

**STANDARDIZATION OF REGENERATION PROTOCOL IN COTTON (** _Gossypium hirsutum_ **L.)**

_Thesis submitted for the award of degree of_ _  
Master of Science (Agriculture)_ _in_ _Plant_ _Breeding and Genetics_ _to the  
Tamil Nadu Agricultural University, Coimbatore-03_

### By

### R.SATHYA

### I.D.No.06-612-014

### CENTRE FOR PLANT BREEDING AND GENETICS

### AGRICULTURAL COLLEGE AND RESEARCH INSTITUTE

### TAMIL NADU AGRICULTURAL UNIVERSITY

### COIMBATORE-641003.

### 2008

### CERTIFICATE

This is to certify that the thesis entitled " _In vitro_ **studies in cotton (** _Gossypium hirsutum_ **L.)** " submitted in part fulfillment of the requirements for the award of the degree of **Master of Science (Agriculture) in Plant Breeding and Genetics** to the Tamil Nadu Agricultural University, Coimbatore is a record of _bonafide_ research carried out by **Ms.R.Sathya,** I.D.No.06-612-014, under my supervision and guidance and that no part of this thesis has been submitted for the award of any other degree, diploma, fellowship or other similar titles or prizes and that the work has not been published in part or full in any scientific or popular journal or magazine.

Date:

Place: Coimbatore

(Dr.A.Nirmalakumari)

Chairman

APPROVED BY

Chairman :

(Dr.A.Nirmalakumari)

Members :

(Dr.T.S.Raveendran)

(Dr.N.Senthil)

Date:

External Examiner :

ACKNOWLEDGEMENT

With Regardful memories.....

I offer my salutations at the feet of the Lord, who kindly provided the energy and enthusiasm through ramifying the paths of thick and thin of my efforts.

I deem it a great pleasure to express my respectful and heartful thanks to **Dr.A.Nirmalakumari** , Chairperson of the advisory committee for her transcendent suggestions, impeccable guidance, and cordial treatment and over willing help throughout the progress of my P.G.programme.

I humbly express my deep sense of gratitude to advisory committee members; **Dr.T.S.Raveendran** , Director, (CPBG) and **Dr.N.Senthil** , Associate Professor, (DPMB& B) for their valuable suggestions and extended help in executing this investigation.

I place my sincere thanks to **Dr.T.S.Raveendran** , Director, Centre for Plant Breeding and Genetics for his constant encouragement and providing me all facilities throughout my curriculum.

I profoundly thank **Dr.S.Shanmugasundaram** , Professor (Genetics); **Dr.C.Babu**. Associate Professor (Genetics) and **Dr.S.Rajeshwari** , Associate Professor (Genetics) for their constant encouragement and providing me all facilities throughout my curriculum.

Words seem inadequate to express my deep sense of gratitude and indebtness to **Uthirakumar** , **Dhayalan** for their constant encouragement throughout this study.

I wish to express my heartful thanks to all my classmates, Junior and Senior friends for their moral support and kindly help rendered during the tenure of study.

I express thanks to glass house and laboratory staff for their help to complete the experiments.

I owe a great deal my beloved parents, brothers **R.Kolanchinathan** and **R.Sivamurugan** , my relatives, for their blessings, continuous encouragement, overwhelming interest and guidance which they showed on me throughout my study.

Last but not least, I thank Vignesh computers for their timely and neat execution of thesis manuscript.

Heart filled with growing love, I submit everything at the feet of my parents **T.Ramalingam and R.Balamani** for carrying me on their shoulders through different phases of my life till now.

(R. Sathya )

ABSTRACT

Studies on in vitro studies in cotton (Gossypium hirsutum L.)

By

R.SATHYA

Degree : Master of Science (Agriculture) in Plant

Breeding and Genetics

Chairman : Dr.A.Nirmalakumari

Professor (Millets),

Centre for Plan Breeding and Genetics,

Coimbatore-641 003.

2008

The present study was undertaken with a view to develop an efficient regeneration protocol in four genotypes Viz., Coker310, SVPR2, MCU12, and MCU13 of cotton ( _Gossypium hirsutum_ L.).The explants of the four genotypes chosen for this study included hypocotyl, cotyledon, and leaf and shoot tip.

Standardization of sterilization procedure for the preparation of the explants was found to be 0.1 per cent mercuric chloride for 10 minutes duration of exposure. The callus induction frequency of the four genotypes was studied in MS supplemented with different growth hormones accounting 33 media combinations.

The callus induction frequency was found to be highest in hypocotyl explants of Coker 310 (86 per cent) in (MS 4) MS medium supplemented with 2, 4-D  
(0.1mgl-1) + Kinetin (0.5 mgl-1) + maltose (30gl-1). The calli so obtained from the MS 4 medium was induced within 7-9 days after inoculation of the explants and this the MS 4 combination was the best with calli characteristics of yellow and brown coloured, smooth and highly friable callus nature.

The 21 days old calli after induction was transferred to both semi solid medium and liquid medium for proliferation and maintenance. The results indicated that the liquid medium proliferated more significantly than the semi solid medium in all the four explants of the four genotypes. The highest callus proliferation percentage (61.15) was recorded in hypocotyl explants of Coker 310 in liquid medium and it was only 58.27 in semi solid medium. The subculturng of proliferated calli was done once in 10 days in liquid medium and once in 5 days in semi solid medium for callus growth and maintenance.

Callus differentiation into somatic embryos was observed only in Coker 310 and was recorded highest in hypocotyl explants (79.66 per cent). Though callus induction, differentiation, and development of somatic embryos were observed in explants of Coker 310 they failed to regenerate into complete plantlet.

The proliferated calli which failed to differentiate into somatic embryos were transferred to different media combinations for development of shoots and roots from the callus cultures Among the four genotypes used, the shoot induction percentage was the highest (74.33) in explants of SVPR2 in media composition of MS with BAP (2.0 mgl-1) + IAA (0.5 mgl-1) + GA3 (1.0 mgl-1) + glucose (30gl-1) taking 14 days for producing maximum number of 6 shoots of 8.95 cm length.

The root induction percentage was highest (87.66) in SVPR2 in media combination of IBA (1.0mgl-1) and IAA (0.5mgl-1) having 4 to 5 roots of 2.20 - 4.60cm average root length.

The regeneration efficiency of SVPR2 (40.52) in hypocotyl explant derived calli was highest among the four genotypes. The transfer efficiency was recorded to be the highest (35.48 per cent) in plantlets obtained from the hypocotyl explants of SVPR2. Among the four genotypes two plantlets from the genotypes SVPR2 (33.00 per cent) and MCU12 (25 per cent) were established well in the field and they are at square setting stage.

With this protocol, the period from callus initiation to callus differentiation will be about 7 months and field transfer of the plantlets could be accomplished in 9-10 months.

### LIST OF ABBREVIATIONS

°C Degree Celsius

µm Micromolar

2, 4,-D 2, 4,-dichlorophenoxyacetic acid

2-iP 6---dimethyl allylamino purine

BAP Benzylaminopurine

CD Critical difference

CM Callus induction medium

cm Centimetre

CV Coefficient of variation

cv Cultivars

et al Co-workers

g Gram

GA3 Gibberellic acid

IAA Indole-3=acetic acid

IBA Indole butyric acid

K Kinetin

LM Liquid medium

mgl-1 Milligam per cent litre

ml Millilitre

MS Murashige and Skoog

NAA α-Napthaleneacetic acid

RD Root differentiation medium

RH Relative humidity

RM Root differentiation medium

SD Shoot differentiation medium

SE (d) Standard error of deviation

SEM Somatic embryo induction medium

var Variety

viz., Namely

w/v Weight/Volume

### CONTENTS

### CHAPTER. NO. | ### TITLE | ### PAGE. NO

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### 6.

### INTRODUCTION

### REVIEW OF LITERATURE

### MATERIALS AND METHODS

### RESULTS

### DISCUSSION

### SUMMARY

### REFERENCES |

### LIST OF TABLES

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Studies on somatic embryogenesis in cotton

Nutrient media and hormonal compositions for tissue culture, Regeneration of cultivated cottons ( _G.hirsutum_ ) via somatic embryogenesis

Composition of the MS medium

Different media composition used for callus induction and proliferation

Somatic embryo induction media combination

Shoot differentiation media combination

Root differentiation media

Effect of concentrations of surface sterilant and durations of exposure on the contamination per cent of the four different explants on four genotypes.

Effect of concentrations of surface sterilant and durations of exposure on contamination per cent of the four explants in Coker 310 and SVPR2

Effect of concentrations of surface sterilant and durations of exposure on the contamination per cent of the four explants in MCU12 and MCU13.

Effect of concentrations of surface sterilant and durations of exposure on the contamination per cent of the hypocotyl and cotyledon explants from different genotypes.

Effect of concentrations of surface sterilant and durations of exposure on the contamination per cent of the leaf and shoot tip explants from different genotypes.

Effect of concentrations of surface sterilant and durations of exposure

on the contamination per cent for different genotypes.

Effect of concentrations of surface sterilant and durations of exposure on the contamination per cent for different explants

Effect of concentrations of surface sterilant and durations of exposure on survival per cent of the four different explants for four genotypes.

Effect of concentrations of surface sterilant and durations of exposure on survival per cent of the four explants in Coker 310 and SVPR2.

Effect of concentrations of surface sterilant and durations of exposure on survival per cent of the explants in MCU12 and MCU13.

Effect of concentrations of surface sterilant and durations of exposure

on survival per cent of the hypocotyl and cotyledon explants of

four different genotypes.

Effect of concentrations of surface sterilant and durations of exposure on survival per cent of the leaf and shoot tip explants for different genotypes.

Effect of concentrations of surface sterilant and durations of exposure on survival per cent for different genotypes.

Effect of concentrations of surface sterilant and durations of exposure on survival per cent for different explants.

Effect of media composition on callus induction of four explants in

four genotypes.

Effect of media composition on callus induction for different explants in Coker 310 and SVPR2.

Effect of media composition on callus induction for different explants in MCU12 and MCU13.

Effect of media composition on callus induction in hypocotyl and cotyledon explants for different genotypes.

Effect of media composition on callus induction in leaf and shoot tip

explants for different genotypes

Effect of growth hormones on callus induction of different explants in

different genotypes

Effect of carbon sources on callus induction of explants in

different genotypes

Calli characteristics

Effect of growth regulators on Callus proliferation on semi-solid medium for different genotypes

Effect of growth regulators on Callus proliferation on semi-solid medium for different explants.

Effect of growth regulators on Callus proliferation on liquid medium for different genotypes.

Effect of growth regulators on Callus proliferation on liquid medium for different explants.

Semi solid vs liquid medium on best media combination in different explants four genotypes.

Effect of media composition on callus differentiation in Coker 310 genotype

Effect of different genotypes on shoot induction from callus cultures.

Effect of different explants on shoot induction from callus cultures.

Effect of growth regulators on days to shoot induction, number of shoots and length of shoots.

Effect of different genotypes on root induction from callus cultures.

Effect of different explants on root induction from callus cultures.

Plant regeneration efficiency of calli from different explants of four different genotypes.

Transfer efficiency of regenerated plants from different explants of four different genotypes |

### LIST OF PLATES

Plate No. | Title | Page No.

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Seed Germination and Explant preparation

Effect of genotypes on callus induction

Effect of explants on callus induction

Effect of Growth hormones on callus induction

Callus proliferation on semi solid medium

Callus proliferation on liquid medium

Shoot induction through callus cultures

Root induction through callus cultures

Plantlet regeneration

Hardening

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### LIST OF FIGURES

Figure No. | Title | Page No

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Effect of contamination per cent on four explants of different genotypes

Effect of contamination per cent on different explants of four genotypes

Effect of survival per cent on four explants of different genotypes

Effect of survival per cent on different explants of four genotypes

Effect of carbon sources n callus induction

Effect of callus proliferation on semi solid medium for different genotypes

Effect of callus proliferation on semi solid medium for different explants of four genotypes

Effect of callus proliferation on liquid medium for four explants of different genotypes

Effect of callus proliferation on liquid medium for different explants of four genotypes

 |

CHAPTER I

### INTRODUCTION

Malvaceae family member cotton ( _Gossypium .hirsutum_ L.) is one of the most commercially important fiber crops in the world. An important renewable resource, cotton ( _Gossypium_ spp.) is the world's leading fibre and the second largest oilseed crop in production. In addition to textile manufacturing, it produces seeds with a potential multi product base such as hulls, and food for animals (Mishra _et a_ l., 2003; Aragao _et al.,_ 2005). There are 51 species of cotton in the world, of these, 46 are diploid (2n = 2x = 26) and other five are teteraploid (2n = 4x = 52) (Fryxell, 1992).  
_G. arboreum_ L. (2n = 2x =26), _G.herbaceum_ L. (2n= 2x = 26), _G. hirsutum_ L. (2n = 4x = 52) and _G.barbadense_ (2n=4x=52) are the four primary cultivars and others are wild species. Cotton was one among the first species to which Mendelian principles were applied (Balls, 1906). _Gossypium_ , being a vast and variable genus, has received considerable attention from eminent botanists starting with Linnaeus who described about six species as early as in 1753 A.D.

One of the chief constraints threatening cotton cultivation is the large number of pests especially the bollworm complex. All the cultivated varieties are susceptible to the bollworms while a few wild species are reported to be resistant. Hence, there is a considerable interest in the development of tissue culture and gene transfer technology for this species. Now a days, tissue culture techniques would be an alternative tool for cotton breeding. Because through this technique the transgenic plants have been produced (Zapta _et al_., 1999; Wilkins _et al_., 2000 and Satyavathi  
_et al.,_ 2002).

Efficient _in vitro_ techniques for regeneration of large numbers of plantlets from cotton are limited when compared to other major commercial crops. Price and Smith (1979) were the first to report somatic embryogenesis in the cotton _. G.klotzchianum_ , although complete plants could not be regenerated. Davidonis and Hamilton (1983) first described plant regeneration from two year old callus of  
_G. hirsutum_ L. cv Coker 310 through somatic embryogenesis.

Although regeneration efficiency has been improved through somatic embryogenesis or organogenesis, genotype dependent regeneration, a prolonged culture period, high frequency of abnormal embryo development, low conversion rate of somatic embryos into plantlets, lack of shoot elongation, difficulties of browning which cause death of tissues of explants are the problems associated with cotton tissue culture (Kumria _et al_., 2003). Some of these problems are related to the plant material such as explant age or genotype and others to the culture conditions such as hormones, medium composition or other physical culture conditions (Ikram -  
Ul-Haq, 2004).

Regeneration efficiency through somatic embryogenesis has been improved (Wilkins _et al_., 2004). Subsequently, high frequency production and development of somatic embryos into normal plantlets through manipulation of nutrient and environmental conditions was reported (Kumria _et al.,_ 2003).

Somatic embryogenesis and plant regeneration in other cultivated cotton ( _G.arboreum_ L) were also reported. (Rajasekaran _et al.,_ 2004). Regeneration in wild cottons through somatic embryogenesis, however, is very poor. Price and Smith (1979) established suspension cultures of wild cotton _G.klotzschianum_ , in which somatic embryos were formed, but not regenerated. Finer and Smith (1984) also obtained somatic embryos of _G.klotzschianum_. Most embryos either failed to mature or developed abnormal leaves and shoots. Efforts to regenerate commercial varieties of cotton through somatic embryogenesis have not been fruitful.

Most of the successful reports of upland cotton regeneration involved Coker lines and showed high regenerative capacity through somatic embryogenesis (Trolinder and Goodin ,1987;Trolinder and Goodin ,1988b; Firoozabady and Deboer,1993;Kumria _et al_.,2003;Wu _et al.,_ 2004).However ,even in the protocols developed for Coker lines are not efficient and reproducible which is evident from recently published reports on Coker regeneration (Wu _et al_., 2004; Kumar and  
Tuli, 2004).

With regard to Indian cotton varieties, success was reported so far only in two varieties; MCU 5 (Kumar and Pental, 1998) and SVPR 2 (Ganesan and Jayabalan, 2004). However, attempts to regenerate other Indian varieties like MCU 5, MCU 7 and MCU 11 have failed (Ganesan, 2003; Ganesan and Jayabalan, 2004; Gopikrishnan, 2005).

Keeping the above back ground in mind, the present study is aimed to investigate the following objectives;

  1. Standardization of protocol for regeneration of cotton through somatic embryogenesis.

  2. Standardization of hardening techniques to establish the regenerated explants.

  3. To study the influence of genotypes in regeneration.

  4. Evaluating the efficiency of liquid culture in somatic embryogenesis and or organogenesis

CHAPTER II

### REVIEW OF LITERATURE

Cotton, the indisputable natural fibre provides raw material for the textile mills in our country. Nearly one third of foreign exchange is earned by export of cotton, yarn and finished products. Due to the alarming increase of population by 2020 AD India's cotton requirement is expected to exceed 240 lakh bales (Ahuja, 2003).

**2.1. Architecture of** _Gossypium_

Malvaceae family member, cotton ( _G.hirsutum_ L.) is an often cross pollinated crop. There are 51 species of cotton in the world, of these, 46 are diploids (2n = 2x = 26) and other five are tetraploids (2n = 4x = 52) (Fryxell, 1992).

2.1.1. Wild species - Germplasm resource

The wild species represent exotic germplasm resources and a source of genetic diversity. There are many valuable agronomic traits and abundant gene resources in wild cotton species. Mating barriers hinder introgression of agronomically important traits, and application of biotechnology may facilitate transfer of alleles among species (Kairon _et al_., 1999).Some wild cotton _G. raimondii_ , _G.davidsonii_ are resistant to bollworm and other pests. These traits are very useful for genetic improvement of the cultivars.

