0.25 mg/l) 27.60±1.47 26.20±0.49 35.20±1.65 43.00±0.83
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T13 BAP (1.0 mg/l + NAA 0.50 mg/l) 24.80±0.37 27.60±0.67 38.00±0.70 44.40±0.51
±SEm
CD at 5% 2.49 2.26 3.87 3.46
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levels of BAP and NAA are presented in Table 12. The in vivo shoot
tips cultured on MS medium + BAP 0.25 + NAA 0.25 mg/l
completed regeneration at the earliest (26.00 days) which was at
par with MS medium + BAP 0.25 + NAA 0.5 mg/l (26.20 days) and
MS medium + BAP 0.25 mg/l (29.60 days) and MS medium + BAP
0.5 mg/l (29.40 days). The maximum time required for completion
of regeneration was noted on MS medium + BAP 1.0 mg/l (28.00
days). The regeneration completed in 31.20 days in control.
4.2.7.2 In vivo nodal segment
From Table 12, it was noted that the in vivo nodal
segments inoculated on MS medium + BAP 0.25 + NAA 0.25 mg/l
completed regeneration at the earliest (27.00 days), which was
statistically at par with MS medium + BAP 0.25 + NAA 0.5 mg/l
(27.80 days) and MS medium + BAP 0.25 mg/l (29.00 days). The
MS medium having BAP 1.0 mg/l took maximum time for
completion of regeneration (44.40 days), amongst the various MS
media compositions, it took 48.00 days in control.
4.2.8Per cent regeneration
4.2.8.1 In vivo shoot tip
The effect of different levels of BAP and NAA on per
cent shoot regeneration is given in Table 13. It was observed that
maximum per cent shoot regeneration was recorded on MS
medium supplemented with BAP 0.25 mg/l (77.77%)
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Table 13: Effect of different levels/concentrations of BAP and NAA on percent regeneration from in vivoshoot tip and nodal segment explants
Treatments Percent regenerationShoot tip Nodal segment
T1 (Control) 24.12±2.99 26.39±2.96
T2 BAP (0.25 mg/l) 77.77±5.15 78.59±4.71
T3 BAP (0.25 mg/l + NAA (0.25 mg/l) 71.95±5.04 73.13±4.56
T4 BAP (0.25 mg/l + NAA (0.50 mg/l) 66.03±3.71 68.42±2.83
T5 BAP (0.50 mg/l) 49.92±2.80 52.87±2.58
T6 BAP (0.50 mg/l + NAA 0.25 mg/l) 46398±2.84 49.47±2.82
T7 BAP (0.50 mg/l + NAA 0.50 mg/l) 45.79±1.40 47.63±1.44
T8 BAP (0.75 mg/l) 44.17±1.06 46.36±1.52
T9 BAP (0.75 mg/l + NAA (0.25 mg/l) 38.11±1.59 38.92±1.92
T10 BAP (0.75 mg/l + NAA 0.50 mg/l) 37.06±1.26 38.73±0.96
T11 BAP (1.0 mg/l) 35.74±1.06 38.26±0.88
T12 BAP (1.0 mg/l + NAA 0.25 mg/l) 30.87±0.96 31.40±0.78
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T13 BAP (1.0 mg/l + NAA 0.50 mg/l) 30.35±0.89 31.25±1.10
±SEm
CD at 5% 7.92 7.33
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which was at par with MS medium + BAP 0.25 + NAA 0.25 mg/l
(71.95%). The minimum per cent regeneration was recorded on
MS medium + BAP 1.0 + NAA 0.5 mg/l (30.35%).
4.2.8.2 In vivo nodal segment
From Table 13, it was observed that in vivo nodal
segment explants showed maximum per cent shoot regeneration
on MS medium having BAP 0.25 mg/l, which was at par with MS
medium + BAP 0.25 + NAA 0.25 mg/l (78.59%). The minimum per
cent shoot regeneration was recorded on MS medium + BAP 1.0 +
NAA 0.5 mg/l (31.25%).
4.2.9Number of shoots per explant
4.2.9.1 In vivo shoot tip
Data on multiple shoot formation from in vivo shoot tip
explants inoculated on MS medium supplemented with BAP and
NAA is depicted in Table 14 (Plate 4). In shoot tip explants,
maximum number of shoots were recorded on MS medium
supplemented with BAP 0.25 + NAA 0.5 mg/l (2.60) which was at
par with MS medium + BAP 0.25+ NAA 0.25 mg/l (2.40) and MS
medium + BAP 0.25 mg/l (2.40). The minimum number of shoots
were observed on MS medium + BAP 0.75 mg/l (1.20) which was
at par with control (1.20).
