GENETIC TRANSFORMATION STUDIES IN COTTON

Thesis submitted to the University of Agricultural Sciences, Dharwad in partial fulfilment of the requirements for the Degree of

Master of Science (Agriculture) in GENETICS AND PLANT BREEDING

By SUMITHRA S.

DEPARTMENT OF GENETICS AND PLANT BREEDING COLLEGE OF AGRICULTURE, DHARWAD UNIVERSITY OF AGRICULTURAL SCIENCES, DHARWAD - 580 005 DECEMBER, 2008

ADVISORY COMMITTEE

DHARWAD DECEMBER, 2008 Approved by : Chairman :

(I. S. KATAGERI) MAJOR ADVISOR ____________________________ (I. S. KATAGERI) 1. __________________________ (P. M. SALIMATH) 2. __________________________ (H. M. VAMADEVAIAH) 3. __________________________ (S. N. CHATTANNAVAR) 4. __________________________ (S. B. PATIL) 5. __________________________ (P. U. KRISHNARAJ)

Members :

CONTENTS
Sl. No. CERTIFICATE ACKNOWLEDGEMENT LIST OF ABBREVATIONS LIST OF TABLES LIST OF FIGURES LIST OF PLATES LIST OF APPENDICES 1 2 INTRODUCTION REVIEW OF LITERATURE 2.1 Transformation studies 2.2 Selection of transformed tissues/plants and confirmation 3 MATERIAL AND METHODS 3.1 Experimental materials 3.2 Experimental conditions 3.3 Methodology 3.4 Transformation and plant regeneration 3.5 Confirmation of presence of transgene 3.6 Studies on induction of axillary shoots 3.7 Statistical procedures and analysis 3.8 Histological studies 4 EXPERIMENTAL RESULTS 4.1 In vitro transformation studies 4.2 In vivo transformation studies 4.3 Induction of axillary shoots 4.4 Determination of kanamycin sensitivity 4.5 Histological studies Chapter Particulars Page No. iii v ix x xi xii xiii 1-4 5-32 5 19 33-47 33 33 35 38 41 44 44 44 48-77 48 61 64 68 71

Contd

Sl. No. 5 DISCUSSION

Chapter Particulars

Page No. 78-93 80 81 88 89 92 93 94-96 97-111 112-113

5.1 In vitro transformation studies 5.2 In vivo transformation studies 5.3 Studies on induction of axillary shoots 5.4 Screening of transformants 5.5 Confirmation of transgene 5.6 Future areas of research 6 SUMMARY AND CONCLUSIONS REFERENCES APPENDICES

LIST OF ABBREVIATIONS

MS PGRs PEG BAP TDZ NAA Kn SAM SDDW YEMA EC ELISA GUS DNA RNA OD mg/l CaCl2

Murashige and Skoog medium (1962) Plant growth regulators Polyethylene glycol 6-benzyl amino purine Thidiazuron Naphthlene acetic acid Kanamycin Shoot apical meristem Sterile double distilled water Yeast extract mannitol agar Embryogenic callus Enzyme linked immuno sorbant assay Glucuronidase Deoxy ribonucleic acid Ribonucleic acid Optical density milligram per litre Calcium chloride

LIST OF TABLES

Table No. 1 2 3 4 5 6 7 8 9 10 11 12

Title Effect of explant preculture on regeneration after colonization and cocultivation Effect of Agrobacterium colonization period on the regeneration of two cotton genotypes Effect of cocultivation duration on regeneration of two genotypes of cotton Effect of scalpel wounding and colonization duration on regeneration of two genotypes of cotton Influence of vacuum infiltration duration on regeneration of two cotton genotypes Effect of blot drying or dessication on the regeneration of genotypes Effect of chilling on regeneration of two cotton genotypes Effect of Sand injury on regeneration of cotton genotypes Effect of scalpel wounding and colonization of Agrobacterium on plant proliferation of Jayadhar genotype in MS media Effect of scalpel wounding at the shoot apex region and colonization of Agrobacterium on regeneration of two cotton genotypes in pots Effect of removal of apical meristem of shoot apex by giving different types of cuts on regeneration Effect of removal of apical dominance of seedlings and grown in MS broth supplemented with PGRs on induction of axillary in Jayadhar genotype Effect of removal of apical meristem of seedlings and grown in MS broth supplemented with PGRs on induction of axillary shoots in Surabhi genotype Effect of different concentration of Kanamycin on the explants of cotton Transformation studies in Jayadhar genotype Transformation studies in Surabhi genotype Gene integration studies in T0 (putative) transgenic plants cotton