2.2. Threat to cotton cultivation

The production of cotton is adversely affected by abiotic stresses, viral and fungal infections and insect predation. Therefore, efforts to improve productivity by biotechnology are critical in insect or pest management, enhancement of fibre quality and protein content of cotton seed oil. (Firoozabady _et al_., 1987). A well defined, reproducible and highly efficient plant regeneration is a pre requisite for crop improvement through biotechnology (Rajasekaran _et al_., 2000).

2.3. A biotechnological approach

Plant tissue culture had progressed immensely from its inception in 1930's, when the scientists used this technique to grow cells in culture. Currently, it is used for many different purposes such as callus induction, protoplast culture, ovule culture and somatic embryogenesis (Baja and Gill, 1986; Altman _et al_., 1987; Trolinder and Goodin, 1988a, b).

2.4. Cotton regeneration studies

Genetic transformation of cotton using foreign genes requires the development of an efficient regeneration system for the transformed tissues (Firoozabady _et al_., 1987; Rajasekaran _et al.,_ 2000). Regeneration through somatic embryogenesis is preferred to organ culture because of probable single - cell origin of somatic embryos (Merkle _et al_., 1995), thus reducing the chimeric transformation events.

More than 7000 cotton accessions at the National Cotton Germplasm Collection at College Station, Texas (Sakhanokho, 2001) and nearly 100 cotton cultivars under cultivation in the USA (Gould _et al_., 1991; Hemphill _et al._ , 1998) were reported to be recalcitrant to regeneration through embryogenesis.

The first ever published report of regeneration in _G. hirsutum_ was made by Davidonis and Hamilton (1983). Nevertheless, the results were not reproducible. Subsequent attempts to regenerate other commercial cotton varieties through somatic embryogenesis were also not successful. Majority of the successful reports of upland cotton regeneration involves the agronomically poor Coker lines (Firoozabady and DeBoer, 1993; Trolinder and Goodin, 1989), which formed the base for current generation of commercial transgenic cotton. Hence, a well defined, reproducible and highly efficient plant regeneration scheme is a pre requisite for transformation in cotton.

2.5. Direct induction of cotton somatic embryogenesis

Compared to indirect somatic embryogenesis, direct somatic embryogenesis appears to be associated with greater genetic and cytological uniformity and it takes less time to induce direct somatic embryogenesis than indirect somatic embryogenesis (Lurie, 1968). Explants _viz_., hypocotyls, cotyledons and roots could produce embryogenic callus and somatic embryos but the frequency of direct embryogenesis was different among explants. Embryogenic callus and the somatic embryos were obtained on all induction medium without 2, 4- dichloro phenoxyacetic acid and highest induction was 11.42 per cent .the frequency of embryogenic callus was 44.4 per cent of induced callus. Compared with cotyledon and root, direct somatic embryogenesis was difficult to appear from hypocotyls (Zhang, 1997).

2.6. Somatic embryogenesis

Somatic embryogenesis is the development of a bipolar structure with both root and shoot primordia from any sporophytic part of the plant. It occurred through the same key stages of embryo development as in zygotic embryogenesis where globular, heart and torpedo stages are another way of exhibiting the totipotency by plant cells where they first dedifferentiate and then redetermine towards embryogenic pathway (Sharma and Millam, 2004).

Somatic embryogenesis was first observed in _Oenanthe aquatica_ (Waris, 1957) and _Daucas carota_ (Reinert, 1958). Since then, it has been studied and described in many important species (Bajaj, 1995).

Several genotypes of cotton have been regenerated through somatic embryogenesis. The first report on cotton somatic embryogenesis was published by Price and Smith (1979) in _G.klotzschianum_ , although they were unable to obtain plantlets Davidonis and Hamilton (1983) reported somatic embryogenesis in two year old callus cultures of _G. hirsutum_ var. Coker 310. Although the plants were regenerated the embryos appeared abnormal.

Agronomically superior varieties had genotype limitation which was overcome in Acalas by Rangan and Rajasekaran (1996), Chinese cultivar CR112 (Zhang _et al_., 1999), simian-3 (Zhang _et al.,_ 2001) and Indian variety MCU5  
(Kumar _et al.,_ 1998). Regeneration in these genotypes was accomplished by cotyledon, hypocotyl, root, immature embryos and young leaves. The studies on somatic embryogenesis using various explants are listed in table 1.

Sakhanokho et al. (2001) observed regeneration in G. hirsutum. cv. deltapine 90 and G. barbadense. Mishra et al. (2003) reported regeneration of Acala cultivars of cotton in 8 months through suspension cultures.

Studies on somatic embryogenesis were carried out in a regeneration friendly _G.hirsutum_ cv.Coker 310 and Coker 312.callus induction and proliferation from hypocotyls and cotyledonary leaves explants were obtained at a high frequency when cultured on MS medium supplemented with 0.1mg/l 2,4,D and 0.5 mg/l kinetin. When calli derived from hypocotyls and cotyledonary leaves were transferred onto MS medium supplemented with 1.9 g/l KNO3, somatic embryos were induced. Though frequency of somatic embryos was high, regeneration frequency was found to be comparatively low. (Rashmi, 2004).

Coker genotypes recorded asynchronous nature of somatic embryogenesis. The medium containing 2,4-D (0.1 mg./l) and kinetin (0.5mg/l) recorded maximum callus induction frequency. The callus when subcultured on medium containing twice the amount of KNO3 (1.9 g/l) produced somatic embryos. Cotyledon was found to exhibit higher frequency of somatic embryogenesis. The Coker genotypes could be regenerated in 9-10 months. _Agrobacterium_ -mediated transformation of cotyledon and embryogenic calli resulted in the generation of kanamycin resistant calli at an average frequency of 35.2 per cent and 51.5 per cent respectively and somatic embryos at 12.2 per cent and 29.9 per cent respectively. Stable _gus_ expression was observed in a randomly selected transformed calli and somatic embryos growing on selection medium. Molecular analysis showed positive amplification for _gus_ _A_ gene in 3 out of 4 putative transformed callus lines whereas none of the regenerants shows amplification for _gus A_ gene (Thiruvengadam, 2006)

_Agrobacterium_ -mediated transformation and regeneration _via_ somatic embryogenesis remains the method of choice for regenerating of transgenic cotton plants. Coker 310 recorded highest frequency of embryogenic calli induction  
(92.7 %), somatic embryo induction (76.7%) and somatic embryo maturation (43.00%) (Ahmad Al Jouma Al Mohamed Al Mousa, 2007).

. The importance of liquid medium over the solid medium for somatic embryogenesis in _G.hirsutum_ was published by Gawel and Robacker (1990) after the work of Finer (1988) on regeneration of cotton explants from somatic embryogenic suspension cultures. Though many published regeneration protocols are available, cotton varieties often get exacerbated by phenolic exudates, lengthy time required to obtain embryogenic cell and poor germination of somatic embryos. Recent research aimed at developing protocols that insisted slow dessication (Chaudhary _et al.,_ 2003), use of activated charcoal (Zhang _et al.,_ 2000) to obtain embryogenesis and maturation in relatively short period. Considerable work was reported especially in cotton, yet there is no standardized protocol established for cotton regeneration that was applicable for all laboratory conditions (Sunil Kumar and Rathore, 2001).

2.7. Factors influencing somatic embryogenesis

2.7.1. Genotype

Trolinder and Xhixian (1989) were the first to report the genotype specificity of somatic embryogenetic response in cotton. Most of the reports indicated that Coker cultivars were the most responsive, for somatic embryogenesis than any other cultivars studied (Trolinder and Goodin, 1988b; Kumar _et al.,_ 1998; Wilkins _et al.,_ 2000). The embryogenic potential is reportedly a genetic trait of low heritability that varies significantly from cultivar to cultivar and seed lot to seed lot.(Aydin _et al_., 2004; Ganesan and Jayabalan, 2004; Sakhanokho _et al._ , 2004; Wilkins _et al_., 2004; Wu _et_ _al.,_ 2004).

2.7.2. Explant type

Explant type influences the frequency of somatic embryogenesis (Zhang _et al_., 2000). The seedling explants such as hypocotyl and cotyledon were used by majority of workers (Rajasekaran 1996; Zhang _et al_., 2000; Zhang _et al._ , 2001; Suresh Kumar _et al_., 2003; Kumar and Tull, 2004; Sakhanokho _et al.,_ 2005). Zhang _et al._ (2001) reported that root was the most responsive explant for production of somatic embryos, the hypocotyl was the next and the cotyledon was the least. Sakhanokho _et al._ (2004) observed that the somatic embryos were more consistently induced from hypocotyl callus than cotyledon explants in Pee Dee and Georgia cotton lines.

2.7.3. Explant age

There are many reports on explant age regeneration response of cotton ( _G.hirsutum L._ ). in a study,3 and 5 days old meristematic shoots of _G.hirsutum_ and _G.barbadense_ were cultured and the best regeneration response was obtained from 5 days old plants (Gould _et al._ ,1991).In another study,5,6,7,8,9and 10 days old cotyledonary nodes of 10 different genotypes were cultured and 6 days old plants gave the best regeneration response.

2.7.4. Callus induction medium

For majority of genotypes including cultivated varieties, the callus induction medium that produced embryogenesis was based on MS containing an auxin (NAA or 2,4-D) and a cytokinin (Kinetin or 2-iP) (Trolinder and Goodin 1987, 1988 a, b; Gawel and Robacker 1990; Nobre _et al_., 2001; Kumria _et al.,_ 2003). Mishra _et al_. (2003) used callus induction medium containing two auxins 2, 4-D and NAA to generate good quality callus in Acala cotton cultivars. Ganesan and Jayabalan (2004) reported that medium containing picloram (0.3 mg/l) and kinetin (0.1 mg/l) induced somatic embryogenesis in Indian variety SVPR2 (Table 2).

2.7.5. Callus characteristics

Callus characteristics _viz.,_ colour, texture, friability and size play a major role in successful regeneration _viz_., somatic embryogenesis (Mishra _et al_., 2003). The growth regulator composition of the culture medium particularly auxin and cytokinin, greatly influence callus morphology (Nobre _et al_., 2001).Finer (1988) reported that NAA and kinetin produced calli which are highly variable in colour and texture. He observed typical colours of callus such as green, yellow, white, brown and red.
2.7.6. Plant growth regulators

Price and Smith (1979) obtained embryoids in suspension cultures supplemented with 2, 4-D (0.1 mg/l). The other hormone combinations reported for somatic embryogenesis were Zeatin at 0.1 mg/l in CCR112 (Zhang _et al_.,1999), a combination of NAA (0.5 mg/l) with kinetin (0.5 mg/l) in _G. barbadense_ accessions (Sakhanokho _et al_., 2001), and a combination of picloram (0.2 mg/l) and 2-iP (0.2 mg/l) in Indian cultivar SVPR 2 (Ganesan and Jayabalan, 2004) have also been used for the induction and proliferation of embryos.

2.7.7. Nitrogen and Carbon sources

Nitrogen and carbon are important factors in _in vitro_ somatic embryogenesis. Price and Smith (1979) observed that somatic embryos of _G. klotzschianum_ differentiated in suspension cultures containing 10-15 mM glutamine. The role of glutamine, NH4NO3 and KNO3 for somatic embryogenesis of cotton had been well documented by number of workers,(Sakhanokho _et al_., 2001; Sunilkumar and Rathore, 2001; Kumria _et al._ , 2003; Wu _et al.,_ 2004; Tohidfar _et al_., 2005).

Regeneration of cotton through somatic embryogenesis was reported on medium containing glucose (Firoozabady _et al_., 1987; Rajasekaran, 1996; Kumria  
_et al.,_ 2003); Maltose (Hussain _et al.,_ 2004) and sucrose (Zhan _et al.,_ 2000). Sakhanokho _et al_. (2004) reported that embryo initiation was higher on medium with sucrose in diploid cotton species _G. arboreum_.

Highly reproducible and simple protocol for cotton somatic embryogenesis was described using different concentrations of maltose, sucrose, glucose and fructose. Maltose 30g/l is the best carbon source for embryogenic callus induction and glucose (30g/l) was suitable for induction, maturation of embryoids and plantlet regeneration. Creamy white embryogenic calli of hypocotyls explants were formed on medium containing MS salts, myo-inositol (100mg/l), thiamine HCl (0.3mg/l), Kinetin (0.1mg/l), and maltose (30g/l).

. During embryo induction and maturation accelerated growth was observed in liquid medium containing NH4NO3 (1g/l), Picloram (2mg/l), 2-iP (0.2mg/), Kinetin (0.1mg/)l, glucose (30mg/l).Before embryoid induction ,large clumps of embryogenic tissues were formed. These tissues only produce embryoids. Completely matured somatic embryos were germinated successfully on the medium fortified with MS salts, myo-inositol (50mg/l), thiamine HCl (0.2mg/l), GA3 (0.2mg/l), BA (1.0mg/l), and Glucose (30g/l). Compared with earlier reports, 65% of somatic embryo germination was observed. The abnormal embryo initiation was highly reduced using Glucose (30g/l) compared to other carbon sources. The generated plantlets were fertile but smaller in height than the seed derived control plants (Firoozabady _et al_., 1987; Rajasekaran, 1996; kumria _et al_., 2003).

2.7.8. Effect of haemoglobin on embryogenic callus induction

The percentage of embryogenic callus formation, the efficiency of somatic embryogenesis, the number of somatic embryos formed and the percentage of plantlet regeneration from somatic embryos were increased with addition of haemoglobin (erythrogen) to the medium. The percentage response for callus induction and proliferation increased to 97 per cent in erythrogen supplemented medium compared to the control (84 per cent). The fresh weight of the callus also increased from 260 mg in the control plants to 342 mg in the haemoglobin (400mg/l) treated cultures. In normal somatic embryogenesis only part of the callus was embryogenic and the non embryogenic calli was progressively removed, otherwise it converted the embryogenic calli into non embryogenic calli. However in haemoglobin treated cultures, 97 per cent embryogenic callus formation was observed, whereas the non embryogenic calli was significantly reduced. The addition of erythrogen to any media induces oxygen uptake by cells and accelerates cell division (Carman 1987).

Only SVPR2 lines were used for somatic embryogenesis studies with haemoglobin supplemented medium because the MCU11 line showed no  
response with protocols of Sakhanokho _et al._ (2000), Zhang et al (2001) and  
Kumria et al. (2003). Mishra et al. (2003) showed a genotype dependent response for cotton somatic embryogenesis in Maxxa cultivars; whereas the response of MCU11 showed that somatic embryogenesis in cotton is genotype dependent.

Only creamy white embryogenic calli produced embryoids. Well developed embryoids were induced on medium containing MS salts, sucrose (30g/l),  
myo-inositol (100mg /l ), PIC (0.2mg/l), 2-iP (0.2mg/l) and NH4NO3 (1g/l). The auxin 2, 4-D has been widely used for the induction of embryogenic calli and embryoids (Zhang and Li, 1992; Mckersie and Brown, 1996; Gonzalez Benito _et al_., 1997; Guis _et al_., 1998; Kumar and Pental, 1998; Choi _et al_ 1999; Zhang 2000).In contradiction MS basal medium was alone used without any growth regulators for embryo induction (Mishra _et al_., 2003)

The regeneration of plantlets from somatic embryos is still a problem in many plant species including cotton. Till to date, to our knowledge upto 65 per cent from somatic embryos has been observed in cotton (Sakhanokho _et al_ , 2000; Wilkins _et al.,_ 2003; Mishra _et al._ , 2003; Kumria _et al_., 2003)

Currently transgenes are delivered to cultured plant tissues of cotton by two methodologies: article bombardment (McCabe and Martinell 1993) and co cultivation with _Agrobacterium tumifaciens_ (Firoozabady _et al_ 1987; Umbeck _et al_ 1987). Both methodologies produced transgenic plants with different degrees of efficiency. The former method produces a means to introduce foreign genes into any elite cotton variety; however the transformation efficiency was reported as 1 transgenic plant per 1000 bombarded explants (McCabe and Martinell 1993). The latter method requires regeneration through somatic embryogenesis which has been successfully applied to only a few regenerable cotton cultivars (Coker lines). Nearly 100 cotton cultivars under cultivation in USA and they are in general not amenable to tissue culture techniques as Coker lines (Trolinder andXhixian1989; Firoozabady and DeBoer 1993;Koonce _et al_ 1996) Moreover plants regenerated from an embryogenic callus phase are sometimes sterile and show signs of somaclonal variation which affected both the phenotype and genotype of the plant (Stelly _et al_ ,1989; Firoozabady and DeBoer 1993).Currently cotton regeneration through somatic embryogenesis remains germplasm dependent (Koonce _et al_., 1996).
2.8. Embryogenic suspension cultures

The establishment of suspension cultures for inducing somatic embryogenesis in cotton has been reported by number of workers. Price and Smith (1979) were the first to observe cultures of _G. klotzchianum._ Trolinder and Goodin (1987) reported that globular and heart shaped embryos were formed in liquid suspension culture from calli initiated from Coker 312. Gawel and Robacker (1990) made a comparison of semi solid and liquid embryo proliferation medium and concluded that liquid based proliferation medium produced greater rate of somatic embryos than semi solid medium. Mishra _et al. (_ 2003) achieved genotype independent regeneration system in Acala cultivars such as Maxxa, Riata using liquid cultures. Carbon source dependent somatic embryogenesis and plant regeneration through suspension culture was reported in Indian cultivar SVPR2 (Ganesan and Jayabalan, 2004).

2.9. Characterization of cotton somatic embryos

Voo _et al_. (1991) identified six different types of mature somatic embryos in Coker312. They were green tulip-shaped embryos, trumpet shaped embryos, fused embryos, small jar shaped embryos, and umbrella shaped embryos and irregular embryos of various sizes.