4.2.9.2 In vivo nodal segment
The effect of different concentrations of BAP and NAA
on number of shoots/explant is presented in Table 14
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Table 14: Effect of different levels/concentrations of BAP and NAA on number of shoots/explant andshoot length from in vivo shoot tip and nodal segment explants
Treatments Number of shoot/explant Shoot length (cm)Shoot tip Nodal segment Shoot tip Nodal segment
T1 (Control) 1.20±0.20 1.60±0.24 1.66±0.08 1.40±0.13
T2 BAP (0.25 mg/l) 2.40±0.24 3.20±0.37 3.12±0.27 4.32±0.15
T3 BAP (0.25 mg/l + NAA (0.25 mg/l) 2.40±0.24 3.20±0.37 2.92±0.22 4.42±0.34
T4 BAP (0.25 mg/l + NAA (0.50 mg/l) 2.60±0.24 3.20±0.37 2.72±0.25 4.50±0.18
T5 BAP (0.50 mg/l) 1.60±0.24 1.80±0.20 3.04±0.07 3.70±0.33
T6 BAP (0.50 mg/l + NAA 0.25 mg/l) 1.40±0.24 2.20±0.37 2.66±0.30 3.46±0.42
T7 BAP (0.50 mg/l + NAA 0.50 mg/l) 1.40±0.24 2.00±0.31 2.42±0.28 2.98±0.24
T8 BAP (0.75 mg/l) 1.20±0.20 1.80±0.20 1.92±0.21 2.10±0.26
T9 BAP (0.75 mg/l + NAA (0.25 mg/l) 1.20±0.20 1.80±0.20 1.74±0.09 3.08±0.32
T10 BAP (0.75 mg/l + NAA 0.50 mg/l) 1.20±0.20 1.60±0.24 1.88±0.03 2.54±0.33
T11 BAP (1.0 mg/l) 1.40±0.24 1.80±0.24 1.86±0.13 2.00±0.22
T12 BAP (1.0 mg/l + NAA 0.25 mg/l) 1.60±0.24 1.60±0.24 1.92±0.20 1.84±0.19
T13 BAP (1.0 mg/l + NAA 0.50 mg/l) 1.40±0.24 1.60±0.24 2.02±0.12 1.90±0.17
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±SEm
CD at 5% 0.66 0.81 0.56 0.77
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(Plate 5). The maximum number of shoots/explant were noted on
MS medium + BAP 0.25 mg/l (3.20) which was at par with MS
medium + BAP 0.25 + NAA 0.25 mg/l (3.20) and MS medium +
BAP 0.25 + NAA 0.5 mg/l (3.20). The minimum number of shoots
were recorded on MS medium + BAP 1.0 + NAA 0.25 mg/l (1.60),
which was at par with control (1.60).
4.2.10 Shoot length
4.2.10.1 In vivo shoot tip explant
The shoot length of multiple shoots regenerated from
shoot tip explants were recorded (Table 14). The maximum shoot
length was observed on MS medium having BAP 0.25 mg/l
(3.12cm) which was at par with MS medium + BAP 0.5 mg/l
(3.04cm) and MS medium + BAP 0.25 (2.92 cm) + NAA 0.25 mg/l
and MS medium + BAP 0.25 + NAA 0.5 mg/l (2.72cm). The
minimum shoot length was observed on MS medium + BAP 0.75 +
NAA 0.25 mg/l (1.74cm).
4.2.10.2 In vivo nodal segment explant
The average length of shoots regenerated from nodal
segment explants ranged from 1.84 to 4.50 cm on different MS
media compositions while it was 1.40cm in control (Table 14). It
was noted that maximum shoot length was observed on MS
medium + BAP 0.25 + NAA 0.5 mg/l (4.50 cm) which was
significantly at par with MS medium + BAP 0.25 + NAA 0.25 mg/l
(4.42 cm) and MS medium + BAP 0.25 mg/l (4.32 cm). The
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minimum shoot length was recorded on MS medium + BAP 1.0 +
NAA 0.25 mg/l (1.84 cm).
4.2.1.1 Number of days for root initiation
The number of days required for root initiation in
shoot lets when cultured on MS medium supplemented with
different levels of NAA and IBA are presented in Table 15. Number
of days taken for root initiation ranged from 13.80 to 25.60 days
on different MS media compositions and 31.40 days in control.
The shootlets cultured on ½ MS medium + IBA 1.0 mg/l induced
the root at the earliest (13.80 days) which was significantly
superior over other MS media compositions. The maximum days
required for root initiation was noted on MS medium + NAA 1.0
mg/l (26.60 days).
4.2.1.2 Number of days for completion of rooting
The effect of different levels of NAA and IBA in MS
media on number of days required for completion of rooting are
depicted in Table 15. The shoot lets cultured on ½ MS medium +
IBA 1.0 mg/l completed the rooting at the earliest (25.60 days)
which was significantly superior over other MS media
compositions. The maximum time taken for completion of rooting
was recorded on MS medium + NAA 1.0 mg/l (40.40 days).