Page No. 49 51 52 54 56 57 59 60 62 63 65 66

13

67

14 15 16 17

69 75 76 77

LIST OF FIGURES

Figure No.

Title

Page No.

Map of the cry2Aa gene construct in Ti plasmid

37

LIST OF PLATES

Plate No.

Title

Page No.

Kanamycin sensitivity test

70

2a

Amplification of cry2Aa transformed plants of Jayadhar and Surabhi genotypes

72

2b

nptII amplification of cry2Aa transformed plants of Jayadhar and Surabhi genotypes

72

Explant preparation for induction of axillary shoots in Jayadhar and Surabhi

73

Axillary bud induction in Surabhi and Jayadhar

74

LIST OF APPENDICES

Appendix No.

Title

Page No.

YEMA medium (Yeast Extract Mannitol Agar)

112

II

Murashige and Skoog medium

112

III

Loading dye (6x) and TAE tris Acetate 50x

113

IV

DNA extraction buffer and PCR Requirements

113

1. INTRODUCTION
Cotton the silver fibre has been the principal commercial and industrial crop of India, providing 65-70 per cent of the raw material for the textile industry of the country. This crop plays a crucial role in the global economy as well as social and industrial infrastructure. Besides being the backbone of the textile industry, cotton and its byproducts are also part of the livestock food, seed-oil, fertilizers, paper and other consumer products. India has the distinction of having the largest cotton area of 9.2 million hectares in the world across ten major states, with a production of 322 million bales. Thus, the productivity is estimated to be 591 kg lint/ha (Anon., 2008). India is the third largest cotton producer in the world next to USA and China. Karnataka produces 10 lakh bales of cotton lint from an area of 3.35 lakh hectares with a productivity of 507 kg/ha (Anon., 2008). The Genus Gossypium comprises 50 species, four of which are cultivated. G. arboreaum L. and G. herbaceum L. are diploid (2n=26), while G. barbadense L. and the most widely grown species G. hirsutum L. are tetraploid with 2n=52. G. hirsutum (or) upland cotton is grown in more than 95 per cent of the world wide acreage; the next important cultivar is G. barbadense (or) the long staple Egyptian cotton (Kumaria et al., 2003). There are certain bottlenecks affecting the productivity of cotton, which would as well be described as the problems of Indian agriculture in general. Almost 65 per cent of the area under cotton is rainfed with erratic and poorly distributed rain during the cropping season. Cotton crop is subjected to severe attack of pests and diseases, which cause substantial yield losses. About 162 species of insects are known to attack cotton crop at various stages of growth, 15 of which are important. Several studies have shown that American bollworm, Helicoverpa armigera (Hubner) is the most prevalent and damaging pest of cotton. Other lepidopteron pests like pink bollworm, Pectinophora gossypilla (Saunders) and spotted bollworm Erias vitella (F.) also cause extensive damage. H. armigera, being a polyphagous insect is present throughout the year in the field. Indiscriminate use of pesticide to control Helicoverpa armigera has led to series of consequences like, insecticide resistance, pest resurgence, outbreak of secondary pests, harmful residual effects, imbalances in natural ecosystem and higher production costs, which have been a concern in India and elsewhere. It is therefore necessary to develop more environment friendly approaches with minimum use of chemical pesticides. Development of resistant varieties is one such strategy. Certain morphological features can serve as a hindrance to the easy multiplication of pests. Genetic control of desirable features has been established. Some of which provide a strong defense, while others may help in minimizing the multiplication of pests. The conflicting plant features desired for resistance to bollworms and sucking pests make it difficult to obtain pest resistant genotypes in cotton. Increasing the density of trichomes will increase the tolerance to jassids (Tidke and Sane, 1962; Yadav et al., 1967) but make the plants more susceptible to bollworms as it attracts female moths for egg laying (Kadapa et al., 1983). There being no source of resistance available against bollworms, the breeder has to develop genotypes that are early with synchronous flowering and fruiting to mitigate the losses due to the pest by escape mechanism. Some morphological characters viz., frego bract, coloured plant body, nectarilessness, thickness of bollrind and bio-chemical factors are said to confer tolerance to bollworms, but attempts to obtain pest resistant genotypes of cotton by conventional breeding methods have not been successful because of limited genetic variability and incompatibility associated with pest resistant wild species. Genetically engineered resistance has been actively investigated in recent years as an alternative for genes from wild relatives. Tools of genetic engineering have however made it possible to transfer genes from even unrelated sources such as microbes. Engineering of plant species to acquire novel traits involves the introduction of foreign genes into plant genome and expression of these transgenes to the desirable extent. It encompasses the techniques to transfer the genes to plant chromosomes and regulate their expression by employing suitable promoters. Various methods have been devised to introduce foreign genes into plants.