2.10. Plant regeneration

Davidonis and Hamilton (1983) first obtained regenerated plants from two year old callus of Coker 310. Cousins _et al._ (1991) reported that the time taken from explant isolation to potting plant in the greenhouse varied from 9 to 12 months for Siokra 1-3 genotype. Kumar and Pental (1998) obtained regenerated plants through somatic embryogenesis from Indian variety MCU 5 in 150 to 180 days. Zhang _et al_. (2000) obtained plant regeneration in 140-160 days. Mishra _et al_. (2003) reported regeneration of Acala cultivars of cotton in 8 months through suspension cultures.
2.11. Somatic embryogenesis on semi-solid versus liquid medium

A comparison of semi-solid versus liquid embryo proliferation media using was made using two _G.hirsutum_ genotypes (Coker 312 and T25) and two callus initiation media. Sections of petioles from matured flowering plants were cultured on two modified Murashige and Skoog medium. Medium 1 included NAA (4.0mg/l) and kinetin (1mg/l); medium II contained 2, 4-D (0.1mg/l) and kinetin (0.1mg/l).After six weeks, callus was removed from each explant and divided in half. One callus portion was placed in liquid proliferation medium and other on semi solid medium proliferation medium. Composition of proliferation medium was identical to that of initiation medium except no growth regulators were added.

The percentage of explants forming callus was influenced by genotype initiation medium combination. Analysis of variance procedures revealed significant variability for callus initiation media, proliferation media (semi-solid or liquid and an initiation medium × genotype interaction. Paired t-tests indicated that more embryos were produced in liquid proliferation medium (227.3embryos/culture) than in semi-solid proliferation medium (134.6 embryos/culture) (Gawel and Robacker, 1990).

2.12. Factors affecting somatic embryogenesis

. Genetic differences in tissue culture competence are still one of the limiting factors for the application of biotechnological methods for the study of cotton (Kumria _et al.,_ 2003; Sanjaya _et al.,_ 2005) Some of these problems are related to the plant material such as explant age or genotype and others to the culture conditions such as hormones, medium composition, or other physical culture conditions  
(Ikram - il -Haq, 2004).

Phenolic acids are intermediates of phenyl propanoid metabolism  
(Cvikrova _et al_., 1996) and precursors of lignin (Lewis and Yamamato, 1990) and phenylpropanoid phyto alexanins (Kessmann _et al_., 1990).Phenolics may act as modulators of plant development by regulating Indole acetic acid (IAA) catabolism (Arnaldos _et al_., 2001). They are very effective in plant growth regulation; cell differentiation and organogenesis (Mato _et al_., 1988).There are two opinions on interactions between phenolics and plant growth and development. One indicates that phenolics are negatively related with plant _in vitro_ proliferation while others mention the opposite (Lorenzo _et al.,_ 2001).

In tissue culture studies, phenolic substances, especially oxidized phenolics generally effect _in vitro_ proliferation negatively (Arnaldos _et al.,_ 2001). When cells are damaged, the contents of cytoplasm and vacuoles are mixed and phenolic compounds can readily become oxidised by air. Oxidized Phenolic compounds may inhibit enzyme activity and results in darkening of the culture medium and subsequent lethal browning of explants (Laukkanen _et al.,_ 1999; Compton _et al.,_ 1986).

Liquid media can be used to reduce phenolic exudation. In addition frequent subculturng, some antioxidants such as citric acid and ascorbic acid, PVP (polyvinyl pyrolidone) and activated carbon which are added into medium, can also reduce phenolic oxidation and contribute to regeneration from explants (Toth _et al.,_ 1994).

Phenolic concentration is frequently affected by several internal and external factors (Zapprometov _et al.,_ 1989). Some nutrients, especially carbohydrate supplies influence the phenolic composition (Lux - Endrich _et al_., 2000). Phenolic concentrations can also be increased and decreased in different stages of germination (Thomas and Ravindra, 1999). Therefore, determination of the lowest phenol concentration phase during germination and isolation and culturing of explants in this phase will increase regeneration response and success in tissue culture studies.

2.13. Shoot apex culture

Shoot apex culture was first described by Morel (1952, 1960) for clonal propagation and virus eradication. This method has been successfully used with many monocot and dicot families (Murashige and Skoog, 1974). Theoretically, the tissues of the apical meristem are best suited for use in plant propagation and regeneration because these tissues are programmed for shoot organogenesis and do not need to differentiate to a meristematic state. The genotypic limitation and incidence of somaclonal variation regenerated plants is low (Murashige, 1974).

2.13.1. Plant phenotype

The plants regenerated from shoot apices produced normal phenotype (Murashige, 1974). Cotton plants produced regenerated by somatic embryogenesis, produced many abnormal phenotypes and cytogenetically aberrant plants. Upon cytogenetic analysis, Stelly and colleagues (Stelly _et al_., 1989) found that loss of chromosomes wee common.

The shoot meristem based method can be applied to plant transformation, either by particle bombardment (Christou _et al_., 1988) or _Agrobacterium_ mediated gene transfer. Compared to callus based regeneration by somatic embryogenesis the advantages to use the meristem based method are: a low incidence of culture induced genetic change and a simple and direct development of transformed plants  
(Gould _et al.,_ 1991).Generally it is necessary to transfer embryogenic cultures in a medium with reduced hormone concentration and nutrient content or with no hormone, to initiate /or mature somatic embryos. Other methods used for this purposes include exertion of stressful conditions, such as heat, cold and temporary starvation. Ventilation of cultures has been shown to increase somatic embryo initiation and germination in carrot (Li _et al_., 1989).Subjecting the cultured tissues to periods of stress such as desiccation (Parrott _et al.,_ 1989; Pomeroy _et al.,_ 1994; Timbert _et al.,_ l996; Bomal and Tremblay 1999), cold (Rajasekaran and Mullins 1989; Ritala _et al.,_ 2001) appears to improve the conversion of somatic embryos into mature embryos and subsequently to plants. It has been reported that high light conditions led to a reduction in callus proliferation and subsequently to tissue death, whereas low light conditions were conducive to callus growth and even to callus differentiation to plantlet stage in _G.arboreum_ (Smith _et al_., 1977). However somatic embryo production and maturation and plantlet acclimatization were sporadic But recently published results suggested that somatic embryogenesis could be improved through manipulation of carbon and nitrogen sources (Sakhanokho _et al.,_ 2001a).

**2.13.2.** _In vitro_ **plant regeneration in cotton by Multiple Shoot Induction**

Besides genotype limitation for regeneration the major impediments to somatic embryogenesis have been the low rate of regeneration, phenotypic abnormalities and cytogenetic changes (Li _et al_., 1989; Stelly _et al_., 1989). To circumvent the problem of somatic embryogenesis, cotton plants have been regenerated from shoot apical meristem (Gould _et al_., 1991; McCabe and Martinell, 1993; Nandeshwar, 1995; Gupta _et al_., 1997). The shoot tip based method of regeneration has been used for _Agrobacterium_ mediated transformation successfully (Zapata _et al_., 1999).

Aside from genotypic limitation many of the plants regenerated from callus as somatic embryos are not normal (Stelly _et al_., 1985).culture induced genetic damage is commonly observed among plants regenerated through a callus intermediate (Murashige and Skoog, 1974).Limited analysis of plant regenerated from callus by somatic embryogenesis of two cotton cultivars (Li _et al_., 1989) revealed extensive phenotypic abnormalities and cytogenetic changes. A protocol was developed for the production offshoots and intact plants from pre-existing shoot meristems of isolated seedling plumules of _G.barbadense_ , Pima S-6 and _G hirsutum_ (Gould and Smith, 1988).Shoot proliferation from different explants of several Indian cultivars of cotton was studied in culture. Cotyledonary nodes taken along with shoot apex of seedling produced multiple shoots (Gupta _et a_ l., 1997).apical meristem was reported by Chappel and Mauney (1967) but they did not obtain plant regeneration. Several literature reports are now available on clonal propagation of cotton cultivars using explants from in vitro grown seedlings (Agrawal _et al_., 1997; Gupta _et al_., 1997; Hemphill _et al_., 1998; Hazra _et al_., 2000, 2001).

2.13.3. Improvements in shoot apex regeneration

Cotton ( _G.hirsutum_ L.) and kenaf ( _Hibiscus cannabinus_ L.) belong to the malvaceae family, and both used as source of fibres. The shoot apices of both crops developed successfully without intervening callus formation and no significant differences among cultivars were found. Average of 58% of the cotton shoot apices initiated root and shoot in full strength Murashige and Skoog medium. All regenerated plants of both crops were phenotypically normal and set seeds.

In recent years protocols involving proliferation of cotton  
shoots (Agrawal _et al_., 1997; Gupta _et al_., 1997) or cotton regeneration  
(Hemphill _et al_., 1998;) have been published. The regeneration was carried out without a callus regeneration phase (Agrawal _et al_., 1997; Hemphill _et al.,_ 1998).

2.14. Prospects of genetic transformation and mass propagation

The ability of the cells within somatic tissues to undergo embryogenesis in culture is a genetic trait in many plants. Genetic components for somatic embryogenesis have been identified in alfalfa (Hernandez-Fernandez and Christie, 1989), soybean (Parrott _et al_.,1989, maize (Willman _et al.,_ 1989), barley (Komatsuda _et al_.,1989). Several groups have reported greater success with callus proliferation and regeneration using the Coker cotton varieties (Kuo _et al_.,1989; Trolinder and Xhixian, 1989) as emphasized by Merkle _et al_ , (1995) that if the embryogenic characteristics of cultured Coker 312 somatic cells was a genetic trait, plant breeding to move the heritable capacity for regeneration into selected elite cotton varieties would streamline procedures for genetic transformation and mass propagation.

The variation with respect to color and texture of calli had been observed by several authors (Gawel _et al_.,1986; Shoemaker _et al.,_ 1986; Firoozabady _et al_.,1987; Finer,1988;Trolinder and Goodin ,1988a,1988b).The use of glucose in alleviating the problem of severe browning of the explants due to phenolic oxidation in cotton tissue culture has been emphasized in the past (Beasley and Ting 1973; Smith _et al_., 1989).No browning of cells was observed at any stage of callus growth or embryogenesis in the presence of maltose (Kumria _et al_.,2003).

### CHAPTER III

### MATERIALS AND METHODS

The present investigation on in vitro studies in cotton was undertaken in the tissue culture laboratory, Centre for Plant Breeding and Genetics, Tamil Nadu Agricultural University, Coimbatore during the year 2006-2008.

### Standardization of explants, required media constituents, suitable combination of growth regulators, and culture conditioning for callus induction, multiplication maintenance, regeneration, and liquid culture were the experimentations programmed.

### 3.1. Materials

### 3.1.1. Seeds

Acid delinted cotton (Gossypium hirsutum L.) seeds of the four genotypes viz., Coker 310, SVPR2, MCU12, and MCU13 were obtained from the Department of Cotton, Centre for Plant Breeding and Genetics, Tamil Nadu Agricultural University, Coimbatore for the present study.

### 3.1.2. Nutrient media

Murashige and Skoog medium (1962) was the most used tissue culture medium and many modifications had been developed in that. Basic medium composition was that of MS supplemented with 3 per cent glucose (w/v) as the carbon source along with 0.8 per cent agar (w/v) as gelling agent. The media compositions suggested by Finer (1988), Firoozabady and Deboer (1993), Trolinder and Goodin (1988a) and Ganesan and Jayabalan (2004) for cotton somatic embryogenesis were tried with certain modifications with respect to vitamin sources and plant growth regulators with an objective to identify a medium that would induce, proliferate and maintain the callus from the explants. The details are given in the table 3.

### 3.2. Methods

### 3.2.1. Preparation of Nutrient media

### 3.2.1.1. Preparation of stock solutions

### The medium consisted of macronutrients, micronutrients, minornutrients,  
iron-EDTA, KI, vitamins, amino acids, sugar, agar and plant growth regulators. All the stock solutions and final media were prepared by following the procedure of Bhojwani and Razdan (2004). For Murashige and Skoog's basal medium, different stock solutions were prepared and used.

### 3.2.1.1.1. Macronutrients

### Each salt was weighed exactly and dissolved separately to the last particle in a small amount of distilled water. Finally all the salt solutions were pooled together and volume was made up with distilled water. Calcium chloride was added finally in order to prevent precipitation.

### 3.2.1.1.2. Micro and Minornutrients

### Each chemical was weighed exactly, dissolved separately, mixed together and finally the volume was made up with glass doubled distilled water.

### 3.2.1.1.3. Iron EDTA

FeSO4.7H2O and Na2EDTA.2H2O were dissolved separately in 200ml distilled water by heating and stirring. The two solutions were mixed, pH adjusted to 5.5,finally volume was made up to 500ml using distilled water and stored in amber coloured bottle.

### 3.2.1.1.4. Potassium iodide

### Potassium iodide was dissolved in distilled water and finally the volume was made up using glass double distilled water and the stock solution was stored in amber coloured bottle.

### 3.2.1.1.5. Vitamins

The components were weighed exactly, dissolved in distilled water and finally the volume was made up.

### 3.2.1.2. Preparation of growth regulators

### Separate stock solutions were prepared for each growth hormone by dissolving it in very minute quantity of the appropriate solvent (0.1N NaOH or 0.1N HCl or Ethanol) and making up to the final volume with distilled water.

### 3.2.1.2.1. Auxins

### Hundred milligram each of IAA, IBA, NAA, and 2,4-D were dissolved separately in 2 to 3 ml of 0.1N NaOH, warmed and gradually diluted to 100ml, using glass double distilled water.

### 3.2.1.2.2. Cytokinin

### Hundred milligram each of kinetin, BAP, and TDZ were dissolved in a small volume of 0.1N HCl, heated slightly and diluted to 100ml, using glass double distilled water.

### 3.2.1.2.3. Gibberellic acid

Hundred milligram of GA3 was dissolved separately in 2 to 3 ml of ethanol, warmed and gradually diluted to 100ml, using glass double distilled water.

### All the stock solutions were stored in appropriate reagent bottles under refrigeration.

### 3.2.1.3.. Preparation of agar and sugars

### Purified agar (0.8 per cent) from Hi-media and sucrose/glucose/ maltose  
(3 per cent) Analar-R grade was used directly.

### 3.2.1.4. Preparation of semi-solid agar media

To fifty per cent of total volume of water, the appropriate quantities of various stock solutions, including growth hormones were pipetted out. The final volume was made up with distilled water and pH was adjusted to 5.8 using 0.1N NaOH or 0.1  
N HCl .Then agar (0.8 per cent) was melted in the above solution for gelling purpose. The media was homogenized. About 10-15ml of medium was dispensed into each of the 25 ×150mm sterilized culture tubes. The culture vessels were plugged with sterilized non-absorbent cotton. The tubes were autoclaved at 15 lbs/sq. inch pressure at 121◦ C for 20 minutes. Subsequently the medium was allowed to cool at room temperature and stored inside the culture room.

### 3.2.1.5. Preparation of liquid medium

The medium was prepared as same as above procedure except that agar was not added. From this liquid media, 20 ml was transferred to 125 ml Erlenmeyer flask and plugged tightly with non-absorbent cotton and autoclaved at 15lbs/sq.inch pressure at 121◦ C for 20 minutes. Subsequently the medium was allowed to cool at room temperature and stored inside the culture room.

### 3.2.2. Surface sterilization and seed germination

Acid-delinted cotton (G hirsutum L.) seeds of the four genotypes viz., Coker 310, SVPR2, MCU12, and MCU13 were wrapped in two layers of cheese cloth and placed in running tap water for 30 minutes to remove surface impurities .The seeds were surface sterilized in 70 per cent ethanol for 1 minute and washed three times in sterile distilled water. The seeds were again surface sterilized with 0.1 per cent (w/v) mercuric chloride for 10 minutes in aseptic condition followed by three washes in sterile distilled water. The surface sterilized seeds were plated onto half strength MS medium supplemented with 0.8 per cent agar.

### 3.2.3. Explant preparation

The explant for the in vitro studies in cotton includes hypocotyls, cotyledonary leaves, leaf bits and shoot tip. Hypocotyl sections (0.5-1.0cm), cotyledon pieces  
(0.5-1.0cm), leaf bits (0.5-1.0cm2) and shoot tips were excised from 7 days old seedlings.

### 3.2.4. Surface disinfection of explants

Surface disinfection of explants was carried out aseptically using 0.1 to  
1.0 per cent (w/v) mercuric chloride with varying time of exposure i.e.1 to 5 minutes. Before treating with mercuric chloride, the explants were washed with 70 per cent ethanol for one minute followed by mercuric chloride exposure. The explants were shaken well to ensure thorough sterilization of the whole explants. Finally the explants were repeatedly washed four to five times in sterile distilled water to get rid of the traces of the sterilant.

### 3.2.5. Isolation and transfer of explants

Isolation and transfer of explants to the culture vessels were carried out inside "Thermo dyne Laminar Clean Air Flow Chamber" under aseptic conditions. The chamber was sterilized with absolute alcohol and ultra-violet radiation (253.7◦ A). The instruments used for incubation were autoclaved at 15lbs/sq.inch pressure at 121°C for 20 minutes and sterilized with 70 per cent ethanol, followed by sterilization with 1.0 per cent mercuric chloride solution. Hands were also swabbed with 70 per cent alcohol before carrying out operations in order to ensure aseptic conditions. Twenty five tubes or five petri plates were inoculated for each treatment and three replications were maintained.

