Master of Science in Horticulture...shoot tip and nodal segment explants 40 10 Effect of different...

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.

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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

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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.

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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

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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

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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

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(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

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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

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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.

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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.

-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







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.



3.6.1Plant 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

-35-

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%

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-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

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-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

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-46-

±SEm

CD at 5% 0.78 0.67 0.68 0.77

-47-

-47-

4.2.2Number of days for completion of regeneration


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


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

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-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

-50-

-50-

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%.


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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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


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.


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


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%).


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


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).


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%).



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.



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.

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