4.2.1.3 Number of roots per plantlet
Data on number of root formation from shoot lets
cultured on MS media supplemented with NAA and IBA is
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Table 15: Effect of different levels/concentrations of NAA and IBA on root initiation and rooting
Treatments Number of days requiredfor root initiation
Number of days requiredfor completion of rooting
T1 (Control) 31.40±2.18 47.80±0.80
T2 MS + NAA 0.25 mg/l 22.80±0.97 37.60±0.98
T3 MS + NAA 0.50 mg/l 21.40±0.81 36.60±1.07
T4 MS + NAA 1.0 mg/l 25.60±0.81 40.40±.0.92
T5 MS + IBA 0.25 mg/l 20.40±0.51 33.30±1.98
T6 MS + IBA 0.5 mg/l 18.40±0.51 33.20±0.80
T7 MS + IBA 1.0 mg/l 21.40±1.07 36.60±0.67
T8 ½ MS + NAA 0.25 mg/l 20.20±0.66 34.40±0.74
T9 ½ MS + NAA 0.5 mg/l 20.60±0.51 35.00±0.83
T10 ½ MS + NAA 1.0 mg/l 21.00±0.44 35.20±0.37
T11 ½ MS + IBA 0.25 mg/l 18.40±1.20 33.00±1.00
T12 ½ MS + IBA 0.50 mg/l 18.00±0.89 34.80±1.28
T13 ½ MS + IBA 1.0 mg/l 13.80±1.24 25.60±0.51
±SEm 2.88 2.84
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CD at 5%
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presented in Table 16. The maximum number of roots/plantlet
were differentiated on ½ MS medium supplemented with IBA 1.0
mg/l (3.80) which was significantly superior over other MS media
compositions. The minimum number of roots were recorded on
MS medium + NAA 1.0 mg/l (1.40) which was at par with control
(1.20).
4.2.1.4 Root length
The effect of different concentrations of NAA and IBA
in MS media on root length is given in Table 16. The maximum
root length was recorded on ½ MS + IBA 1.0 mg/l (3.88cm) which
was significantly at par with MS medium + IBA 1.0 mg/l (3.48cm).
The minimum root length was observed on MS medium + NAA 1.0
mg/l (1.48cm) which was at par with control (1.15cm). The
average length of roots ranged from 1.48 to 3.88 cm on different
media compositions.
4.2.1.5 Per cent rooting
The effect of different levels of NAA and IBA on per
cent rooting is depicted in Table 17 (Plate 6). It was observed that
all the modified medium induced rooting which ranged from 67.60
to 100 per cent. The maximum per cent rooting was recorded on
½ MS medium + IBA 1.0 mg/l (100%) which was significantly
superior over other MS media compositions. The minimum per
cent rooting was observed on MS medium + NAA 1.0 mg/l
(67.60%).
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Table 16: Effect of different levels/concentrations of NAA and IBA on number of roots/plantlet and rootlength (cm)
Treatments Number of roots/plantlet Root length (cm)
T1 (Control) 1.20±0.20 1.15±0.12
T2 MS + NAA 0.25 mg/l 1.80±0.20 1.90±0.17
T3 MS + NAA 0.50 mg/l 1.60±0.24 1.68±0.18
T4 MS + NAA 1.0 mg/l 1.40±0.24 1.48±0.16
T5 MS + IBA 0.25 mg/l 2.40±0.24 2.56±0.13
T6 MS + IBA 0.5 mg/l 2.60±0.24 2.88±0.17
T7 MS + IBA 1.0 mg/l 3.00±0.31 3.48±0.20
T8 ½ MS + NAA 0.25 mg/l 2.20±0.20 2.62±0.21
T9 ½ MS + NAA 0.5 mg/l 1.80±0.10 2.34±0.17
T10 ½ MS + NAA 1.0 mg/l 1.80±0.37 2.00±0.13
T11 ½ MS + IBA 0.25 mg/l 2.80±0.37 2.96±0.14
T12 ½ MS + IBA 0.50 mg/l 3.00±0.31 3.24±0.17
T13 ½ MS + IBA 1.0 mg/l 3.80±0.20 3.88±0.18
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±SEm
CD at 5% 0.75 0.48
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4.2.1.6 Per cent survival of rooted plantlets
The effect of different levels of NAA and IBA in MS
media on per cent survival of rooted plantlets is depicted in Table
17. Highest per cent survival of rooted plantlets (100%) was
recorded on ½ MS medium + IBA 1.0 mg/l which was at par ½
MS medium + IBA 0.5 mg/l (91.40%). The minimum survival per
cent of rooted plantlets was noted on MS medium + NAA 1.0 mg/l
(70.40%). In control, the per cent survival of rooted plantlets was
very low (48.20%).
4.2.1.7 Hardening
Well rooted micropropagated plantlets having height
more than 3.0 cm were transplanted to plastic bags having
different potting mixtures. These were later transferred under
green house for acclimatization (Plate 7 and 8) and results are
described below:
4.2.1.8 Height of plants (cm)
The effect of different combinations of potting mixtures
on plant height at 15 and 30 days after transplanting is presented
in Table 18. The maximum height of plants at 15 days (6.28cm)
and at 30 days (8.24cm) after potting were recorded in potting
mixture having soil, sand and FYM in 1:1:1 ratio by volume which
was significantly at par with soil rite mix (6.04 and 7.98cm,
respectively). The minimum plant height at 15 and 30 days after
potting (4.54 and 6.36cm) were observed in potting mixture
having soil and vermiculite in 1:2 ratio by volume.