Bacillus thuringiensis (Bt) is a gram positive, aerobic, sporulating bacterium, which synthesise crystalline proteins which are highly insecticidal even at very low doses. These proteins called -endotoxins are highly toxic to lepidopteron, dipteran and coleopteran insects. With the advent of genetic engineering techniques, it is now possible to transfer useful genes from other species or organisms. Genes responsible for these crystalline proteins (cry) have been identified and cloned to transfer into plants. These proteins are harmless to human beings, other vertebrates and also to non-target insects. A number of methods such as microprojectile bombardment, electroportation, sonication, Agrobacterium mediated transfer, etc. are available to transfer desirable foreign genes into plants of these, the most widely used method for genetic transformation in plants is the one based on Agrobacterium tumefaciens. Agrobacterium tumefaciens, a gram negative soil dwelling micro-organism that causes crown gall disease in many species of dicotyledonous plants with its disarmed Ti plasmid can be used as a vector to transfer genes into plant cells without altering their capacity to regenerate whole plant in vitro. Use of Agrobacterium as a vector is technically simple and gene transfers are often low copy, permanent and heritable. Introduction of foreign genes in elite genotypes is limited by the genotype specific nature of gene transfer in cotton. Cocker genotypes, which are amenable for regeneration in vitro by somatic embryogenesis, are widely used in genetic transformation experiments (Firoozabady et al., 1987; Umbeck et al., 1987 and Finer and Mcmullen et al., 1990). However, alternate procedures to transform non-cocker genotypes have been reported (Gould and Cedeno, 1998, Zapata et al., 1999, Satyavathi et al., 2002). Katageri et al. (2007) reported successful introduction of Bt-cry1Ac gene in an elite Indian genotype of cotton (Bikaneri nerma) following a modified shoot apical meristem procedure with an efficiency of 0.2 per cent. In many cases, the production of transgenic plants is prevented due to inability to regenerate plants from those tissues susceptible to transformation. In view of the above, current investigations were aimed at developing a protocol to increase the regeneration and transformation efficiency with the following objectives, Standardization of protocol to enhance genetic transformation efficiency in cotton Transfer of cry2Aa gene to cotton

2. REVIEW OF LITERATURE
Genetic transformation systems have been described for many plant species. The technology involves the delivery of defined foreign genes into plant cells, obtaining integration of the genes into plant genomes and observing expression of the genes in the regenerated plants. This successful application of plant transformation technology is due to advances in rDNA technology and the development of techniques which allow plant regeneration from cells or tissues. Genes are introduced either with the help of Agrobacterium tumefaciens or through direct delivery of DNA (Davey et al., 1989). However, the most popular and efficient method in dicotyledonous plants is Agrobacterium mediated transformation. Relevant literature on the previous attempts that have been made to transform cotton and other related dicot species are reviewed in this chapter.