### 3.2.5.1.. Inoculation of the sterilized explants to the culture tubes

### Inside the pre-sterilized laminar air flow chamber under aseptic condition, the explants were implanted in the culture media containing semi solid agar media using sterile forceps. It was inoculated as one explant per test tube or five explants per petri plate with one fourth of its bottom portion inserted into the medium for maintaining polarity. For leaves, the adaxial surface was made in full contact with medium. For each treatment 25 explants were inoculated with three replications.

### 3.2.5.2. Culture incubation

### The inoculated test tubes and petri plates were kept in racks inside the culture room protected from any source of contamination. The temperature and relative humidity were maintained at 24±2˚C and 50 to 60 per cent respectively. The photoperiod given was sixteen hours of light and eight hours of darkness, illuminated by cool white fluorescent lamps producing an intensity of 2000 lux. For culture induction, the culture tubes were kept in dark.

### 3.2.5.3. Callus Growth

### After three weeks of culture, calli initiated from the explants. The pro-embryogenic portions from the callus were identified and isolated. The selected pro-embryogenic calli were subcultured once in three weeks for the induction of embryogenic callus. Embryogenic calli were identified by creamy white colour, friability and by the presence of small cells, less vacuolated with a densely filled cytoplasm under microscopic examination.

### 3.2.5.4. Callus subculture

### The dull white, nodular and embryogenic calli obtained from the callus induction media were subcultured at every three weeks interval on the same medium for callus proliferation, maintenance and maturation.

### 3.2.5.5. Somatic Embryogenesis

### After the maturation of calli, they were transferred onto different media compositions for two months for the induction of somatic embryogenesis. The  
pro-embryoids were observed and were subcultured on the same medium for maturation. The somatic embryos (heart and torpedo shaped) were transferred to somatic embryo regeneration medium.

### 3.2.5.6. Organogenesis

### The proliferated calli which failed to differentiate into somatic embryos were transferred to different media combinations for development of shoots and roots from the callus cultures.

### 3.2.5.6.1. Shoot induction

### The proliferated calli was transferred to shoot differentiation medium for the development of shoots for a period of 15-20 days.

### 3.2.5.6.2. Root induction

### The shoot induced calli was transferred to the root induction medium for the development of roots for 15 days.

### 3.2.5.7. Hardening

### After the complete regeneration of the embryoids through calli or organogenesis to plantlets (80-100 days), they were transferred to plastic pots for hardening.

### 3.2.6. Transfer technology

### Fully developed plantlets with shoots and roots were removed from the cultures and washed gently to remove the adhering nutrient medium completely without causing any damage to the roots. They were then transferred to plastic micro pots filled with sterilized hardening rooting media of various compositions and were covered with perforated polythene bag to maintain enough humidity for easy establishment. They were maintained in the culture room for two weeks. The plantlets were then kept in mist chamber at 27-29˚C with 70-80 per cent RH for three weeks. Well established plantlets were later transferred to larger mud pots and kept in net house for further growth. The plants were watered everyday during the first week and once in two days afterwards.

### 3.3. Experimental details

### 3.3.1. Standardization of surface sterilization

In order to study the effects of different concentration of mercuric chloride and exposure time, the explants viz., hypocotyls, cotyledonary leaves, leaves and shoot tip were surface sterilized with 0.1 to 1.0 per cent mercuric chloride and the time of exposure studied was 1 to 10 minutes, the effect of sterilization on contamination percentage, explant survival and mortality were observed.

### 3.3.2. Callus induction and proliferation

### In order to study the effects of different growth regulators alone and in combination, on the induction of callus from hypocotyls, cotyledonary leaves, leaves and shoot tip explants the experiment was carried out in the following media combinations with glucose and maltose in common. (Table 4).

### The culture vessels were kept in dark for callus induction. Callus initiation was evaluated one month after plating of the explants on the culture medium. The frequency of callus induction was expressed in terms of number of explants cultured and was calculated as given below;

### Number of explants produced calli

F requency of callus induction (per cent) = х 100

### Total number of explants cultured

### The embryogenic calli were sub cultured at every 15 days interval on the same media for proliferation and maintenance. Sub culturing was done both in semisolid media and liquid media. Subculturing on semi solid media contains the same combinations whereas the liquid medium includes the following combinations;

### Medium | ### Media composition

---|---

### LM1 | MS+2,4-D (0.1 mgl-1) + kinetin(0.1 mgl-1) + maltose(30 gl-1)

### LM2 | MS+2,4-D (1 mgl-1) + kinetin( 0.5 mgl-1) + glucose (30 gl-1)

### LM3 | MS+2,4-D (1 mgl-1) + kinetin( 0.5 mgl-1) + maltose (30 gl-1)

### LM4 | MS+2,4- D (0.5 mgl-1) \+ NAA(1 mgl-1) \+ maltose(30 gl-1)

### LM5 | MS+NAA (0.5 mgl-1)+ BAP (2 mgl-1)+ glucose (30 gl-1)

### LM6 | MS+NAA (0.5 mgl-1)+ BAP (2 mgl-1)+ maltose(30 gl-1)

### Number of Embryogenic calli produced

### E mbryogenic calli induction (per cent) = х 100

### Total number of calli cultured

### 3.3.3. Effect of Explants

### To test the effect of age of the explants on callusing response, hypocotyls, cotyledon leaf and shoot tips were excised from 4, 7, 10, and 12 days old seedlings. The callus induction frequency of hypocotyls and cotyledon was assessed using the combination of 2, 4-D (0.1mg/l) + kinetin (0.5mg/l).

### 3.3.4. Callus maturation

### After two months the calli were separated from the explants and were transferred onto the MS basal medium to allow them for maturation. The calli were maintained on the same medium for about two months before transferring onto the somatic embryo induction medium (Table 5).The observations on embryogenic calli induction and somatic embryo induction were recorded after six and seven months of culture respectively. The somatic embryo maturation was assessed after eight months of culture.

### Number of somatic embryo produced calli

S omatic embryo induction (per cent) = х 100

### Total number of pro-embryo cultured

### Table.5.Somatic embryo induction media

### Medium | ### Media composition

---|---

### SEM1 | ### MS basal

### SEM2 | MS + IAA (0.5 mgl-1) \+ BAP (1 mgl-1)

### SEM3 | MS + IAA (0.5 mgl-1) \+ BAP (2 mgl-1)

### SEM4 | MS + IBA (0.5 mgl-1) + BAP (1 mgl-1)

### SEM5 | MS + IBA(0.5 mgl-1) + BAP(2 mgl-1)

### SEM6 | MS + NAA (0.5 mgl-1) + BAP (1 mgl-1)

### SEM7 | MS + NAA (0.5 mgl-1) + BAP (2 mgl-1)

### SEM8 | MS + IAA (0.5 mgl-1) \+ BAP(2 mgl-1)

### SEM9 | MS + IAA (0.5 mgl-1) \+ BAP (1 mgl-1)

### 3.3.5. Plantlet regeneration from somatic embryo cultures

### The bipolar torpedo stage and cotyledonary stage somatic embryos (more than 5 mm) with well developed cotyledons or with just an apical dome and with or without root system were transferred to semi-solid and liquid regeneration medium for shoot elongation and root development in both semi-solid and liquid medium separately.

### 3.3.6. Plantlet regeneration from callus cultures

### The response of different explants and growth regulators for regeneration was studied through following experiment combination.

### 3.3.6. 1.Shoot induction

### Hypocotyls, cotyledon and shoot tip explants were cultured with BAP alone (1, 1.5, 2, 2.5 and 3 mg/l) and in combination with IBA (0.1 and 2 g/l).the parameters such as percentage of explants responding for shoot differentiation were measured (Table 6).

### Number of plants with shoots

 Shoot induction (per cent) = х 100

### Total number of calli cultured

### Table.6. Shoot differentiation media combination

### Medium | ### Media composition

---|---

### SD1 | ### MS basal

### SD2 | MS + BAP (1 mg-1) +GA3 (1.0 mgl-1)

### SD3 | MS + BAP (2 mg-1) +GA3 (2.0 mgl-1)

### SD4 | MS +IAA(0.1mg-1) +GA3 (1.0 mgl-1)

### SD5 | MS+ IAA(0.1mg-1)+ BAP (1 mg-1) +GA3 (2.0 mgl-1)

### SD6 | MS+ IAA(0.5mg-1)+ BAP (2 mg-1) +GA3 (1.0 mgl-1)

### SD7 | MS+ IAA(0.5mg-1)+ BAP (1 mg-1) +GA3 (2.0 mgl-1)

### SD8 | MS+ IAA(0.1mg-1)+ BAP (2 mg-1) +GA3 (1.0 mgl-1)

### 3.3.6.2. Root induction

The shoots obtained from the regeneration medium were transferred to the following rooting media for in vitro rooting. The response for in vitro derived roots was studied (Table7).

### Table.7. Root differentiation media

### Medium | ### Media composition

---|---

### RM1 | ### MS basal

### RM2 | MS+ IBA(1.0 mg-1)

### RM3 | MS+ IBA(2.0 mg-1)

### RM4 | MS+IAA(0.1 mg-1)

### RM5 | MS+IBA(1.0 mg-1)+ IAA(0.1 mg-1)

### RM6 | MS+IBA(2.0 mg-1)+ IAA(0.5 mg-1)

### RM7 | MS+IBA(1.0 mg-1)+ IAA(0.5 mg-1)

### RM8 | MS+IBA(0.5 mg-1)+ IAA(2.0 mg-1)

### Number of plants with roots

 Root induction (per cent) = х 100

### Total number of calli cultured

### 3.4. Hardening of regenerated plants

### The plants with well developed roots and shoots were subjected to hardening and the parameters like Regeneration efficiency, Transfer efficiency and Establishment efficiency were also recorded.

When the plantlets attained adequate growth for producing 4-6 leaves with sufficient root system, they were removed from the jam bottles and washed in running tap water carefully to remove the media adhering to the roots.  They were subsequently transferred to pots containing sterilized soil. The plants were covered with polythene bags and kept in culture room. After 15 days, the plants were removed and well established plants were transferred to transgenic house.

### Number of plants Regenerated

 Regeneration efficiency (per cent) = х 100

### Total number of plants with roots

### Number of plants established

### E stablishment efficiency (per cent) = х 100

### Total number of Regenerated plants

### 3.5. Statistical methods

### The data from various experiments conducted for the study were analysed statistically by following the procedures developed by Panse and Sukhatme (1954).The experiments were laid out by he design of CRD. The per cent observations were transformed before statistical analysis (Arcsine transformation) and given in the tables along with original numbers. The significance of the mean difference between treatments was determined by computing the standard error and critical difference.

### .

### Table.3. Composition of the MS medium

### Constituents | ### w/v

### (g/l) | ### Volume of stock solution (ml)

---|---|---

### Prepared

### ml | ### Taken

### ml/l

### Macro nutrients

NH4 NO3

KNO3

MgSO4 .7H2O

KH2PO4

CaCl2

### Micro nutrients

MnS4O .7H2O

ZnSO4.7H2O

H3BO3

### Minor nutrients

Na2MoO4.2H2O

CuSO4.5H2O

CoCl2.6H2O

### Iron stock

Na2EDTA.2H2O

FeSO4.7H2O

### KI

### Vitamins

### Nicotinic acid

### Pyridoxine

### Thiamine HCl

### Glycine

### Myo-inositol

 |

### 16.50

### 19.00

### 3.70

### 1.70

### 4.40

### 2.23

### 0.86

### 0.62

### 0.125

### 0.012

0.012

### 1.8625

### 1.3925

### 0.083

### 0.050

### 0.050

### 0.010

### 0.200

### 10 |

### 500

### 250

### 250

### 250

### 250

### 100

 |

### 50

### 2.5

### 1.0

### 5.0

### 2.5

### 1.0

### Table.4.Different media composition used for callus induction and proliferation

### Medium | ### Media composition

---|---

### CM1 | MS+2,4-D (0.1mgl-1) + kinetin(0.1 mgl-1) + maltose(30gl-1)

### CM2 | MS+2,4-D (0.1 mgl-1) + kinetin(0.1 mgl-1) + glucose (30 gl-1)

### CM3 | MS+2,4-D (1 mgl-1) + kinetin( 0.5 mgl-1) glucose + (30 gl-1)

### CM4 | MS+2,4-D (0.1 mgl-1) + kinetin( 0.5 mgl-1) + maltose (30 gl-1)

### CM5 | MS+2,4- D( 0.1 mgl-1) +kinetin ( 0.4 mgl-1) + maltose(30 gl-1)

### CM6 | MS+2,4-D (0.1 mgl-1) + kinetin( 0.4 mgl-1) + glucose(30 gl-1)

### CM7 | MS+2,4-D (1 mgl-1) + IBA (0.5 mgl-1) + maltose (30 gl-1)

### CM8 | MS+2,4-D (1 mgl-1) + IBA(0.5 mgl-1) + glucose (30 gl-1)

### CM9 | MS+2,4- D (1 mgl-1) \+ NAA(0.5 mgl-1) \+ maltose(30 gl-1)

### CM10 | MS+2,4-D (1 mgl-1) + NAA(0.5 mgl-1) + glucose(30 gl-1)

### CM11 | MS+2,4-D (1 mgl-1) + maltose(30 gl-1)

### CM12 | MS+2,4-D (1.0mgl-) + glucose (30 gl-1)

### CM13 | MS+ NAA(0.4 mgl-1) +kinetin (0.1 mgl-1) + glucose(30 gl-1)

### CM14 | MS+ NAA(0.4 mgl-1) +kinetin (0.1 mgl-1) maltose (30 gl-1)

### CM15 | MS+NAA( 4.0 mgl-1)+ kinetin 1.0 mgl-1)+ maltose(30 gl-1)

### CM16 | MS+NAA (4.0 mgl-1)+ kinetin 1.0 mgl-1)+ glucose(30 gl-1)

### CM17 | MS+NAA (3.0 mgl-1)+ kinetin 1.0 mgl-1)+ maltose30 gl-1)

### CM 18 | MS+NAA (3.0 mgl-1) + kinetin (1.0 mgl-1)+ glucose(30 gl-1)

### CM 19 | MS+NAA (2.0 mgl-1) + kinetin (1.0 mgl-1)+ maltose(30 gl-1)

### CM 20 | MS+NAA (2.0 mgl-1)+ kinetin (1.0 mgl-1)+ glucose(30 gl-1)

### CM 21 | MS+NAA (1.0 mgl-1)+ kinetin (1.0 mgl-1)+ maltose(30 gl-1)

### CM 22 | MS+NAA( 1.0 mgl-1)+ kinetin (1.0 mgl-1)+ glucose(30 gl-1)

### CM 23 | MS+NAA (0.5 mgl-1)+ kinetin (1.0 mgl-1) + maltose(30 gl-1)

### CM 24 | MS+NAA (0.5 mgl-1)+ kinetin (1.0 mgl-1) + glucose3(0 gl-1)

### CM 25 | MS+NAA (1.0 mgl-1)+ kinetin (0.5 mgl-1) + maltose(30 gl-1)

### CM 26 | MS+NAA (1.0 mgl-1) + kinetin (0.5 mgl-1) + glucose(30 gl-1

### CM27 | MS+NAA (0.5 mgl-1)+ BAP (2 mgl-1)+ maltose(30 gl-1)

### CM28 | MS+NAA ( 0.5 mgl-1)+ BAP (1.0 mgl-1) + glucose (30 gl-1)

### CM 29 | MS+NAA( 0.5 mgl-1)+ BAP (2 mgl-1) + maltose (30 gl-1)

### CM 30 | MS+NAA (0.5 mgl-1)+ BAP (1.0 mgl-1)+ glucose(30 gl-1)

### CM31 | MS+NAA 0.1 mgl-1)+ BAP (1.0 mgl-1)+ maltose(30 gl-1)

### CM 32 | MS+NAA( 0.1 mgl-1)+ BAP (1.0 mgl-1)+ glucose(30 gl-1)

CHAPTER IV

### RESULTS

Given the increasing per cent of transgenic cotton being grown worldwide, it is imperative that genotype independent methods for regenerating cotton be developed to maintain diversity in the gene pool. There is a need at this time to develop a protocol to attain an efficient callus induction, its proliferation and regeneration system for cotton as there is an increase of transgenic cotton being grown world wide.

Experiments were conducted to study the effect of growth regulators, response of explants (hypocotyl, cotyledon, leaf and shoot tip) from the seedlings of four different genotypes of cotton _viz._ , Coker 310, SVPR2, MCU12, and MCU13 for callus induction proliferation, somatic embryogenesis, regeneration of plantlets, shoot and, root induction and their growth through semi-solid and liquid media. The results are presented in this chapter.

4.1. Seed germination

The acid delinted seeds showed 90-95 per cent successful germination rate from all the four genotypes following 7 days of culture in half strength MS medium. The explants chosen for the study included hypocotyl, cotyledon, leaf and shoot tip. The explants were inoculated in various media combinations and the observations on various parameters were recorded (Plate 1).

4.2. Sterilization

The effect of mercuric chloride (0.1, 0.5. and 1.0 per cent) at different durations of exposure (1-10 minutes) in establishing contamination free and surviving culture was studied and the results are presented.

4.2.1. Contamination

Significant differences for the contamination per cent of the different explants of different genotypes were recorded at various sterilization treatments and are presented in the tables.
4.2.1.1. Genotypes

The effect of concentrations of the surface sterilant and durations of exposure on the contamination per cent on the four genotypes was recorded (Table.8)  
(Figure 1).

The mean per cent of contamination of different explants in different sterilization treatments ranged from 54.12 in MCU13 to 60.11 percent in Coker 310.