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Table 17: Effect of different levels/concentrations of NAA and IBA on percent rooting and percentsurvival of rooted plantlet
Treatments Percent rooting Percent survival of rootedplantlets
T1 (Control) 34.80±2.57 48.20±8.93
T2 MS + NAA 0.25 mg/l 81.80±3.52 80.20±5.38
T3 MS + NAA 0.50 mg/l 74.40±2.04 77.40±3.21
T4 MS + NAA 1.0 mg/l 67.60±2.13 70.40±3.55
T5 MS + IBA 0.25 mg/l 76.20±2.55 79.60±2.92
T6 MS + IBA 0.5 mg/l 84.40±2.80 85.80±2.20
T7 MS + IBA 1.0 mg/l 87.20±1.15 90.00±1.51
T8 ½ MS + NAA 0.25 mg/l 73.40±1.07 75.80±1.90
T9 ½ MS + NAA 0.5 mg/l 74.40±1.03 78.80±2.81
T10 ½ MS + NAA 1.0 mg/l 78.20±2.59 80.20±2.57
T11 ½ MS + IBA 0.25 mg/l 85.80±1.90 87.60±1.56
T12 ½ MS + IBA 0.50 mg/l 90.40±1.83 91.40±1.83
T13 ½ MS + IBA 1.0 mg/l 100±0.00 100±0.00
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±SEm
CD at 5% 6.05 10.33
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4.2.1.9 Number of leaves per plant
The number of leaves per plant at 15 and 30 days
after potting were counted in different potting mixtures and
depicted in Table 18. The maximum number of leaves/plant at 15
(17.20) and 30 (27.40) days after potting were noted in potting
mixture having soil rite mix which was at par with soil: sand:FYM
1:1:1 v/v (16.80 and 26.40). The minimum number of
leaves/plant at 15 and 30 days after potting (9.00 and 18.40)
were noted in potting mixtures having soil and vermiculite in 1:2
ratio by volume.
4.2.1.10 Per cent survival of plantlets under green house
condition
The effect of different potting mixtures on per cent
survival of plantlets under green house is presented in Table 19.
Highest per cent survival of plantlets (100%) was found under
green house in the potting mixture having soil, sand and FYM in
equal proportion 1:1:1 by volume which was at par with soil rite
mix (95.90%). The minimum per cent survival (52.20%) was
recorded in potting mixture having soil and vermiculite 1:2 ratio
by volume.
4.2.1.11 Per cent survival of plantlets in open condition
After four weeks of hardening under green house,
plantlets were transferred to the open condition in different
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potting mixtures and survival percentage of plantlets are depicted
in Table 19. The maximum survival per cent of plantlets
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Table 18: Effect of different combinations of potting mixtures on plant height and number ofleaves/plant at 15 and 30 days after potting
Treatments Height of plants (cm) at 15 and30 days after potting
Number of leaves at 15 and 30days after potting
15 days 30 days 15 days 30 days
T1 (Soil: sand 1:1 v/v) 5.22±0.29 7.28±0.29 15.40±0.92 24.40±1.43
T2 (Soil: sand: FYM 1:1:1 v/v) 6.28±0.20 8.24±0.33 16.80±0.73 26.40±0.81
T3 (Soil: sand FYM 2:1:1 v/v) 5.36±0.28 7.40±0.27 16.00±0.63 26.40±1.03
T4 (Soil: sand : vermicompost 1:1:1 v/v) 5.50±0.23 7.56±0.32 15.60±0.81 24.80±1.39
T5 (Soil: sand : vermicompost 2:1:1 v/v) 5.12±0.24 6.78±0.36 14.00±0.70 23.40±1.16
T6 (Soil: sand : vermiculite 1:1:1 v/v) 5.20±0.24 7.28±0.24 13.60±0.24 23.80±0.66
T7 (Soil: sand : vermiculite 2:1:1 v/v) 4.92±0.18 6.98±0.21 13.00±0.31 22.80±0.73
T8 (Soil: vermiculite 1:1 v/v) 4.64±0.14 6.72±0.16 12.00±0.31 22.40±0.67
T9 (Soil: vermiculite 1:2 v/v) 4.54±0.14 6.36±0.11 9.00±0.60 18.40±0.81
T10 (Soil rite mix) 6.04±0.25 7.98±0.297 17.20±0.58 27.40±0.60
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±SEm
CD at 5% 0.64 0.78 1.79 2.80Table 19: Effect of different combinations of potting mixtures on percent survival of plantlets under
green house and field condition
Treatments Percent survival ofplantlets under green
house condition
Percent survival ofplantlets under field
condition
T1 (Soil: sand 1:1 v/v) 82.20±2.93 90.20±3.23
T2 (Soil: sand: FYM 1:1:1 v/v) 100±0.00 100±0.00
T3 (Soil: sand FYM 2:1:1 v/v) 83.20±2.74 91.20±2.65
T4 (Soil: sand : vermicompost 1:1:1 v/v) 87.40±3.84 91.40±2.31
T5 (Soil: sand : vermicompost 2:1:1 v/v) 83.60±3.07 85.00±2.95
T6 (Soil: sand : vermiculite 1:1:1 v/v) 77.40±0.81 85.00±4.74
T7 (Soil: sand : vermiculite 2:1:1 v/v) 73.80±1.85 80.40±3.18
T8 (Soil: vermiculite 1:1 v/v) 62.40±1.12 71.60±2.24
T9 (Soil: vermiculite 1:2 v/v) 52.20±2.61 70.40±0.40
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T10 (Soil rite mix) 95.40±1.28 95.20±1.42
±SEm
CD at 5% 6.68 7.66
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(100%) was found in potting mixtures having soil: sand: FYM
(1:1:1 v/v) which was at par with soil rite mix (95.20%). The
minimum survival per cent of plantlets in open condition was
observed in potting mixture having soil and vermiculite 1:2 ratio
by volume (70.40%).