2.1 TRANSFORMATION STUDIES

The discovery of gene transfer mechanism between Agrobacterium and the plant kingdom (Chilton et al., 1977) has led to its use of facilitating the insertion of desirable genes into the plant genome, irrespective of the origin of these genes. Transformation systems are now available for a large number of plant species and examples of transgenic plant species for pests, viral, fungal, herbicide resistance and environmental stress tolerance exist. Several procedures have been reported to accomplish gene transfer which can be achieved mainly through two broad methods, direct DNA transfer and vector mediated transfer.

2.1.1 Direct DNA transformation

Direct delivery of free DNA molecules into plant protoplast has been well documented by Davey et al. (1989). It is applicable to both stable and transient gene expression studies and utilizes a range of vectors. Physical as well as chemical methods have been developed to facilitate DNA delivery across the plasma membrane. Although the frequency of stable transformation is low, direct DNA uptake is applicable to those plants not amenable to Agrobacterium tumefaciens transformation, particularly monocotyledons. The choice of transformation method is dependent on a number of factors, such as (1) The efficiency of DNA delivery (2) Subsequent protoplast survival (3) Ease with which these can be handled Direct uptake of DNA by protoplast a. Electroporation The naked DNA molecules can be introduced into protoplast by electrically induced changes in the membrane permeability. This has been used successfully for transformation of a wide range of crop species (Christou et al., 1987; Fromm et al., 1985). Shillito et al. (1985) has reported highest plant transformation efficiencies in tobacco with 0.2 per cent of electroporated leaf mesophyll protoplast which gave rise to transgenic, regenerative competent cell. b. PEG mediated transformation This involved use of chemicals like PEG and divalent cations designed to increase the membrane permeability in freshly isolated protoplasts to DNA. PEG mediated DNA uptake typically transforms 0.1 per cent 0.4 per cent of the total protoplast treated (Hayashimoto and Murai, 1990). Absolute protoplast transformation efficiencies ranging from 0.7-1.0 per cent have been achieved with tobacco protoplasts electroporated in presence of PEG (Negrutiu et al., 1987).

Mechanical transformation a. Microinjection This involves immobilization of protoplast and microinjecting DNA directly into the nucleus. Transformation efficiencies have ranged from 12-66 per cent (Miki et al., 1987) with the highest efficiency being obtained in tobacco. Toyoda et al. (1988) have obtained Kanamycin resistant callus clones of tomato, one month after injection, from single cells whose nuclei were microinjected with nptII (Neomycin Phosphotransferase II) DNA fragment of the PE2KX plasmid. b. Sonication Mild sonication (20 KHz ultrasound) has been used to facilitate the uptake of CAT gene in tobacco. Upto 81 per cent of maximum transient expression was achieved with no significant loss of viability (Joersbo and Brunstedt, 1990). c. Particle bombardment Another method of physical transfer of DNA is through biolistics. Klein et al. (1985) reported a method where in vitro cultured cells were bombarded with tungsten particles coated with DNA. Microprojectile mediated transformation of pre-existing meristem, followed by micropropagation can also be employed as it has several advantages over de novo organogenesis. It can be used in species where de novo organogenesis does not occur, or it is very difficult to achieve. Further, it is less subject to somaclonal variation and less time consuming than regeneration from callus (Christou et al., 1990). Transgenic tobacco carrying coding sequences for npt II and gus were produced following micro-projectile bombardment of tobacco leaves and subsequent culture (Tomes et al., 1990). Interestingly, micro projectile bombardment of plant tissues has been used, to increase transformation frequency by Agrobacterium tumefaciens (Bidney et al., 1992). Cotyledons of immature peanut (Arachis hypogea L.) and zygotic embryos were transformed via particle bombardment with a plasmid containing a chimeric hph gene confering resistance to hygromycin. Under continuous selection, hygromycin resistant plantlets were regenerated from somatic embryos and were recorded from nearly 1.6 per cent of bombarded cotyledons (Deng Xiang Yang et al., 2001).