Among the four genotypes used, the lowest contamination per cent of 34.49 was recorded in Coker 310, when the explants of Coker 310 was treated in 0.1  
per cent mercuric chloride for 10 minutes and the highest contamination per cent (80.99) was recorded in Coker 310 when the explants of the same genotype was treated with 0.1 per cent mercuric chloride for 5 minutes

4.2.1.1.1. Coker 310

The mean per cent of contamination of different explants of the genotype Coker 310 ranged from 54.12 in shoot tip to 60.66 in leaf explant. (Table 9).

Among the different explants used, the lowest contamination per cent of 28.00 was recorded in hypocotyl explants, when the explants were treated in 0.1 per cent mercuric chloride for 10 minutes and the highest contamination per cent of 88.33 was recorded in cotyledon explants treated in 0.1 per cent mercuric chloride for 5 minutes.

The individual interaction effects between the concentration of mercuric chloride, duration of exposure and the explants were significant.

4.2.1.1.2. SVPR2

The mean per cent of contamination of different explants of SVPR 2 ranged from 57.29 in hypocotyl explant to 64.44 in shoot tip explant (Table 9).

Among the different explants used, the lowest contamination per cent of 30.00 was recorded in hypocotyl explants, when the explants were treated in 0.1 per cent mercuric chloride for 10 minutes and the highest contamination per cent (80.00) was recorded in hypocotyl explants treated in 0.1 per cent mercuric chloride for 5 minutes.

The individual and interaction effects between the concentration of mercuric chloride, duration of exposure and the explants were significant.

4.2.1.1.3. MCU12

The mean per cent of contamination of different explants of the genotype MCU12 ranged from 55.66 in hypocotyl explant to 62.58 in shoot tip (Table.10).

Among the different explants used, the lowest contamination per cent of 33.66 was recorded in hypocotyl explants, when the explants were treated in 0.1 per cent mercuric chloride for 10 minutes and the highest contamination per cent of 77.66 was recorded in hypocotyl explants treated in 0.1 per cent mercuric chloride for 5 minutes.

The individual and interaction effects between the concentration of mercuric chloride, duration of exposure and the explants were significant.

4.2.1.1.4. MCU13

The mean per cent of contamination of different explants ranged from 48.21 in cotyledon explant to 57.21 in shoot tip explant (Table.10).

Among the different explants used, the lowest contamination per cent of 30.46 was recorded in hypocotyl explants, when the explants were treated in 0.1 per cent mercuric chloride for 10 minutes and the highest contamination per cent of 80.33 was recorded in hypocotyl explants treated in 0.1 per cent mercuric chloride for 5 minutes.

The individual and interaction effects between the concentration of mercuric chloride, duration of exposure and the explants were significant.

4.2.1.2. Explants

The effect of concentrations of surface sterilant and durations of exposure on the contamination per cent of four explants _viz_., hypocotyl, cotyledon, leaf and shoot tip of four genotypes was recorded (Table.8) (Figure 2).

The mean per cent of contamination of four explants of four genotypes in different sterilization treatments ranged from 59.25 in hypocotyl explants to 64.44 in shoot tip explant.

Among the four explants used, the lowest contamination per cent of 30.53  
per cent was recorded in hypocotyl explants treated in 0.1 per cent mercuric chloride for 10 minutes and the highest contamination per cent (79.91) was recorded in hypocotyl explants treated in 0.1 per cent mercuric chloride for 5 minutes.

4.2.1.2.1. Hypocotyl

The mean per cent of contamination of hypocotyl explants of different genotypes in different sterilization treatments ranged from 55.41 in the genotype MCU 13 to 59.25 in the genotype in Coker 310 (Table.11).

The lowest contamination per cent of 28.00 was recorded in hypocotyl explants of Coker 310 when treated in 0.1 per cent mercuric chloride for 10 minutes and the highest contamination per cent (81.66) was recorded in hypocotyl explants of the same genotype treated in 0.1 per cent mercuric chloride for 5 minutes.

4.2.1.2.2. Cotyledon

The mean per cent of contamination of cotyledon explant of four genotypes in different sterilization treatments ranged from 48.21 in MCU13 to 62.77 in SVPR2 (Table.11).

Among the four genotypes used, the lowest contamination per cent of 30.00 was recorded in cotyledon explants of Coker 310 when treated in 0.1 per cent mercuric chloride for 10 minutes and the highest contamination per cent (88.33) was recorded in cotyledon explants of Coker 310 treated in 0.1 per cent mercuric chloride for 5 minutes.

4.2.1.2.3. Leaf

The mean per cent of contamination of leaf explants of different genotypes in different sterilization treatments ranged from 50.40 in MCU13 to 60.66 in the genotypes Coker 310 and MCU12 (Table.12).

Among the four genotypes used, the lowest contamination per cent of 36.33 was recorded in leaf explants of Coker 310 when treated in 0.1 per cent mercuric chloride for 10 minutes and the highest contamination per cent (82.00) was recorded in leaf explants of the same genotype treated in 0.1 per cent mercuric chloride for  
5 minutes.

4.2.1.2.4. Shoot tip

The mean per cent of contamination of shoot tip explants of four genotypes in different sterilization treatments ranged from 57.21 in MCU 13 to 64.44 in SVPR2 genotype (Table.12).

The lowest contamination per cent of 43.66 was recorded in shoot tip explants of Coker 310 when treated in 0.1 per cent mercuric chloride for 10 minutes and the highest contamination per cent (72.66) was recorded in shoot tip explants of the SVPR2 treated in 0.1 per cent mercuric chloride for 5 minutes.

4.2.1.3. Concentrations and durations of exposure of the sterilant

The per cent of contamination for different explants of the four genotypes at various concentrations (0.1, 0.5, and 1.0 per cent) and durations of exposure (5, 7, 10 minutes) of the sterilant was recorded.

4.2.1.3.1. Genotypes

The mean per cent of contamination in four genotypes with different explants in different concentrations of the sterilant ranged from 57.40 in SVPR2 to 62.77 in MCU 12 (Table.13).

The lowest contamination per cent (30.00) was recorded in Coker 310 when the explants were treated in 0.1 per cent mercuric chloride for 10 minutes and the highest contamination per cent (88.33) was recorded in the explants of the same genotype when treated with 0.1 per cent mercuric chloride for 5 minutes.

4.2.1.3.2. Explants

The mean per cent of contamination of four explants in different sterilization treatments ranged from 56.91 in hypocotyl explants to 60.98 in shoot tip explant (Table.14).

Among the different explants used, the lowest contamination per cent of 30.53 was recorded in hypocotyl explants when treated in 0.1 per cent mercuric chloride for 10 minutes and the highest contamination per cent was recorded in hypocotyl (79.91) explants treated in 0.1 per cent mercuric chloride for 5 minutes.

4.2.2. Survival per cent

The survival per cent of four different explants from four different genotypes of cotton treated with various concentrations of mercuric chloride at different durations of exposure was recorded and presented in the tables

4.2.2.1. Genotypes

The effect of concentrations of the surface sterilant and durations of exposure on the survival per cent of different explants on the four genotypes was recorded (Table.15) (Figure 3).

The mean per cent of survival of explants of four different genotypes in different sterilization treatments ranged from 46.70 in SVPR2 to 47.87 in MCU12.

Among the four genotypes used, the lowest survival per cent (21.49) was recorded in MCU 13, when the explants were treated in 0.1 per cent mercuric chloride for 5 minutes and the highest survival per cent of 80.49 was recorded in Coker 310, when the explants were treated with 0.1 per cent mercuric chloride for 10 minute

4.2.2.1.1. Coker 310

The mean per cent of survival of different explants of Coker 310 ranged from 44.55 in cotyledon explant to 49.66 in shoot tip (Table 16).

The lowest survival per cent (17.66) was recorded when the leaf explants were treated in 1.0 per cent mercuric chloride for 5 minutes and the highest survival per cent (80.33) was recorded when the hypocotyl explants were treated with 0.1  
per cent mercuric chloride for 10 minutes.

The individual and interaction effects of different concentrations of sterilant and different durations of exposure on the survival per cent of four different explants from Coker 310 cotton genotype were significant.

4.2.2.1.2. SVPR2

The mean per cent of survival of different explants of SVPR2 genotype ranged from 46.44 in hypocotyl explants to 48.95 in leaf explants (Table 16).

Among the different explants used, the lowest survival per cent (15.00) was recorded when the shoot tip explants were treated in 0.1 per cent mercuric chloride for 5 minutes and the highest survival per cent (81.66) was recorded when the hypocotyl explants were treated with 0.1 per cent mercuric chloride for 10 minutes.

The individual and interaction effects of different concentrations of sterilant and different durations of exposure on the survival per cent of four different explants from SVPR2 cotton genotype were significant.

4.2.2.1.3. MCU 12

The mean per cent of survival of different explants ranged from 46.07 in cotyledon explants to 49.62 in shoot tip explants (Table 17).

Among the different explants used, the lowest survival per cent (17.66) was recorded when the shoot tip explants were treated in 0.1 per cent mercuric chloride for 5 minutes and the highest survival per cent (78.33) was recorded when the hypocotyl explants were treated with 0.1 per cent mercuric chloride for 10 minutes.

The individual and interaction effects of different concentrations of sterilant and different durations of exposure on the survival per cent of four different explants from MCU12 cotton genotype were significant.

4.2.2.1.4. MCU13

The mean per cent of survival of different explants of MCU13 ranged from 46.19 in hypocotyl explants to 46.44 in cotyledon and shoot tip explants (Table 17).

Among the different explants used, the lowest survival per cent (24.66) was recorded when the shoot tip explants were treated in 0.1 per cent mercuric chloride for 5 minutes and the highest survival per cent (82.33) was recorded when the hypocotyl explants were treated with 0.1 per cent mercuric chloride for 10 minutes.

The individual and interaction effects of different concentrations of sterilant and different durations of exposure on the survival per cent of four different explants from Coker 310 cotton genotype were significant.

4.2.2.2. Explants

The survival per cent for four explants of different genotypes was recorded based on their performance with respect to genotype, concentrations of the sterilant and durations of exposure.

The mean per cent of survival of four explants ranged from 46.07 in cotyledon explants to 48.44 in leaf and shoot tip explants. (Table 15) (Figure 4).

Among the four explants used, the lowest survival per cent (21.49) was recorded when the hypocotyl explants were treated in 0.1 per cent mercuric chloride for 5 minutes and the highest survival per cent (71.41) was recorded when the hypocotyl explants were treated with 0.1 per cent mercuric chloride for 10 minutes.
4.2.2.2.1. Hypocotyl

The mean per cent of survival of hypocotyl explants of different genotypes ranged from 46.29 in Coker 310 and MCU12 to 46.44 in MCU13 (Table 18).

Among the different genotypes used, the lowest survival per cent (20.66) was recorded when the hypocotyl explants of MCU 13 were treated in 0.5 per cent mercuric chloride for 5 minutes and the highest survival per cent (80.33) was recorded when the hypocotyl explants of MCU 13 were treated with 0.1 per cent mercuric chloride for 10 minutes.

. **4.2.2.2.2. Cotyledon**

The mean per cent of survival of cotyledon explants of different genotypes ranged from 44.55 to 48.21 percent. The mean per cent of survival of different explants in different concentrations of the sterilant and durations of exposure ranged from 21.91 to 72.91 percent (Table 18).

The lowest survival per cent (23.33) was recorded when the cotyledon explants of MCU 13 were treated in 0.1 per cent mercuric chloride for 5 minutes and the highest survival per cent (80.66) was recorded when the cotyledon explants of Coker 310 were treated with 0.1 per cent mercuric chloride for 10 minutes.

4.2.2.2.3. Leaf

The survival per cent for leaf explants of different genotypes was recorded based on their performance with respect to genotype, concentrations of the sterilant and durations of exposure (Table 19).

The mean per cent of survival of leaf explants ranged from 46.33 in Coker 310 and MCU12 to 48.95 in MCU 13 and SVPR2.

Among the four genotypes used, the lowest survival per cent (17.66) was recorded when the leaf explants of MCU 13 were treated in 0.1 per cent mercuric chloride for 5 minutes and the highest survival per cent (78.00 per cent ) was recorded when the leaf explants of Coker 310 were treated with 0.1 per cent mercuric chloride for 10 minutes.

4.2.2.2.4. Shoot tip

The survival per cent for shoot tip explants of four genotypes was recorded based on their performance with respect to genotype, concentrations of the sterilant and durations of exposure (Table 19).

The mean per cent of survival of shoot tip explants ranged from 43.87 in Coker 310 to 49.66 in MCU12.

Among the different treatments used, the lowest survival per cent (15.00) was recorded when the shoot tip explants of SVPR2 were treated in 0.1 per cent mercuric chloride for 5 minutes and the highest survival per cent (76.00) was recorded when the shoot tip explants of Coker 310 were treated with 0.1 per cent mercuric chloride for 10 minutes.

4.2.2.3. Concentrations of the sterilant and durations of exposure

The per cent of survival for different explants of the four genotypes at different concentrations of the sterilant (0.1, 0.5, and 1.0 per cent) and durations of exposure (1 to 10 minutes) was recorded.

4.2.2.3.1. Genotypes

The mean per cent of survival of different explants of the four genotypes at different concentrations of the sterilant at different durations of exposure ranged from 47.63 in MCU12 to 52.96 in MCU13 (Table 20).

Among the four different genotypes used, the lowest survival per cent 21.49 was recorded in MCU13 when the concentration of in 0.1 per cent mercuric chloride was used to sterilize the explants for 5 minutes and the highest survival per cent of 80.49 was recorded in Coker 310 when the explants were exposed to 0.1 per cent mercuric chloride for 10 minutes.

4.2.2.3.2. Explant

The per cent of survival of four different explants of the four genotypes at different concentrations of the sterilant (0.1, 0.5, and 1.0 per cent) and durations of exposure (1 to 10 minutes) was recorded (Table 21).

The mean survival per cent of the four explants of different genotypes ranged from 44.90 in shoot tip explant to 47.63 in hypocotyl explant.

Among the different explants used, the lowest survival per cent (21.49) was recorded in hypocotyl when treated in 0.1 per cent mercuric chloride for 5 minutes and the highest survival per cent of 76.41 was recorded in hypocotyl explants when exposed to 0.1 per cent mercuric chloride for 10 minutes.

4.3. Callus induction

Experiment was conducted to standardize the media composition with different concentrations of growth regulators for callusablity of four different explants from four genotypes of cotton. Four explants _viz_., hypocotyl, cotyledon, leaf and shoot tip from seven days old seedlings of cotton were inoculated in MS medium containing different growth regulator combinations for callus induction.

4.3.1. Effect of genotypes on callus induction

The callus induction per cent was recorded in four genotypes _viz.,_ Coker 310, SVPR2, MCU12, and MCU13 for different explants. The callus induction per cent of different explants of the four genotypes was recorded with response to various growth regulator combinations, days taken for callus induction and type of calli (Plate 2).

The mean per cent of callus induction of the four genotypes ranged between 53.99 in MCU12 to 60.69 in SVPR2 (Table22).

The highest callus induction per cent was recorded in Coker 310 genotype (80.33 per cent) in media composition (MS 4) containing MS + 2, 4-D (0.1mgl-1) +Kinetin (0.5 mgl-1) +maltose (30gl-1) and the lowest callus induction per cent (31.33) was recorded in MCU13 in media combination (MS 30) containing MS + NAA  
(0.5 mgl-1) +BAP (1mgl-1) + glucose (30gl-1).

The callus obtained from MS 4 took 7 to 9 days for callus induction and the type of callus obtained was smooth, yellow and brown coloured with high friability.

The callus obtained from MS 30 took 12 to 15 days for callus induction and the type of callus obtained was compact with various callus colour of green, yellow, brown and white with low to medium friability.

4.3.1.1. Coker 310

The mean per cent of callus induction of the different explants of Coker 310 ranged between 54.00 in hypocotyl explant to 62.82 in leaf explant (Table 23).

The highest callus induction per cent (86.00) was recorded in hypocotyl explants in MS4 [MS + 2, 4-D (0.1mgl-1) + Kinetin (0.5 mgl-1) + maltose (30gl-1)] and the lowest callus induction per cent (23.33) was recorded in shoot tip explants cultured in MS 30 [MS +NAA (0.5 mgl-1) +BAP (1mgl-1) + glucose (30gl-1)] and the callus initiated within 12 to 15 days from the day of explant inoculation.

**4. 3.1.2. SVPR2**.

The mean per cent of callus induction in SVPR 2 of different explants ranged between 58.97 in shoot tip explant to 61.58 in cotyledon explant (Table 23).

The highest callus induction per cent (78.66) was recorded in hypocotyl explants in the media combination of MS 4 containing MS + 2, 4-D (0.1mgl-1) + Kinetin (0.5 mgl-1) + maltose (30gl-1) and the lowest callus induction per cent (27.33) was recorded in shoot tip explants cultured in MS 30 media combination containing MS +NAA (0.5 mgl-1) +BAP (1mgl-1) + glucose (30gl-1).

4. 3.1.3. MCU12

The mean callus induction per cent of the different explants of MCU12 ranged between 51.00 in leaf explant to 56.66 in shoot tip explant (Table 24).

The highest callus induction per cent (78.00) was recorded in the media composition of MS 4 containing MS \+ 2, 4-D (0.1mgl-1) + Kinetin (0.5 mgl-1) + maltose (30gl-1) in hypocotyl explants and the lowest callus induction per cent (34.00) was recorded in shoot tip explants cultured in MS 30 [MS +NAA (0.5 mgl-1) + BAP (1mgl-1) + glucose (30gl-1)].

4. 3.1.3. MCU13

The mean per cent of callus induction of the different explants of MCU13 ranged between 58.21 in hypocotyl explant to 61.81 leaf explant (Table 24).