4.3 Agrobacterium-mediated transformation in acid
lime (Citrus aurantifolia Swingle) var. Kagzi Lime
Effect of different co-cultivation and selection
conditions on Agrobacterium mediated transformation frequencies
in the acid lime internodal segments is presented in (Table 20).
Explants inoculated with A. tumefaciens EHA 105 for 15-30
minutes were transferred to MSB1 medium or to tomato feeder
plates, with or without geneticin at 10mg/l (G10) or kanamycin at
100 mg/l (Km100). Transformation frequency (%) was defined as
the number of explants with transformation events (blue spots per
total number of Agrobacterium-inoculated explants).
4.3.1 Sensitivity of acid lime explants to different amino
glycoside antibiotics
Before transformation experiments, the effect of
various concentrations of kanamycin (0, 100, 200, 300, 500 mg/l)
and geneticin (0, 10, 20, 50 mg/l) were evaluated on acid lime
internodal stem segments culture on MSB1 medium (90 explants
per treatment), to determine the appropriate dose of the selection
agent on shoot regeneration. The kanamycin at 100 mg/l or
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higher concentration did not permit development and gradually
reduced callus formation to zero with 500 mg/l kanamycin.
Geneticin was more effective, since on a concentration of 10mg/l,
only one shoot regenerated which soon bleached and died. Higher
concentrations of geneticin even killed most of the acid lime
explants. Kanamycin at 100 mg/l and geneticin at 10mg/l were
choosen as selective agent in the experiments.
4.3.2 Establishment of optimal co-cultivation and
selection conditions for transformation
The co-cultivation of the acid lime explants was carried
out on tomato feeder plates and on MSB1 medium in order to
choose the most efficient conditions for transformation was
carried out. Tomato cell suspensions were used in the feeder
plates as transformation enhancers. The use of tomato feeder
plates for co-cultivation of acid lime explants with Agrobacterium
resulted not only in a higher transformation frequency but also in
a higher frequency of transformation events per explant (Table
20). The average number of blue spots per explant were higher on
tomato feeder plate with geneticin (9.5) whereas it was least on
MSB 1 with or without antibiotics. The transformation frequency
ranged from 23.4 to 83.3%.
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Table 20: Effect of different cocultivation and selection conditions on Agrobacterium mediatedtransformation frequencies in the acid lime internodal stem segments
Treatments Total number ofAgrobacterium
inoculated explants
Transformationfrequency (%)
Maximum/minimum
number of bluespots perexplant
Averagenumber ofblue spots
per explant
Feeder plate/no antibiotics 76 68.4 23/0 6.1±1.15y
Feeder plate/G10 66 83.3 38/0 9.5±4.18y
Feeder plate /Km100 78 82.0 26/0 6.1±0.95y
MSBI/no antibiotics 81 23.4 10/0 2.1±0.15z
MSBI /G10 71 38.0 18/0 2.3±0.85z
MSBI /Km100 83 42.2 13/0 1.7±1.09z
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Different letter (y, z) indicate significant differences at the P = 0.05 level.±SEm
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CHAPTER-5
Discussion
Among the various fruit crops, citrus is an important
one in India. The genus Citrus includes more than 150 species.
Acid lime (C. aurantifolia Swingle) is an important species sharing
23 per cent area and production in the country. Commercially
acid lime is propagated by seeds. However, the progress through
this method has been slow due to long juvenility and frequent
occurrence of nucellar embryos. Recent advances in plant tissue
culture offer great potential for faster multiplication and
developing superior germplams of citrus (Chaturvedi and Mitra,
1974). In tissue culture, million fold increase in the rate of
multiplication over conventional method has been achieved very
recently. A few scientists have tried to obtain differentiation of
shoot buds from various explants in vitro culture of different
species (Grimbat, 1972; Grosser et al., 1993; Baruah et al., 1996,
Thirumalai and Thamburaj, 1996; Desai et al., 1996).
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However, for quicker propagation using tissue culture
technique, it is essential to develop reliable protocol for high
frequency of plant regenerated from cell organ culture. It also
hastens the timely increase and availability of planting material of
newly evolved variety as well as quicker spread. Though a large
number of viruses are not transmitted through seeds (Bitters and
Murashige, 1967), but the plants raised by seeds often show
genetic variability. So the only way to get true-to-type and
pathogen free plants on a large scale is to use the in vitro
techniques for multiplication.