2.1.2 Agrobacterium mediated DNA transfer

Agrobacterium mediated DNA transfer is the most common and widely used method for the transformation of dicotyledonous plants. The first transgenic plants of Nicotiana tabaccum were produced via Agrobacterium mediated transformation (Horsch et al., 1984). With this success, many crop plants were transformed via Agrobacterium. This is the most simple and widely used method now available for transferring genes into intact plant tissue (Horsch et al., 1985) with host range being its limitation. Agrobacterium tumefaciens is a gram negative bacteria and an useful plant pathogen which induces crown gall disease in plants by transferring a discrete portion of its plasmid DNA (Binns and Thomashow, 1988, Klee and Rogers, 1987, Zambrysky, 1992). Crown gall occurs when the bacteria invade through the wound and in the stem allows A. tumefaciens bacteria to invade the plant and cause cancerous proliferation of cells. Two important regions are essential for transformation : oncogenic T-DNA and the virulence (vir) genes which encode proteins for T-DNA transfer. T-DNA transfer is dependent on direct repeat flanking sequence of 25 bp. The T-DNA is capable of inducing tumour in a transformed plant, accomplished by products of three genes which codes for auxins (Shroeder et al., 1984 and Thomashow et al., 1984) and cytokinin (Akiyoski et al., 1984). Agrobacterium tumefaciens plasmid transfers only T-DNA. In principle, it is possible to intentionally introduce any gene into plant genome (Zambrysky et al., 1983). The oncogenic T-DNA region is deleted from Agobacterium strain in order to prevent the over production of phytohormones which interferes with normal plant development. The disarmed strain was only the vir region and is suitable host for plant transformation.

The Ti plasmid of Agrobacterium strains have been disarmed and are used as vehicles for gene transfer. Some examples are pLBA4404 (Hoekema et al., 1983), pGV3850 (Zambrysky et al., 1983), and pEHA 101 (Hood et al., 1986). Transformation vectors are designed such that they have the following basic features. a. They should function in three organisms : E. coli, Agrobacterium tumefaciens and plant cells. b. Should have directly repeated T-DNA direct repeat border sequences in proper orientation. c. A suitable plant selection marker gene and convenient restriction enzyme sites.

Two vector systems have been developed, co-integrating and binary vectors. Cointegrate vector rely upon recombination and co-integration with the disarmed Ti-plasmid, hence can integrate into a limited number of Ti plasmid. Binary vectors replicate autonomously in Agrobacterium. Agrobacterium harbouring T-region and the vir region of the Ti plasmid on separate replicons can efficiently transfer their T-DNA to plants. Thus, binary vector systems are easiest to handle and widely used than cointegrate types (Klee and Rogers, 1989).

2.1.3 Agrobacterium mediated in vitro transformation

In vitro transformation is possible either through one of the following pathways; direct or callus mediated regeneration, somatic embryogenesis or through protoplasts. Gynheung (1985) has reported that tobacco calli were transformed at levels upto 50 per cent by cocultivation of tobacco cultured cells with Agrobacterium tumefaciens harbouring the binary transfer DNA vector, PGA-472, containing a kanamycin resistance marker. Transformation frequency was dependent on the physiological state of the tobacco cells, the nature of Agrobacterium strain and less so on the expression of vir genes of the tumour inducing plasmid. Maximum transformation frequency was obtained with exponentially growing plant cells, suggesting that rapid growth of plant cells is an essential factor for efficient transformation of higher plants. Firoozabady et al. (1987) have efficiently transformed and regenerated cotton cotyledon tissues. Transformation was confirmed by opine production, kanamycin resistance, immuno assay and DNA blot hybridization. This process for producing transgenic cotton plants facilitates transfer of genes of economic importance to cotton. Umbeck et al. (1987) reported preliminary results on cotton (G. hirsutum L.) transformation via Agrobacterium using hypocotyls as explants. Selection and regeneration were not thoroughly characterized. Chee et al. (1989) have confirmed that about 0.01 per cent of the total seeds infected with Agrobacterium tumefaciens harbouring npt II gene have shown transformation in case of germinating seeds of Glycine max L. Potrykus (1990) has reported that the shoot apical meristem of plants regenerates the whole green part of the plant body, including the flowers, thus it is a very important tissue in the developmental biology of plant in cereals. In particular, the meristem is a tissue with a very high regeneration potential. Gene transfer to meristem cells could therefore, overcome many of the regeneration problems due to difficult tissues culture systems in many important crop species. Medford (1992) has reported that the meristem is often confused with the complete shoot apex, which also contains the leaf primordia and the young leaves in Brassica olerecea. Furthermore, the meristem as a tissue may represent a complicated pattern of cells. Each of these cells may differ physiologically due to its unique position in the meristem.