The highest callus induction per cent (80.33) was recorded in hypocotyl explants in the media composition of MS4 [MS + 2, 4-D (0.1mgl-1) + Kinetin  
(0.5 mgl-1) + maltose (30gl-1)] and the lowest callus induction per cent (26.00) was recorded in shoot tip explants cultured in MS 30 [MS + NAA (0.5 mgl-1) + BAP (1mgl-1) + glucose (30gl-1)].

4. 3.2. Effect of explants

The callus induction per cent of different explants of the four genotypes was recorded. The mean per cent of callus induction of the explants ranged between 58.13 in hypocotyl explant to 60.28 in cotyledon explant (Table 22) (Plate 3).

Among the four explants used, the highest callus induction per cent (82.49) was recorded in hypocotyl explants of the genotype in the callus induction media  
MS 4 [MS + 2, 4-D (0.1mgl-1) + Kinetin (0.5 mgl-1) + maltose (30gl-1)] and the lowest callus induction per cent (30.60) in the shoot tip explants in the callus induction medium MS 30 [MS + NAA (0.5 mgl-1) + BAP (1mgl-1) + glucose (30gl-1)].

4. 3.2.1. Hypocotyl

The callus induction per cent of hypocotyl explant of the genotypes was recorded. The mean per cent of callus induction of hypocotyl explant of the genotypes ranged between 52.66 in the genotype MCU12 to 61.40 in SVPR2 (Table 25).

The hypocotyl explant recorded highest callus induction per cent (86.00) in Coker 310 in the media combination of MS + 2, 4-D (0.1mgl-1) + Kinetin (0.5 mgl-1) + maltose (30gl-1) [MS4] and the shoot tip explant recorded the lowestcallus induction per cent (37.66) in the callus induction medium MS 30 [MS + NAA (0.5 mgl-1) \+ BAP (1mgl-1) + glucose (30gl-1)].

4. 3.2. 2. Cotyledon

The callus induction per cent of cotyledon explant of the four genotypes was recorded. The mean per cent of callus induction of the cotyledon explant ranged from 54.20 in Coker 310 to 61.58 in SVPR2 (Table 25).

The cotyledon explant recorded highest callus induction per cent (83.33) in Coker 310 in the callus induction media MS 4 [MS + 2, 4-D (0.1mgl-1) + Kinetin (0.5 mgl-1) + maltose (30gl-1)] and the lowest callus induction per cent (31.00) in the cotyledon explants of MCU13 was recorded in the callus induction medium MS 30[MS + NAA (0.5 mgl-1) + BAP (1mgl-1) + glucose (30gl-1)].

4. 3.2.3. Leaf

The callus induction per cent of leaf explant of the four genotypes was recorded. The mean per cent of callus induction of the leaf explants ranged between 51.00 in MCU 12 to 61.81 in MCU13 (Table 26).

The leaf explants of Coker 310 recorded highest callus induction per cent (76.33) in the callus induction media MS 4 containing MS + 2, 4-D (0.1mgl-1) + Kinetin (0.5 mgl-1) + maltose (30gl-1) and the lowest callus induction per cent (22.66) was recorded in the leaf explants of SVPR2 in the callus induction medium MS 30 containing MS + NAA (0.5 mgl-1) + BAP (1mgl-1) + glucose (30gl-1).

4. 3.2. 4. Shoot tip

The shoot tip explants recorded the mean per cent of callus induction from the four genotypes that ranged from 56.66 in MCU12 to 60.39 in MCU13 (Table 26).

The shoot tip explants of the genotype Coker 310 in the callus induction media MS 4[MS + 2, 4-D (0.1mgl-1) + Kinetin (0.5 mgl-1) + maltose (30gl-1)] recorded highest callus induction per cent (80.33) and the same explants of MCU13 recorded the lowest callus induction per cent (26.00) in the callus induction medium MS 30 [MS \+ NAA (0.5 mgl-1) + BAP (1mgl-1) + glucose (30gl-1)].

4.3.3. Effect of growth hormones on callus induction

Effect of growth hormones was exhibited differently in the callus induction of different explants of four genotypes (Table 27) (Plate 4).

The mean value of callus induction per cent for various growth hormonal combinations ranged from 57.46 in combination of NAA with BAP to 62.88 in combination of 2, 4-D with Kinetin.

The highest callus induction per cent (74.78) was recorded from the growth hormonal combination of 2, 4-D (0.1 mgl-1) and kinetin (0.5 mgl-1) .The callus so obtained from this combination was smooth and highly friable with varying callus colour of yellow and brown and took 7-9 days for callus induction from the day of explant inoculation.

Though calli was also induced from other hormonal combination such as  
2, 4-D with IBA, NAA, and BAP and NAA with Kinetin having different calli characteristic, the calli obtained from 2, 4-D (0.1 mgl-1) and kinetin (0.5 mgl-1) was best.

4.3.4. Effect of carbon sources

Two different carbon sources were used as one of the important component in callus induction media _viz.,_ maltose and glucose each at 30gl-1.Among the two sources used maltose gave significantly better mean values of callus induction in different genotypes (61.28 per cent) and for different explants (63.28 per cent) compared to glucose in general (Table28) (Figure 5).

Table.28.Effect of carbon sources on callus induction of explants  
in different genotypes

Carbon source

(30gl-1) | Callus induction frequency (%) |

Mean

---|---|---

Genotypes | Explants

Maltose | 61.28 | 63.28 | 62.28

Glucose | 60.58 | 61.27 | 60.92

Mean | 60.93 | 62.27 | 61.60

4.3.5. Types of calli

Different types of calli were observed from different explants of four genotypes in different media compositions. The calli characteristics such as callus colour, texture, friability, days taken for callus induction in different growth regulator combinations were observed (Table 32).

Table.29.Calli characteristics

Media composition | Days taken for callus initiation |

Colour |

Texture |

Friability

---|---|---|---|---

2,4-D+IBA | 7-10 | Yellowish green | Compact | Medium

2,4-D+NAA | 10-12 | Green | Compact | Low

2,4-D+Kinetin | 7-9 | Yellow, brown | Smooth | High

2,4-D+BAP | 10-12 | Green, white, brown | Compact | Low

NAA+ Kinetin | 12-15 | Green, yellowish .brown, white | Compact | low to medium

The calli obtained from the MS 4 medium with the hormonal combination of 2, 4-D (0.1mgl-1) \+ kinetin (0.5mgl-1) induced within 7-9 days after inoculation of the explants was yellow, and brown coloured, smooth and highly friable and was the best among the calli obtained from other hormonal combinations.

4.4. Callus proliferation

After the callus induction and growth, the highly responding calli were sub cultured in callus proliferation medium after 21 days on the same best callus induction medium and their frequency of proliferation was recorded. The callus proliferation followed similar trend of callus induction for the source of variations _viz.,_ genotypes, explants and hormones. The callus induction medium that responded well was chosen for proliferation in both liquid and semi solid medium.

4.4.1. Callus proliferation in semi solid medium

The calli was transferred to the semi solid medium for proliferation after  
21 days of callus induction and maintained by sub culturing once in 15 days. The effect of growth regulators on callus proliferation was recorded for both genotypes and explants (Plate 5).

4.4.1.1.Genotypes

The mean callus proliferation per cent of the genotypes ranged from 58.74 in SVPR 2 to 61.49 in MCU 13 (Table 30) (Figure 6).

The highest callus proliferation per cent was found in genotype Coker 310 (75.00 per cent) in the combination of 2, 4-D (0.1mgl-1) and kinetin (0.1mgl-1) and the lowest was recorded in SVPR2 (46.33) in the combination of 2, 4-D (0.1mgl-1) and IBA (0.5mgl-1).

**4.4.1.2.** **Explants**

The mean callus proliferation per cent of the explants ranged from 53.77 in shoot tip explant to 56.81 in hypocotyl explant (Table 31) (Figure 7).

The highest callus proliferation per cent was found in hypocotyl explants (73.00 per cent)) in the combination of 2, 4-D (0.1mgl-1) and kinetin (0.1mgl-1) and the lowest was recorded in cotyledon explants (44.30) in the combination of NAA (2.0 mgl-1) and kinetin (0.1mgl-1).

4.4.2. Callus proliferation in liquid medium

The well developed calli from callus induction medium was transferred to the liquid medium for proliferation on the same best callus induction medium but without agar after 21 days with frequent sub culturing of once in 10 days (Plate 6).

4.4.2.1. Genotypes

The mean callus proliferation per cent of the genotypes ranged from 60.82 in MCU13 to 64.33 in SVPR2 (Table 32) (Figure 8).

The callus of Coker 310 genotype obtained from the media combination of  
2, 4-D (0.1mgl-1) and kinetin (0.1mgl-1) with maltose(30 gl-1) as the carbon source recorded highest callus proliferation per cent (76.33), followed by MCU13 (68.00 per cent) in the same medium followed by SVPR2 genotype in combination of 2,4-D (0.1mgl-1) with kinetin (0.4mgl-1), and MCU12 in combination of 2,4-D (0.1mgl-1) with kinetin (0.1mgl-1) and the lowest was recorded in SVPR2 in the combination of kinetin (0.1mgl-1) with NAA (2.0mgl-1).

4.4.2.2. Explants

The mean callus proliferation per cent of the explants ranged from 53.91 in cotyledon explant to 59.25 in shoot tip explant (Table 33) (Figure 9).

The highest callus proliferation per cent was found in hypocotyl explants (74.63) in the combination of 2, 4-D (0.1mgl-1) and kinetin (0.1mgl-1) and the lowest was recorded in cotyledon explants (45.33) in the combination of kinetin (0.1mgl-1) and NAA (2.0mgl-1).

4.4.2.3. Semi solid versus liquid medium

The efficiency of liquid medium over solid medium was compared for its proliferation. The 21 days old calli after induction was transferred to both semi solid medium and liquid medium for proliferation and maintenance.

The mean values of callus proliferation of the four different genotypes ranged from 52.73 per cent (MCU12) in semi solid medium to 58.29 per cent (Coker 310) in liquid medium. (Table.37)

The range of callus proliferation per cent of the four different explants was from 52.57 (shoot tip) in semi solid medium to 58.29 per cent (hypocotyl) in liquid medium.

The results indicated that the liquid medium proliferated more significantly than the semi solid medium in all the four explants of the four genotypes. The highest callus proliferation per cent (61.15) was recorded in hypocotyl explants of Coker 310 in liquid medium and it was only 58.27 in semi solid medium.

4.5. Callus sub culturing and maintenance

The proliferated calli was maintained by frequent subculturing. This was done in the same media combinations which were used for proliferation. Significant difference was observed on subculturing. The callus obtained from liquid medium was smooth and highly friable and in semi solid medium it showed low to medium friability. Phenolic exudation was found to be reduced in liquid media than solid media. This was achieved by frequent subculturing. Callus sub culturing in semi solid medium was done once in 5 days in semi solid medium and once in 10 days in liquid medium for callus growth and maintenance.

4.6. Callus differentiation

The proliferated calli after sub culturing and maintenance was transferred to the medium containing various levels of IAA, IBA, NAA, and BAP and sub cultured for two months at an interval of 15 days. Of the four genotypes cultured only Coker 310 induced somatic embryos and mature further. After two months of callus maintenance, the callus was sub cultured on MS basal medium at fifteen days interval for a period of two months to achieve callus maturation. During callus differentiation the callus became smooth and turned into greyish brown or black progressively.

4.6.1. Somatic embryogenesis

The mean per cent of calli differentiation into somatic embryos of the various explants of Coker 310 ranged from 54.36 in shoot tip explant to 69.16 in leaf explant. The calli differentiation was the highest in hypocotyl explants (79.66) (Table 35).
**4.6.1.** **1..Plantlet regeneration**

Though callus induction, differentiation, and development of somatic embryos were observed in explants of Coker 310 they failed to regenerate into complete plantlet.

4.6.2. Organogenesis

The proliferated calli which failed to differentiate into somatic embryos were transferred to different media combinations for organogenesis. It included development of shoots and roots from the callus cultures in various growth regulator combinations

4.6.2.1. Shoot induction

All the eight media combinations for shoot induction were based on MS. Two levels of (1.0 mgl-1l and 2.0 mgl-1) of cytokinin (BAP) with auxins (IAA and NAA) (0.1 mgl-1and 0.5 mgl-1) and GA3 (1.0 and 2.0 mgl-1) were tried for shoot induction. The shoot induction was visible within 12-23 days of sub culturing. The MS basal medium without any growth regulator failed to induce shoots .However the calli remained green for a short duration (up to 10 days) and then dried (Plate 7).

4.6.2.1.1. Genotypes

The effect four different genotypes in the effect of mean induction of shoots ranged from 44.28 per cent in Coker 310 to 56.03 per cent in SVPR2 (Table 36).

Among the four genotypes the shoot induction per cent was the highest (74.33) in SVPR2 in media composition of MS with BAP (2.0 mgl-1) + IAA  
(0.5 mgl-1) + GA3 (1.0 mgl-1) + glucose (30gl-1), 66.00 per cent in Coker 310, 71.33 per cent in MCU12, and 73.50 per cent in MCU13 in MS and growth  
regulator combination of BAP (1.0 mgl-1) + IAA (0.1 mgl-1l) + GA3 (2.0 mgl-1) +  
glucose (30gl-1).
Table 36. Effect of different genotypes on shoot induction from callus cultures

Growth regulators(mgl-1) | Shoot induction (%)

---|---

BAP | IAA | GA3 | Coker 310 | SVPR2 | MCU12 | MCU13 | Mean

- | - | - | - | - | - | - | -

1.0 | - | 1.0 | 35.33

(36.46) | 32.66

(34.85) | 27.33

(31.51) | 26.00

(30.65) | 30.33

(33.41)

2.0 | - | 2.0 | 34.33

(35.86) | 25.66

(30.43) | 31.66

(34.24) | 31.66

(34.20) | 30.82

(33.72)

- | 0.1 | 1.0 | 55.66

(48.24) | 61.33

(51.54) | 53.33

(46.90) | 48.66

(44.23) | 54.74

(47.71)

1.0 | 0.1 | 2.0 | 66.00

(54.33) | 58.00

(49.60) | 71.33

(57.62) | 73.50

(66.03) | 69.70

(56.60)

2.0 | 0.5 | 1.0 | 46.66

(43.05) | 74.33

(59.55) | 52.33

(46.33) | 52.66

(46.52) | 56.49

(48.72)

1.0 | 0.5 | 2.0 | 35.33

(36.46) | 61.00

(51.35) | 51.00

(45.57) | 40.00

(39.23) | 49.33

(44.61)

2.0 | 0.5 | 1.0 | 34.33

(35.86) | 66.00

(54.33) | 41.33

(40.00) | 53.66

(47.09) | 48.83

(44.32)

Mean | 44.28 (41.71) | 56.03 (49.62) | 47.12

(43.34) | 48.35

(44.05) | 48.94

(44.39)

SE(d)

1.605 | CD (5%)

3.269 | CD (1%)

4.390

The values in parentheses are arc sine transformed values

4.6.2.1.2. Explants

The mean shoot induction per cent in different explants of four genotypes ranged from 58.96 in cotyledon explant to 61.57 in shoot tip (Table 37).

The hypocotyl explant recorded highest shoot induction per cent (73.99) in the media composition containing BAP (2.0 mgl-1) + IAA (0.5 mgl-1) \+ GA3 (1.0mgl-1).
Table.37.Effect of different explants on shoot induction from callus cultures

Growth regulators (mgl-1) | Shoot induction (%)

---|---

BAP | IAA | GA3 | hypocotyl | cotyledon | leaf | Shoot tip | Mean

- | - | - | - | - | - | - | -

1.0 | - | 1.0 | 47.33

(43.46) | 58.24

(49.74) | 53.41

(46.95) | 55.58

(48.20) | 53.64

(47.08)

2.0 | - | 2.0 | 69.33

(56.37) | 60.66

(51.15) | 53.33

(46.90) | 55.49

(48.15) | 59.70

(50.59)

- | 0.1 | 1.0 | 63.58

(52.87) | 59.91

(50.71) | 53.07

(46.76) | 63.74

(52.97) | 60.08

(50.81)

1.0 | 0.1 | 2.0 | 62.83

(52.43) | 58.83

(50.08) | 73.49

(59.01) | 64.91

(53.67) | 65.02

(53.74)

2.0 | 0.5 | 1.0 | 73.99

(59.33) | 53.33

(46.90) | 60.41

(51.00) | 59.91

(50.71) | 61.91

(51.89)

1.0 | 0.5 | 2.0 | 57.07

(49.06) | 58.58

(49.940 | 59.16

(50.27) | 65.41

(53.97) | 60.06

(50.80)

2.0 | 0.5 | 1.0 | 52.41

(46.38) | 60.24

(50.90) | 62.58

(52.28) | 64.33

(53.32) | 59.89

(50.70)

Mean | 58.98 (50.17) | 58.96 (50.16) | 60.00

(50.76) | 61.57

(51.68) | 60.72

(51.11)

The values in parentheses are arc sine transformed values

4.6.2.2. Shoot growth

The shoots were induced within 12 -23 days with a mean number of shoots ranging from 1-5 and length ranging from 3.45 to 8.95 cm. The number of days for optimum shoot induction was 14 days producing maximum number of 6 shoots of 8.95 cm length in growth regulator combination of BAP (1.0 mgl-1) + IAA (0.5 mgl-1) + GA3 (1.0 mgl-1) (Table 38).
Table.38.Effect of growth regulators on days to shoot induction, number of   
shoots and length of shoots

Growth regulators(mgl-1) | Days to shoot induction | Number of shoots | Average length of

shoots (cm)

---|---|---|---

BAP | IAA | GA3

- | - | - | - | - | -

1.0 | - | 1.0 | 23.00 | 1.30 | 3.45

2.0 | - | 1.0 | 21.00 | 2.20 | 3.75

- | 0.1 | 2.0 | 18.00 | 2.43 | 4.88

1.0 | 0.1 | 1.0 | 17.00 | 4.37 | 6.34

2.0 | 0.5 | 2.0 | 16.00 | 4.57 | 6.95

1.0 | 0.5 | 1.0 | 14.00 | 6.35 | 8.95

2.0 | - | 0.5 | 12.00 | 5.40 | 6.20

Mean | 17.00 | 4.08 | 5.33

4.6.2.3. Root induction

The roots were initiated at the basal portion of the shoots in the root induction medium. The time taken for root induction varied between 10-15 days and the roots gradually grew leading to complete development of plantlets (Plate 8).