5.1 In vitro production of acid lime seedlings
Three different sterilization durations (5, 10 and 15
minutes) with (0.1%) HgCl2 were used on per cent contamination.
The minimum contamination was recorded with 10 minutes
(12.00%), while with 5 and 15 minutes it was 31.00% and 40.00%
respectively. HgCl2 is the most effective surface sterilizing agent
which act at very low concentration and kill the germ completely.
Similar results have been reported by Ashari et al. (1988) gave
three treatments of sterilization duration with (0.1%) HgCl2 (5, 10
and 15 minutes) observed that there was minimum (12.5%)
contamination with 10 minutes sterilization. Aseptic production of
acid lime seedlings was achieved on basal MS medium containing
GA3 along with BAP at different concentrations. The results
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depicted in Table 7 and 8, revealed significant role of gibbrellin in
germination process supplemented with different concentrations
of BAP (0.5, 1.0 mg/l). Which ultimately lead to faster growth of
seedlings. However, increased concentration of GA3 with BAP
showed no beneficial result. Inoculated seeds showed the earliest
germination (15.40 days) and germination (72.16 per cent) on MS
medium supplemented 0.5 mg/l GA3. However, maximum
seedlings length was observed on MS medium + BAP 0.5 + GA3
0.5 mg/l (5.40cm). The germination percentage decreased due to
increasing the concentrations of GA3 and BAP. The role of
gibberllin in seed germination is well documented. Nourishment
for the embryo during the first few days of germination comes
from endosperms by degradation and solubilization of the starch
and proteins presented in this region. The embryo is a rich source
of gibberellin which diffuses to endosperms and initiates the
solubilization process. The maximum seedling length was
recorded on MS medium supplemented with BAP and GA, which
might be due to the synergistic effect of these two growth
hormones. Gibberllings are well known for inter nodal cell
elongation while cytokinins enhance cell division, thereby leading
to increase in seedling length. Similarly, Singh et al. (1994)
observed wide variation among the six citrus species with regard
to germination. In vitro condition helped in early germination of
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rough lemon (14.2 days) followed by sour orange (15.1 days) and
Rangpur lime (15.3 days). Germination frequency was maximum
for rough lemon (98.2%) followed by sour orange (94.6%),
Rangpur lime and rough lemon (85.7%).
5.2 Micropropagation
Recently, tissue culture approach is being employed in
few horticultural crops for micropropagation. In 1962, Murashige
and Skoog developed the MS media, which is the most popular
medium used in tissue culture of various plant species. Several
scientists developed other media formulations specifically for a
particular crop or for a particular purpose. The shoot tip and
nodal segment culture are the important methods of
micropropagation for faster multiplication and production of
disease free planting material. Hence, for micropropagation
studies these explants were inoculated on different media
compositions.
5.2.1Shoot differentiation
Direct regeneration of shoot, roots and whole plant
without intervention of callus was observed from both in vitro and
in vivo shoot tip and nodal segment on basal MS media containing
BAP along with NAA at different concentrations.
In shoot proliferation process, supplementation with
lower concentration of BAP (0.25 mg/l) resulted in higher number
of shoots. However, increasing concentration of BAP with NAA
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above 0.25 mg/l showed no beneficial results. Maximum four
shoots per explant were obtained on the MS medium + BAP 0.25
mg/l. The difference in number of shoots among both the
explants might be attributed to difference in the physiological
data as well as the number of cells undergoing differentiation.
Similar results have been reported by Mas et al. (1994) that the
best multiplication rate of 5:1 achieved by culturing on MS
medium + BA 1.0 + Kinetin 0.5 + NAA 0.5 mg/l. The number of
days required for shoot initiation ranged from 8.40 to 24.80 days
on different MS media supplemented with BAP and NAA. It might
be due to stimulated bud growth with cytokinin (BAP) resulting in
early shoot initiation. Similarly, Shah et al. (1999) reported that
earliest shoot initiation took place on MS medium + BAP 0.25
mg/l. The benefit of using a combination of cytokinin and auxin is
that it stimulate shoot proliferation and organogenesis in tissue
culture.
5.2.2Length of multiple shoots
Shoot length was considered as growth parameter of
multiple shoots regenerated from different explants. Maximum
shoot length (3.82cm) was recorded on medium supplemented
with BAP 0.5 mg/l, whereas shoot regenerated from in vitro nodal
segment observed maximum shoot length (4.58 cm) on MS
medium supplemented with BAP 0.25 + NAA 0.25 mg/l. However,
shoots regenerated from in vivo shoot tip explant recorded
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maximum length (3.12 cm) on MS medium + BAP 0.25 mg/l.
whereas shoot regenerated from in vivo nodal segment observed
maximum length (4.50 cm) on MS medium + BAP 0.25 + NAA 0.5
mg/l. Cytokinins are generally referred as the regulators of cell
division. The supplementation with BAP media resulted in
enhanced rate of cell division, thereby leading to increase in shoot
length in case of in vitro and in vivo shoot tip explant, however,
NAA supplements were required for attaining increase in shoot
length in inter nodal explants, perhaps, due to synergistic effect of
NAA due to its simulative cell elongation. On other hand, Kamble
et al. (2005) reported that maximum shoot length was observed on
MS medium + BAP 0.25 mg/l.