Rhodora and Thomas (1996) confirmed genetic transformation of rice (Oryza sativa L.) mediated by Agrobacterium tumefaciens for japonica varieties and extended to include the more recalcitrant indica varieties. Immature embryos with sizes ranging from < 1.5 to >2.5 mm isolated from sterile immature panicles of indica rice varieties were inoculated with either A. tumefaciens At656 (pCHL56) or LBA4404 (pTOK233). Transformation was based upon resistance to hygromycin (pTOK233 contains the hpt gene), the presence of GUS activity (from the gusA gene), southern blots for the integrated gusA gene, and transmission of GUS activity to progeny in a Mendelian 3:1 segregation ratio. Key factors facilitating the transformation of rice by Agrobacterium tumefaciens appeared to be the use of embryos as the explant, the use of hygromycin as selection agent (which, doesnt interfere with rice regeneration), the presence of extra copies of vir genes on the binary vector of pTOK233 and maintaining high concentrations of acetosyringone for inducing the vir genes during cocultivation of embryos with Agrobacterium. Dillen et al. (1997) indicated that temperature plays an important role in transformation with A. tumefaciens. They observed the best transformation efficiency at 22 C in both Phaseolus acutifolius callus and tobacco leaves, irrespective of type of helper plasmids. Although 25 C is commonly employed for in vitro cocultivation experiments, they showed that a lower temperature (19-22 C) is more optimal because in planta tumour formation occurs more frequently at 22 C. Mahalakshmi and Khurana (1997) have studied the age and physiological status of the cereals and reported that the meristematic tissues, explants from the young plants and cells undergoing differentiation are the choicest material for Agrobacterium mediated transformation. The age and physiological status of the plant plays an important role during plant microbial interactions. Usually, 3-4 days old seedlings were used for agro infection in maize, wheat and other cereals than dry dessicated seeds with exposed apical meristems. Immature embryos of maize were differentially susceptible to Agrobacterium. The best stage corresponded to 12-22 days after pollination when the two leaf initials were formed indicating a specific window of competence in the host plant. It was also observed that leaves were more competent than roots, scutellum or seed remnants in maize, rice wheat and barley. Cervera et al. (1998) and Cao et al. (1998) have suggested that, low efficiency of transformation in citrange explants was due to an insufficient length of cocultivation. However, they have also reported that it was more difficult to eliminate Agrobacterium after longer periods of cocultivation. Further, they have also reported that the 5 day culture period resulted in over growth of Agrobacterium and a subsequent decrease in the cocultivation was the most effective for increasing the frequency of transient GUS-expression in citrange explants. Therefore, the period of cocultivation should be optimized. Gould and Cedeno (1998) have presented a protocol for rapid genotype independent transformation and regeneration of cotton (Gossypium spp.) from shoots isolated from germinating seedlings. They have inoculated the isolated shoots with a super virulent strain of Agrobacterium tumefaciens, subjected them to a mild antibiotic selection and directly regenerated as shoots in vitro. They have reported that by this method the shoots do not differentiate and mutation rates are low. Rooted shoots can be obtained within 6-10 weeks of isolation and inoculation depending on the cotton cultivar. Hemphill et al. (1998) reported a clonal propagation system to regenerate mature cotton (G. hirsutum L.) plants from pre-existing meristem from in vitro grown tissues. Shoot apices, lateral nodes and cotyledinary nodes were cocultivated with A. tumefaciens and grown to a two-leaf stage by this in vitro culture system. They, further reported that, this procedure resulted in 121 kanamycin selected shoots and 40 mature viable plants, which produced viable T1 seeds. Mature T1 plants expressed GUS activity in pollen grains that suggested that the transgene was inherited by progeny. Pawan et al. (1998) achieved stable transformation and expression of transgenes was achieved in mungbean using Agrobacterium rhizogenes mediated system. A. rhizogenes strains containing Ri plasmid and a binary vector (pBin9GUSint) with neomycin phosphotransferase II and GUS (with a intron in coding region) as marker genes, produced roots which showed GUS expression and rapid growth on hormone free medium containing