4.6.2.3.1. Genotypes

The frequency of root induction of shoots derived from different explants of four genotypes ranged from 38.12 in SVPR2 and MCU12 to 47.12 in MCU13. The root induction per cent was highest (87.66) in SVPR2 in media combination of IBA (1.0mgl-1) and IAA (0.5mgl-1) .The number of roots ranged from 4 to 5 in all the genotypes and root length varied from 2.20 - 4.60cm (Table 39).
Table 39. Effect of different genotypes on root induction from callus cultures

Growth regulators(mgl-1) | Root induction (%)

---|---

IBA | IAA | Coker 310 | SVPR2 | MCU12 | MCU13 | Mean | No: of

Roots | Root length

(cm)

- | - | - | - | - | - | - | - | -

1.0 | - | 24.66

(29.77) | 19.66

(26.32) | 26.00

(30.65) | 64.33

(53.32) | 33.66

(35.46) | 2.45 | 2.78

2.0 | - | 20.33

(26.80) | 24.00

(29.33) | 33.66

(35.46) | 54.33

(35.86) | 33.08

(35.11) | 3.45 | 3.56

- | 0.1 | 37.33

(37.66) | 39.66

(39.03) | 38.00

(38.05) | 32.00

(34.44) | 36.75

(37.31) | 4.00 | 3.35

1.0 | 0.1 | 39.33

(38.83) | 44.00

(41.55) | 47.00

(43.28) | 77.33

(61.56) | 51.92

(34.40) | 2.75 | 2.59

2.0 | 0.5 | 19.00

(25.84) | 18.66

(25.59) | 13.66

(21.69) | 13.66

(21.69) | 16.25

(23.77) | 4.15 | 4.25

1.0 | 0.5 | 82.00

(68.86) | 87.66

(64.89) | 80.00

(63.43) | 67.33

(55.13) | 79.25

(62.90) | 4.60 | 5.00

0.5 | 2.0 | 68.66

(55.95) | 66.00

(54.33) | 52.66

(46.52) | 49.00

(44.42) | 59.08

(50.23) | 3.78 | 3.62

Mean | 38.70

(38.46) | 38.12

(38.12) | 38.12

(38.12) | 47.12

(43.34) | 40.52

(44.15) | 3.42 | 3.40

The values in parentheses are arc sine transformed values

4.6.2.3.2. Explants

The mean root induction per cent of shoots derived from different explants of the four genotypes ranged from 56.43 in shoot tip explant and 64.57 in leaf (Table40).

The highest root induction per cent was recorded in hypocotyl explant derived shoot (72.00) in the media combination with IBA (1.0mgl-1) and IAA (0.5 mgl-1) having maximum of five roots with root length of 5.65 cm.

Table.40. Effect of different explants on root induction from callus cultures

Growth regulators(mgl-1) | Root induction (%) | No: of

Roots | Average root length (cm)

---|---|---|---

IBA | IAA | Hypocotyl | cotyledon | leaf | Shoot tip | Mean

- | - | - | - | - | - | - | - | -

1.0 | - | 39.00

(38.64) | 55.00

(47.86) | 55.33

(48.06) | 55.33

(48.06) | 48.73

(44.27) | 2.25 | 3.21

2.0 | - | 55.33

(48.05) | 63.33

(52.73) | 62.00

(51.94) | 63.00

(52.53) | 59.80

(50.12) | 3.65 | 3.43

- | 0.1 | 62.00

(51.94) | 63.33

(52.73) | 65.33

(53.92) | 61.00

(51.35) | 62.73

(52.37) | 4.00 | 3.27

1.0 | 0.1 | 68.33

(55.75) | 62.00

(51.94 | 69.33

(56.37) | 66.00

(54.33) | 67.53

(55.26) | 3.25 | 2.85

2.0 | 0.5 | 62.33

(52.14) | 65.00

(53.72) | 66.00

(54.33) | 64.66

(53.52) | 64.06

(53.16) | 4.75 | 3.85

1.0 | 0.5 | 72.00

(58.05) | 70.33

(56.99) | 65.00

(53.72) | 62.66

(52.33) | 66.73

(54.77) | 5.60 | 5.65

2.0 | 0.5 | 40.66

(39.61) | 55.00

(47.86) | 69.00

(56.16) | 61.33

(51.54) | 53.33

(46.90) | 4.12 | 3.85

Mean | 57.09

(49.07) | 62.00

(51.94) | 64.57

(53.47) | 62.00

(51.94) | 56.43

(48.69) | 3.94 | 3.18

The values in parentheses are arc sine transformed values

4.6.2.4.. Plantlet regeneration

Among the four genotypes, only the callus cultures of three genotypes resulted into plantlets. The regeneration efficiency was more in SVPR2 (40.52) in hypocotyl explant derived calli and the lowest per cent (10.00) was observed in leaf explant derived calli of Coker 310(Table 41) (Plate 9).
**4.6.2.** **5. Hardening**

The hardening process involves the transfer of the _in vitro_ grown plantlets from aseptic cultured tailored environment to the free living environment of the mist chamber, net house and ultimately to the field conditions.

The rooted plants were removed from the culture tubes, washed thoroughly in running tap water to remove the agar which might be the potential source of contamination. Plantlets were then transplanted to the perforated pots containing sterilized mixture of establishment media. The plantlets were protected from desiccation in the mist chamber for two weeks.

The transfer efficiency was recorded to be the highest (35.48 per cent) in plantlets obtained from the hypocotyl explants of SVPR2 and lowest (12.00 per cent) in plantlets obtained from leaf explants of Coker 310 (Table 42).

After sufficient establishment in mist chamber, they were gradually exposed to the lower humidity and higher light intensity under net house environment and then to normal field condition. Watering was done based on the requirement.

Among the four genotypes two plantlets from the genotypes SVPR2 (33.00 per cent) and MCU12 (25 per cent) were established well in the field. They are now in flowering stage (Plate10).

Table.41. Plant regeneration efficiency of calli from different explants of  
four different genotypes.

Genotypes

Explants | Plant regeneration (%)

---|---

Coker 310 | SVPR2 | MCU12 | MCU13 | Mean

Hypocotyl | 25.00 | 40.52 | 36.00 | 23.45 | 42.38

Cotyledon | - | - | - | - | -

Leaf | 10.00 | 32.00 | 26.00 | 19.00 | 21.75

Shoot tip | - | - | - | - | -

Mean | 17.5 | 36.26 | 31.00 | 21.22 | 26.49

Table.42.Transfer efficiency of regenerated plants from different explants of four different genotypes.

Genotypes Explants | Transfer efficiency (%)

---|---

Coker 310 | SVPR2 | MCU12 | MCU13 | Mean

Hypocotyl | 28.00 | 35.48 | 29.62 | 20.50 | 28.40

Cotyledon | - | - | - | - | -

Leaf | 12.00 | 25.62 | 19.25 | 15.25 | 18.03

Shoot tip | - | - | - | - | -

Mean | 20.00 | 30.55 | 24.43 | 23.9 | 24.72

CHAPTER V

### DISCUSSION

Cotton, _Gossypuim_ spp., is the chief fibre crop of the world and is an important source of oil and high quality protein meal. Genetic engineering of cotton is prudent for at least the following three reasons.

  1. Intensive management of cotton, since it is susceptible to attack by several insect pests, nematodes and fungal pathogens. Engineering cotton to express anti-pathogen compounds would make cotton production more cost effective.

  2. Improvement of cotton by conventional plant breeding practices has limitations due to time consuming selection programs and lack of availability of wild relatives with desired agronomic and fiber traits. Most of the commercial varieties currently available have been produced by hand emasculation and crossing and recurrent selection in progeny rows, which requires approximately 6-7 years for production. Hybrid cotton can only be produced currently by hand emasculation and pollination due to lack of availability of methods for inducing male sterility and pollen restoration (Jenkins, 1993). The procedure is laborious and expensive; it is used on a limited scale in China and India.

  3. Cotton is primarily cultivated in arid regions with little or no irrigation; thus it is subjected to environment and other stress factors.

Hence, it is no surprise that cotton becomes one of the first crops to be genetically engineered for improved agronomic and fibre traits.

Within a decade, transgenic cotton varieties, such as insect-resistant and herbicide tolerant cottons, have become commercially available for cultivation. This remarkable achievement is largely due to improvements in recombinant DNA technologies and cotton tissue culture regeneration.

The production of genetically modified cotton has been one of the greatest success stories of plant biotechnology (Wilkins _et_ al., 2000).Genetically modified cotton, one of the first transgenic crops in commercial production, now accounts for the vast majority of cotton acreage in cotton producing countries around the world (Wilkins _et al._ , 2004).

Standardization of _in vitro_ techniques is an important preliminary step which will precede its application to crop improvement and commercial exploitation

**5.1.** _In vitro_ **regeneration of plants through somatic embryogenesis**

Several genotypes of cotton have been regenerated through somatic embryogenesis. Regeneration _via_ somatic embryogenesis is preferred over organogenesis because of probable single cell origin of the somatic embryos  
(Merke _et al_., 1995) thus reducing the chimeric transformation events.

Price and Smith (1979) published the first report of cotton somatic embryogenesis in the species _Gossypium klotzschianum_ although they were unable to obtain plants. Davidonis and Hamilton (1983) reported somatic embryogenesis in two years old callus cultures of _G.hirsutum_ var. Coker 310.

Though there are a few reports on transformation of Indian cotton cultivars through shoot apex cultivar (Satyavathi _et al_., 2002; Balasubramani _et al_., 2003; Sanjaya _et al_., 2005), no published reports are available on transformation and regeneration of Indian cultivars through somatic embryogenesis.

Therefore, the present investigation was aimed at developing simple, efficient and reproducible regeneration protocols through somatic embryogenesis in the genotypes of Coker 310, SVPR2, MCU12 and MCU13 using four different explants _viz._ , hypocotyl, cotyledon, leaf and shoot tip.

5.1.1. Seed germination

The acid delinted seeds of the four genotypes _viz_., Coker 310, SVPR2, MCU12, and MCU13 showed 90-95 per cent successful germination rate following 7 days of culture in half strength MS medium. The explants chosen for the study included hypocotyl, cotyledon, leaf and shoot tip isolated from the 7 days old seedling. It had already been established that younger explants exhibited greater morphogenic potential than older explants (Welander, 1988) as they might have more metabolically active cells with hormonal and nutritional conditions that were responsible for organogenesis (Famiani _et al.,_ 1994). Hence the explants were derived from seedlings for the study.

5.1.2. Surface sterilization

The explants were inoculated in various media combinations after following a set of sterilization treatments with various concentrations of sterilant at different durations of exposure.

General sterilization procedures were reported by many workers (Dodds and Robberts, 1982; Dixon, 1985).In this experiment, mercuric chloride was used as the sterilant with different concentrations.

Among the four genotypes used, the lowest contamination per cent of 34.49 was recorded in Coker 310, when the explants of Coker 310 were treated in 0.1 per cent mercuric chloride for 10 minutes. Among the four explants used, the lowest contamination per cent of 30.53 per cent was recorded in hypocotyl explants treated in 0.1 per cent mercuric chloride for 10 minutes

Survival per cent of explants was high when the exposure time was 10 minutes in 0.1per cent mercuric chloride. Among the four genotypes studied, the highest survival per cent of 80.49 was recorded in hypocotyl explants of Coker 310. Lowest survival in cotyledon was due to higher rate of contamination.

5.1.3. Callus induction

In the present study, MS medium with distinct differences in various compositions of hormones were used for callus induction. Callus induction was observed on all media compositions in all the four explants. Callus was initiated on MS medium (Murashige and Skoog, 1962) supplemented with different growth hormone combinations _viz_., 2, 4-D, IBA, IAA, Kinetin and BAP (Dixon,1985).

**5.1.3.1.** **Effect of Genotypes on callus induction**

Out of the four genotypes studied, the genotype Coker 310 showed maximum response for all the _in vitro_ studies _viz._ , callus induction, proliferation, shoot and root induction and growth, somatic embryogenesis and plantlet development. Coker varieties appeared to be superior to other varieties in their embryogenic response (Davidonis and Hamilton, 1983; Shoemaker _et al_., 1986). Several authors had reported that the capacity of regeneration was limited to Coker cultivars (Trolinder and Goodin, 1988 a, b; Trolinder and Xhixian, 1989; Zhang _et al_., 1993).

The highest calli induction per cent (86.00) was recorded in Coker 310 genotype followed by SVPR2 (76.74), MCU13 (73.49) and MCU12 (68.58).

5.1.3.2. Effect of explants on callus induction

Among the four explants of the genotypes used, the hypocotyl explants (82.49 per cent) produced more callus in the present study as reported by Trolinder and Goodin (1988a) and Sakhanokho _et al_.(1998,2001,2004).Among the four explants used, the hypocotyl explants of Coker 310 recorded highest callus induction per cent (86.00) after 7 to 9 days. Rapid callus development in hypocotyl tissue might shorten the culture duration, thus reducing the occurrence of somaclonal variation, a major problem in cotton tissue culture (Sakhanokho _et al_., 2004).

5.1.3.3. Effect of media composition on callus induction

Callus is an undifferentiated mass of proliferating parenchymatous cells. Using tissue culture techniques, callus can be induced in plant organs and tissues that usually develop callus in response to injury. It is known that the suitability of a particular medium for specific culture system depends not only on the mere presence of various nutrients but also on the actual and relative quantities of those nutrients.

The highest callus induction per cent was recorded in Coker 310 genotype (80.33) in media composition (MS4) containing MS + 2, 4-D (0.1mgl-1) +Kinetin (0.5 mgl-1) + maltose (30gl-1) among the four genotypes.

Among the four explants used, the highest callus induction per cent (82.49) was recorded in hypocotyl explants in the callus induction media MS 4 [MS + 2, 4-D (0.1mgl-1) + Kinetin (0.5 mgl-1) + maltose (30gl-1)].

Most of the published works have also reported MS based medium containing an auxin (NAA or 2,4-D) and a cytokinin (kinetin or 2-iP) is suitable for callus induction (Trolinder and Goodin,1987,1988a,b;Gawel and Robacker, 1990; Firoozabady and DeBoer,1993; Kumar _et al_., 1998; Sakhanokho _et al_.,1998;  
Nobre _et al_.,2001; Sunilkumar and Rathore,2001; Kumria _et al_.,2003).

5.1.3.4. Effect of plant growth regulators on callus induction

Hormones, in intact plants, act to regulate and co-ordinate processes which led to normal development, growth as well as differentiation of tissue and cells. The major growth regulators generally used in tissue culture include auxins and cytokinins. Auxins differ in their physiological activity and the extent to which they move within tissues or bound within cells or metabolized (George and Sherrington, 1993). Auxins and cytokinins alone and in combination could be the cause to promote callus induction, proliferation and accelerated growth. These hormones influence cell growth and duration of cell cycle which in turn is influenced by hormonal concentrations (King, 1980 a). The duration of cell cycle is influenced by hormone concentration (Baylise, 1977) and varies with cell types (King.1980). Wernicke and Brettel (1982) observed that longer persistence of proliferating activity around vascular bundles could be due to growth regulators and their transporting activity within the plant. Each explant, however, differed in the requirement of growth regulators in inducing callus and its growth.

A perusal of growth regulators involved for callus induction readily achieved in all growth levels in all the explant of four genotypes, but with varying frequency from treatment to treatment. The auxin, 2, 4-D at 0.1mgl-1 was found encouraging for the maximum callus induction and growth in all the explants. The addition of Kinetin along with 2, 4-D was also found to favour profuse callusing.

The superiority of the combination of 2, 4-D (0.1mgl-1) and Kinetin (0.5mgl-1) when compared to other treatments resulted in good callus induction efficiency which was evident from the highest per cent of callus induction (80.33 per cent), earliness in callus induction (7-9 days of inoculation) with calli characteristics of smooth, yellow and brown and friable .

The callus induced in 2, 4,-D containing had good subculturing (once in  
15 days) character such as friable and viable for subsequent subculturing and differentiation. These observations had indicated that callus growth hardly occurred in the absence of growth regulators. This is an agreement with the findings of  
Eskes _et al_. (1974); Stamp (1987) and other earlier workers, who reported that there was no callus growth in the basal media without growth regulators.

Mishra _et al._ (2003) reported that using two auxins, 2, 4,-D and NAA, generated good quality callus that supported somatic embryogenesis in Acala cultivars. Earlier studies by Finer (1988), Trolinder and Goodin (1987) and Katageri and Khadi (1998) indicated that the callus induction was more at lower concentrations of auxins and cytokinins.

5.1.3.5. Effect of carbon sources

The role of carbon sources on somatic embryogenesis, embryo maturation and organogenesis through calli had been investigated in several plant species including cotton. In the present study, maltose gave better mean values of callus induction in genotypes (61.28 per cent) and explants (63.28 per cent) compared to glucose. The earlier investigators reported regeneration of cotton plantlets on medium containing glucose (Trolinder and Goodin, 1987; Firoozabady et al., 1987; Rajasekaran, 1996; Kumar et al., 1998), maltose (Cousins et al., 1991; Kumria et al., 2003; Hussain  
et al., 2004; Sun et al., 2005a) and sucrose (Zhang and Li, 1992; GonzalezBenito  
et al., 1997; Zhang et al., 2000 ; Sakhanokho et al., 2004).