5.2.3Root differentiation
Regenerated shoots of 2.5 cm or more height and
minimum four leaves obtained earlier were considered as criteria
for evaluating the rooting response and the shoots were
transferred for rooting. Root differentiation was noticed on all MS
basal media supplemented with NAA or IBA. The medium ½ MS
medium + IBA 1.0 mg/l induced 100 per cent rooting followed by
½ MS medium + IBA 0.5 mg/l (90.40%).
IBA is most commonly used auxin for in vitro rooting.
The effectiveness of IBA in promoting in vitro rooting has been
earlier reported also. IBA might have increased the
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biosynthesis of IAA or could act as synergist to IAA. The another
possible reason might be the involvement of IBA in ethylene
biosynthesis (Arteca, 1990). It has been suggested that auxin
induced ethylene may induce adventitious root formation instead
of auxin itself (Hudge, 1989).
5.2.4Hardening
The survival percentage of micropropgated plantlets
under green house condition varied from 52.20 to 100 per cent in
different potting mixtures. The maximum survival percentage
(100%) was recorded in potting mixture having soil, sand and
FYM in an equal proportion (1:1:1 v/v) followed by soil rite mix
(95.40%). It might be due to the availability of optimum moisture,
aeration and nutrients to the plantlets for sufficient length of acid
lime plants. The survival percentage of plantlets after hardening
period of four weeks in open condition ranged from 70.40 to 100%
in same potting mixtures. Similar results were reported by Desai
et al. (1996) in acid lime and Omura and Hidaka (1992) in
Satsuma mandarin.
5.3 Agrobacterium-mediated transformation in acid
lime (Citrus aurantifolia Swingle) var. Kagzi Lime
The effects of various concentration of kanamycin (0,
100, 200, 300, 500 mg/l) and geneticin (0, 10, 20, 50 mg/l) were
evaluated on acid lime internodal segments cultured on MSB1
medium to determine the appropriate dose of the selection agent
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on shoot regeneration. Kanamycin at 100 mg/l and geneticin at
10mg/l proved to be the best selection agents. In previous citrus
transformation reports, a high number of escapes were obtained
from Agrobacterium inoculated explants when kanamycin at 100
mg/l was used as selective agent. Moore et al. (1992), Kaneyoshi
et al. (1994), Pena et al. (1995 a,b) suggested that kanamycin was
not a reliable selective agent for transformation. On the contrary,
it has been reported that a concentration of 100 mg/l kanamycin
prevented shoot development, therefore, regeneration of escapes
may not be attributable only to the use of kanamycin as a
selective antibiotics in transformation.
The inclusion of suspension cell feeder layers during
cocultivation, separated from the explants by a layer of filter
paper, has been reported to improve transformation frequencies
in some species (Horsch et al. 1985; McCormick et al. 1986;
Fillatti et al. 1987a, b; Mullins et al. 1990; Newell et al. 1991;
Janssen and Gardner 1993) by supplying A. tumefaciens
vir-inducing compound such as a acetosyringone and other
phenolic compounds, by reducing the effects of bacterial- induced
stress and by providing a moisture environment beneficial to the
explants. The use of tomato feeder plates for co-cultivation of acid
lime explants with Agrobacterium resulted higher frequency of
transformation events (occurrence of blue spots showing GUS
(positive) as compared to MSB1 medium (Table 20). Feeder plates
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seemed to be stimulate not only Agrobacterium tumefaciens
virulence but also wound response in the acid lime explants.
Potrykus (1995) suggested that feeder plate offered cells where
the explant had cut a better treatment to sift them to a competent
state for transformation, involving dedifferentiation, induction of
cell division and consequently callus proliferation.
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CHAPTER-6
Summary and Conclusion
The present study entitled “In vitro studies in acid lime
(Citrus aurantifolia Swingle) var. KagziLime” was conducted during
2004 to 2006. The objectives of this study were to standardize the
seed sterilization duration, in vitro germination media and the
protocol for in vitro multiplication of acid lime.
The experimental material comprised of seeds and
different explants (in vitro shoot tip and nodal segment, in vivo
shoot tip and nodal segment) with different combinations of
growth regulators (GA3, BAP, NAA, IBA) supplementing the MS
basal medium.
For axenic production of seedling through in vitro seed
germination, the best medium was MS medium + GA3 0.5 mg/l
whereas for seedling growth, best medium was MS medium + GA3
0.5+BAP 0.5 mg/l.