The positive effect of maltose as carbon source was also proved in a number of plant species. In Hordeum vulgare, somatic embryogenesis was initiated by replacing the sucrose with maltose (Scott and Lyne, 1994). Maltose as carbon source had been shown to be more efficiently utilized than glucose in rice (Ghosh-Biswas and Zapata, 1993). Maltose increased the number of developed somatic embryos and improved their morphology and viability in several other species (Kunitake et al., 1997; Norgaard, 1997; Blanc et al., 1999).

Browning of callus occurred after two or three subcultures was reduced by the addition of maltose to the callus induction medium. The per cent of non embryogenic calli formation was also reduced by maltose when compared to other carbon sources (Ganesan and Jayabalan, 2004)

5.1.4. Calli characteristics

Callus morphology is an important step in cotton tissue culture considering diverse nature of callus morphologies (Sakhanokho _et al_., 2001). Calli characteristics such as callus colour, texture, and friability play a major role in the successful regeneration of cotton through somatic embryogenesis (Mishra _et al.,_ 2003).The calli types are distinguished based on their physical appearance (Shoemaker _et al_., 1986).Trolinder and Goodin (1987) reported that callus morphology differed with growth regulator combinations.

The calli characteristic in the present study depended on the media compositions and the explants used as reported by Nobre _et al_ (2001). The calli obtained from the hormonal combination of 2, 4-D (0.1mgl-1) \+ Kinetin (0.5mgl-1) within 7-9 days after inoculation of the explants were yellow and brown coloured, smooth and highly friable as reported by Gawel _et al_.(1986),Shoemaker _et al_.(1986), and Firoozabady _et al_.(1987).

The calli obtained from 2, 4-D and NAA was light green, compact, less friable and the callus growth was slow as reported by Nobre _et al_. (2001).
5.1.5. Calli proliferation

In the present study, it was observed that the co-existence of embryogenic and non-embryogenic calli significantly affected the embryogenic nature of the calli by rendering them into non-embryogenic. During subculturng, the non-embryogenic calli were removed from the cultures and only embryogenic calli were allowed to proliferate. Failure to separate embryogenic calli from non-embryogenic ones often resulted in the embryogenic calli turning into non-embryogenic calli (Sakhanokho et a/., 2001).

Callus proliferation was observed both on semi solid and liquid medium. The slurry of cells formed in the liquid cultures had more ready access to nutrients and were exposed to a more gradual pH change during culture time than those on semi-solid medium. Furthermore, any toxic metabolites that might have been produced by tissue would have been diluted by the liquid medium and endogenous hormones may have leached more quickly when proliferated in liquid medium (Gawel and  
Robacker, 1990).

The results indicated that the liquid medium recorded highest proliferation (76.33 per cent) in Coker 310 than solid medium (74.63 per cent) in the same genotype. Similar results were also obtained by Gawel and Robacker (1990) in cotton. Due to the high medium to tissue contact in the liquid culture systems, media effects were rapid and embryo development could be better controlled than with solid-support system (Finer, 1988). Many plant species exhibited improved performance when cultured on liquid based medium as compared to solid medium.

For example, when cultured in liquid media, Triticum aestivum yielded more somatic embryos (Jones and Petolino, 1988) and had higher somatic embryo germination (Wei, 1982). Zouine et al. (2005) reported that the production of somatic embryos in Phoenix dactylifera was high in liquid media than on solid media.
5.1.6. Calli differentiation

There are two ways to regenerate plants from callus, through organogenesis and initiation of shoots and roots by somatic embryos (Murashige, 1976 b).

Regeneration of plants from cultured cells could occur through somatic embryogenesis and had been reported for over 300 plants _in vitro_ (Tisserat, 1976). Somatic embryos which often lack a suspensor are morphologically indistinguishable from sexual embryos. The responses of different explants _viz_., hypocotyl, cotyledon, leaf and shoot lip were tried for somatic embryogenesis. The response could be obtained from all explants. Calli, after maturation, were cultured on calli differentiation media to induce somatic embryos.

In the present study, though callus induction was found in all explants of the four genotypes, the calli differentiation into somatic embryos were observed from callus induced from Coker 310 only.

The calli differentiation was highest in hypocotyl explants (79.66 per cent) in MS + IAA (0.5mgl-1) + BAP (1.0mgl-1) as reported by as reported by Zhang et al. (1999, 2001). Sakhanokho et al. (2004) observed that more somatic embryos were consistently induced from hypocotyl-derived callus than cotyledon explants in Pee Dee and Georgia lines.It was observed that full strength MS salts produced higher somatic embryogenic response than half strength MS salts (Kumria et al., 2003).

However, plant growth regulators such as zeatin (Zhang et al., 2000; Zhang et al., 2001), a combination of NAA with Kinetin (Sakhanokho et al., 2001), a combination of picloram and 2ip (Ganesan and Jayabalan, 2004) and a combination of BAP and Kinetin (Aydin et al., 2004) have also been used to induce callus which exhibit embryogenic response later in appropriate medium.
5.1.7. Plantlet regeneration

Though callus induction in different explants of four genotypes and differentiation for somatic embryos in Coker 310 were observed, they failed to regenerate into complete plantlet except in Coker 310 as reported by Trolinder and Goodin (1988b), Voo et al., (1991), Zhang et al. (1991), Firoozabady and DeBoer (1993), Zhang et al. (2000) Sakhanokho et al. (2001) Kumria et al. (2003),  
Hussain et al. (2004) and Ganesan and Jayabalan (2004).

Although more than 70 genotypes other than Coker cultivars have now been identified to be capable of differentiating from callus into somatic embryos (Gawel and Robacker, 1990; Kumar and Pental 1998; Zhang _et al_., 1999, Zhang _et al_ ., 2001; Sakhanokho _et al_., 2001; Mishra _et al_., 2003; Wu _et al_ ., 2004; Ganesan and Jayabalan, 2004; Sakhanokho _et al_ ., 2004; Ayidin _et al_., 2004; Hussain _et al_., 2004), only a few have been successfully regenerated into mature plants.

**5.2** _. In vitro_ **regeneration of plants from callus cultures through organogenesis**

If the plant cells are totipotent, that is capable of regenerating a whole plant from a single cell or a small group of cells, then their ability to undergo organogenesis depends on their differentiation to meristematic cells and then their further differentiation to specialized cells.

5.2.1. Shoot induction

Regeneration of shoots is generally accomplished in the presence of light and in a culture medium of high osmolarity containing chelating agents (Murashige, 1977). An exogenous supply of carbohydrates is usually necessary for shoot initiation (Thorpe and Murashige, 1968). The maintenance of shoot cultures usually requires the presence of exogenous cytokinins as well as auxins. (Murashige, 1977 b).The single most important factor determining organ formation in tissue culture is the relative quantities of auxin and cytokinin (Skoog and Miller, 1957). As stated by Fujimora and Komamine (1980) cytokinin in combination with an auxin appears essential for the onset of the growth and induction of organogenesis. For induction of multiple shoots earlier workers had used Murashige and Skoog's (1972) medium having high mineral salt concentrations which was often modified by the addition of nutrients of the growth regulators like 2, 4-D, IAA, IBA, NAA, BAP or Kinetin

Since the MS basal medium only favoured callus induction and proliferation, in the present study the eight media combinations for shoot induction were based on MS only. Two levels of (1.0 mgl-1l and 2.0 mgl-1) of cytokinin (BAP) with auxins (IAA and NAA) (0.1 mgl-1and 0.5 mgl-1) and GA3 (1.0 and 2.0 mgl-1) were tried for shoot induction.. The MS basal medium without any growth regulator failed to induce shoots .However the calli remained green for a short duration (up to 10 days) and then dried.

Among the four genotypes, the highest shoot induction per cent was 74.33 in SVPR2 in media composition with BAP (2.0 mgl-1) + IAA (0.5 mgl-1) + GA3 (1.0 mgl-1) with glucose (30gl-1).These results are in corroboration with the observations of Stephen _et al_. (1997).

In the present study, the IAA would have enhanced the action of BAP. Further, IAA induced the sustained growth of multiple shoots. However, it was likely that the synergistic action of IAA with BAP also might be the plausible cause for the sustained shoot growth.

The shoots were induced within 12 -23 days with 1-5 numbers of shoots and average length of shoots ranging from 3.45 to 8.95cm. The number of days for optimum shoot induction was 14 days producing maximum number of (6) shoots with a shoot length of 8.95 cm in growth regulator combination of BAP (1.0 mgl-1) + IAA (0.5 mgl-1) + GA3 (1.0 mgl-1).

It was seen that GA3 when fortified along with BAP and IAA resulted in marginal increase of shoot production and shoot length. The stimulatory effect of GA3 on the promotion of shoot growth in these genotypes is understandable because it is well known that GA3 stimulates cell division and elongation simultaneously at the sub apical zone of the shoots which ultimately resulted in effective shoot proliferation and shoot elongation (Sache, 1961).

In the present study calli cultured on shoot induction medium in response to IAA in combination with BAP induced greenish protrubences at the end of two weeks. After four weeks they were sub cultured on the same medium for further development into the shoots.

**5.2.2. Rooting of** _in vitro_ **derived shoots**

The shootlets obtained from _in vitr_ o culture need to be transferred to a medium for proper root deployment before they are transferred to hardening medium.

In the present investigation MS basal medium at full and half strength along with IAA, IBA and NAA at (0.5mgl-1) and (1.0mgl-1) individually and in combination for rooting of _in vitro_ derived multiple shoots was studied..

The root induction per cent was maximum (87.66) in Coker 310 in auxin combination of IBA (1.0mgl-1) and IAA (0.5mgl-1) .The number of roots ranged from 4 to 5 in all the genotypes and root length varied from 2.20-4.60cm.

The shoots after root induction were hardened and established in pots. It was seen that gradual acclimatization with decreased humidity was required for plant survival during transition from culture vessels to pots and then to the field as reported by Sommer _et al_. (1986).

**5.3. Transfer technology for** _in vitro_ **derived plants**

Many valuable propagules can be lost if care is not taken when transferring them to soil. The change from the heterotrophic to autotrophic state has to be some what gentle. A good root system should help propagules to withstand some moisture stress or establishment shock. Placing the small plants in shaded green house and under an intermittent mist spray can reduce excessive transpiration (Staba, 1980).

Sterilizing the soil in an autoclave can reduce pathogens, particularly those which cause damping off. Soil sterilized in an autoclave should be allowed to remain covered, at room temperature, for1 to 2 weeks prior to use. The plantlets transferred to the paper cups containing sterilized soil mixture covered with perforated polythene bags and kept under shaded net house showed better establishment and acclimatized successfully.

Among the four genotypes, only the callus cultures of three genotypes resulted into plantlets. The regeneration efficiency was more in SVPR2 (40.52) in hypocotyl explants and the lowest per cent (10.00) was observed in leaf explants of Coker 310.

The main problem associated with transplanting of tissue cultured plants might be, because of their proneness to desiccation due to poor development of surface wax on the leaves, an impaired photosynthetic apparatus, leaves formed _in vitro_ are not well adopted, they are thin, soft and photosynthetically less active, either absence of root hairs or unutility, unsuitability of roots and absence of sterile conditions and one or more of their physiological aspects being ill-developed are most likely to be responsible for slow growth and poor establishment of tissue cultured plants as reported by George and Sherrington (1993) and Prakash and Pierik (1993).

The regenerated plantlets produced 4-6 leaves and sufficient root system within 8 weeks period. They were maintained in the culture room for about 15 days to allow for gradual acclimatization before their transfer to the greenhouse for seed production. Among the four genotypes two plantlets from the genotypes SVPR2 (33.00 per cent) and MCU12 (25 per cent) established well in the field. The regenerated plants showed normal growth, morphology, flowering and boll setting as compared to seed-derived cotton plants. Some investigators have reported the presence of aberrant phenotypes and sterility in the plants regenerated in vitro (Trolinder and Goodin, 1988b; Stelly et al., 1989).

The complete protocol optimized in the present study for successful in vitro regeneration of cotton genotypes through callus culture is presented. With this protocol, the period from callus initiation to plantlet development will be about 7 months and field transfer of the plantlets could be accomplished in an additional period of 2-3 months. Earlier workers had also suggested 9-10 months duration for establishment of plants from tissue culture (Firoozabady and DeBoer, 1993; Kumar and Pental, 1998; Sunilkumar and Rathore, 2001: Sakhanokho et al., 2001;  
Mishra et al., 2003; Wilkins et al., 2004) (Figure 10).

Flow chart for plantlet regeneration through callus culture in cotton

**Explant Preparation** (7 days old seedling)

(Hypocotyl, Cotyledon, Leaf and Shoot tip)

  (7-10 days)

Callus induction

[MS + 2, 4-D (0.1mgl-1) + Kinetin (0.5 mgl-1) + maltose (30gl-1)]

  (25days)

Callus proliferation

[MS + 2, 4-D (0.1mgl-1) + Kinetin (0.1 mgl-1) + maltose (30gl-1)]

  (21-25days)

Callus subculture

  (2months, 4 subcultures once in 15 days)

Callus maturation

  (60days)

Shoot induction

  [MS+ BAP (2.0 mgl-1) + IAA (0.5 mgl-1) + GA3 (1.0 mgl-1) + glucose (30gl-1)]

(15-20 days)

Root induction

[MS+ IBA (1.0mgl-1) +IAA (0.5mgl-1) + glucose (30gl-1)]

  (15 days)

T ransfer

(15 days)

N et house/mist chamber

(15 days)

**Field condition** (9-10 months)
CHAPTER VI

### SUMMARY

Investigations were undertaken to develop an efficient protocol for regeneration of cotton genotypes _viz.,_ Coker 310, SVPR2, MCU12, MCU13 through somatic embryogenesis, The present study was undertaken in Tissue Culture Laboratory, Centre for Plant Breeding and Genetics, Agricultural College and Research Institute, TNAU, Coimbatore.

6.1. Seed germination

The acid delinted seeds showed 90-95 per cent successful germination rate from all the four genotypes following 7 days of culture in half strength MS medium.

6.2. Preparation of explants

The seven days old seedlings were chosen for preparation of explants. The explants chosen for the study included hypocotyl, cotyledon, leaf and shoot tip.

6.3. Sterilization

Among the five explants of four genotypes subjected to different sterilization treatments, the hypocotyl explants of the genotype Coker 310 recorded the lowest contamination percentage and as a result the highest survival percentage. The individual interaction effects between the concentration of mercuric chloride, duration of exposure and the explants were also significant.

6.4. Callus induction

The four explants from seven days old seedlings of the four genotypes of cotton were inoculated in MS medium containing different growth regulator combinations for callus induction. The calli characteristics such as callus colour, texture, friability, days taken for callus induction in different growth regulator combinations were observed

The highest callus induction percentage was recorded in hypocotyl explants of Coker 310 genotype (80.33 per cent) in media composition (MS 4) containing MS + 2, 4 D (0.1mgl-1) + Kinetin (0.5 mgl-1) + maltose (30gl-1). The calli so obtained was yellow and brown coloured, smooth and highly friable and took 7-9 days for induction and was the best among the calli obtained from other hormonal combinations.

6.5. Callus proliferation and maintenance

The callus induction medium that responded well was chosen for proliferation in both liquid and semi solid medium. The callus proliferation followed similar trend of callus induction for the source of variations _viz.,_ genotypes, explants and hormones.

The efficiency of liquid medium over solid medium was compared for its proliferation. The 21 days old calli after induction was transferred to both semi solid medium and liquid medium for proliferation and maintenance. The highest callus proliferation percentage (61.15) was recorded in hypocotyl explants of Coker 310 in liquid medium and it was only 58.27 in semi solid medium.

Callus sub culturing in semi solid medium was done once in 5 days in semi solid medium and once in 10 days in liquid medium for callus growth and maintenance.

6.6. Callus differentiation

Of the four genotypes cultured only Coker 310 induced somatic embryos and mature further. The calli differentiation was the highest in hypocotyl explants (79.66) of Coker 310 but failed to regenerate into complete plantlet

6.7. Organogenesis

The proliferated calli which failed to differentiate into somatic embryos were transferred to different media combinations for development of shoots and roots from the callus cultures

Among the four genotypes used, the shoot induction percentage was the highest (74.33) in explants of SVPR2 in media composition of MS with BAP  
(2.0 mgl-1) + IAA (0.5 mgl-1) + GA3 (1.0 mgl-1) + glucose (30gl-1) taking 14 days for producing maximum number of 6 shoots of 8.95 cm length.

The root induction percentage was highest (87.66) in SVPR2 in media combination of IBA (1.0mgl-1) and IAA (0.5mgl-1) having 4 to 5 roots of 2.20 - 4.60cm average root length.

**6.8. Plantlet regeneration** **and Hardening**

The regeneration efficiency of SVPR2 (40.52) in hypocotyl explant derived calli was highest among the four genotypes.

The transfer efficiency was recorded to be the highest (35.48 per cent) in plantlets obtained from the hypocotyl explants of SVPR2.

The rooted plants from the culture tubes were transferred to the perforated pots and were protected from desiccation in the mist chamber for two weeks. They were gradually exposed to the lower humidity and higher light intensity under net house environment and then to normal field condition.

Among the four genotypes two plantlets from the genotypes SVPR2  
(33.00 per cent) and MCU12 (25 per cent) were established well in the field and they are at square setting stage.

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