Direct regeneration of shoots, roots and whole plant
without intervention of callus from both in vitro and in vivo shoot
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tip and nodal segment on MS basal medium containing BAP along
with NAA at different concentrations was observed. The MS
medium + BAP 0.25 mg/l was observed to be the best treatment
for shoot proliferation in both in vitro and in vivo explants. The
absolute value of cytokinin and auxin was found to be important
rather than their ratio. The MS medium + BAP 0.25 mg/l was
found to be the most effective one for morphogenic growth
parameters like number of shoots and shoot length. The number
of shoots emerging ranged from 1.46 to 3.40 shoots for in vitro
shoot tip explant whereas it was 1.74 to 3.80 shoots for in vitro
nodal segment explant. However, number of shoots per explant
ranged from 1.20 to 2.60 and 1.60 to 3.20 in vivo shoot tip and
nodal segment explant.
The maximum length of shoot was 3.82 cm and 4.58
cm on the MS medium + BAP 0.5 mg/l and MS medium + BAP
0.25 + NAA 0.25 mg/l regenerated from in vitro shoot tip and
nodal segment explants respectively. However, maximum length
of shoots regenerated from in vivo shoot tip and nodal segment
was observed 3.12 cm and 4.50 cm on the MS medium + BAP
0.25 mg/l and MS medium + BAP 0.25 + NAA 0.5 mg/l
respectively. Regenerated shoots of 2.5 cm height with minimum
of four leaves were considered as criteria for estimating the root
differentiation response. These shoots were transferred for
rooting. Root differentiation took place on full and ½ MS medium
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supplemented with NAA or IBA. Medium ½ MS + IBA 1.0 mg/l
was found to be best for different aspects of root differentiation
like number of days required for root initiation (13.80), completion
of rooting (25.60), per cent rooting (100%), number of
roots/plantlet (3.80), root length (3.88 cm) and per cent survival
of rooted plantlets (100%). Well rooted plantlets were transferred
into different potting mixtures for hardening. The maximum
survival percentage of micro propagated plantlets under green
house condition and open condition after hardening period of four
weeks was 100 per cent in the potting mixture having soil, sand
and FYM in an equal proportion (1:1:1 v/v)
Improved transformation frequencies were obtained by
co-cultivation of the explants with Agrobacterium on feeder plates
using kanamycin at 100 mg/l as selective agent. Attempts to use
geneticin as selection antibiotic were not successful. The presence
and expression of transferred genes was verified by
-glucuronidase histochemical expressed as blue spots in
co-cultivated explants.
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ABSTRACT
Title of thesis : In vitro studies in acid lime (Citrusaurantifolia Swingle) cv. Kagzi Lime
Name of the degreeholder
: Raj Kumar Verma
Title of degree : Master of Science
Name and address ofMajor Advisor
: Dr. Sultan SinghAssociate Professor (Horticulture)Chaudhary Charan Singh Haryana Agricultural University, Hisar-125 004
Degree awardingUniversity
: Chaudhary Charan Singh Haryana Agricultural University, Hisar-125 004
Year of award of degree : 2006
Major subject : Horticulture
Total number of pagesin thesis
: 76 +xi
Number of words in theabstract
: Approx. 350
The experiment was conducted during 2004-2006 on invitro studies in acid lime (Citrus aurantifolia Swingle) var. Kagzi Lime inthe laboratory of Centre for Research and Application in Plant TissueCulture CRATPC CCS HAU, Hisar Haryana. The experiment consistedof in vitro raising of seedlings and direct shoot regeneration in acid limevar. Kagzi Lime. The experimental material comprised of seeds anddifferent explants (in vitro shoot tip and nodal segment, in vivo shoot tipand nodal segment) with different combinations of growth regulators(GA3, BAP, NAA, IBA) supplementing the MS basal medium. For axenic
production of seedling through in vitro seed germination, the bestmedium was MS medium + GA3 0.5 mg/l and for seedling growth best
medium was MS medium + GA3 0.5 + BAP 0.5 mg/l. Direct
regeneration of shoots, roots and whole plant without intervention ofcallus from both in vitro and in vivo shoot tip and nodal segment on MSbasal medium containing BAP along with NAA at differentconcentrations was observed. The MS medium + BAP 0.25 mg/l was
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observed to be the best medium for shoot regeneration inducing 90percent in both in vitro shoot tip and nodal segment and 77.77 and78.59 percent in in vivo shoot tip and nodal segment respectively.
The half MS medium + IBA 1.0 mg/l was found to be bestfor different aspects of root differentiation like number of days requiredfor root initiation (13.80) and completion of rooting (25.60), percentrooting (100%), number of roots/plantlet (3.80), root length (3.88cm)and percent survival of rooted plantlets (100%). The maximum survivalpercentage of micro propagated plantlets under green house conditionand open condition after hardening period of four weeks was 100percent in the potting mixture having soil, sand and FYM in an equalproportion (1:1:1 v/v). Improved transformation frequencies wereobtained by co-cultivation the explants with Agrobacterium on feederplants by using kanamycin at 100 mg/l as selective agent. Attempts touse geneticin as selection antibiotic were not successful. The presence
and expression of transferred genes was verified by -glucuronidasehistochemical expressed as blue spots in co-cultivated explants.
MAJOR ADVISOR SIGNATURE OF DEGREE HOLDER
HEAD OF THE DEPARTMENT