50 mg/l kanamycin and 500 mg/l cefotaxime. Transformation frequency varied with the age of the explants, duration of pre-induction of explants, cocultivation time and medium, concentration and strain of Agrobacterium but did not vary too much with different genotypes of mungbean. Hypocotyl explants of 2-day-old seedlings were more responsive compared to those from 4 to 6-days-old seedlings to infection with strain LBA 9402. Bacteria at a 8 -l concentration of 10 cells ml and cocultivation for higher transient transformation rates and controllable bacterial growth. Cocultivation medium also affected transformation. Explants cultured on B5 with high BAP concentration (1.0 mg-l). Prior to cocultivation, inoculation of explants in bacterial suspension for 1 to 12 h also affected the transient transformation frequency. Six hours preinduction was found to be optimal. Moralejo et al. (1998) have described a procedure for genetic transformation of Eucalyptus globulus (Labill) and they have studied the influence of explant pre-cultivation and reported that when seedlings were precultured for 4-6 days, the level of GUS transient expression was significantly greater than that of control (i.e. without pre-culture) and that the seedlings precultured for 6 days seemed to be more suitable for stable integration of transgenes. They further reported that the improvement of DNA uptake could be due to stimulation of cell division by the hormones in the pre-cultivation medium, since mitotic cells would be more susceptible to Agrobacterium (or) would have a higher level of transcription. However, they also opined that the physiological status optimal for DNA uptake (transient expression) is not necessarily optimal for integration for foreign DNA in the host genome. Infact, division of cells would not be sufficient for transformation and integration may primarily depend on the availability of DNA repair enzymes. These proteins, although not yet identified, are thought to play a key role in the last steps of T-DNA integration (Tinland, 1996). Wang et al. (1998) have constructed a plant expression vector, pBINK, carrying two insecticidal genes: pea lectin gene and soybean kunitz trypsin inhibitor gene and successfully transferred into upland cotton cultivars, via Agrobacteirum mediated transformation, hypocotyls segments from aseptic seedlings were used as transgene recipients. After cocultivation, kanamycin resistant calli were screened and somatic embryos and regenerated plants were obtained using various media. Transgenic cotton plants with two insecticidal genes were confirmed by ELISA, PCR and southern analysis. Bioassays demonstrated that the transgenic plants had significant resistance to larvae of cotton bollworm. Zapata et al. (1999) reported that transgenic cotton (G. hirsutum) plants of texas cultivar were obtained using Agrobacterium mediated transformation coupled with the use of shoot apex explants. Regeneration of primary plants was carried out in a medium containing 100 mg/l of kanamycin and the progeny obtained by selfing T0 plants were subjected to kanamycin screening. Surviving plants showed more than one copy of T-DNA. The use of shoot apex circumvents the problem of genotype dependent regeneration of cotton. Donaldson and Simmonds (2000) have reported that the overall rate of in vitro transformation for co-cultural explants placed on selection media ranged from 27-92 per cent depending on the cultivar. However, transformation was predominantly confined to nonregenerable hypocotyls callus (or) other non-regenerable tissue, in regeneration, competant tissue of the cotyledonary node or in differential tissues was rare. Satyavathi et al. (2002) have given a protocol for consistent production of transgenic cotton plants in three Indian varieties. Shoot tip explants were transformed by cocultivation with A. tumefaciens, strain LBA4404. Among the different combinations of BAP and NAA tested, 0.1 mg/l of BAP and NAA in the medium influenced efficient regeneration of shoots by organogenesis. Shoot bud proliferation and elongation were achieved in 3-4 weeks time on medium supplemented with GA3. The putatively transformed shoots were harvested and placed for rooting on medium containing IBA and 75 mg/l kanamycin. Transgenic plantlets were obtained in 12-16 weeks from the time of gene transfer to the establishment in pots. Sunilkumar and Rathore (2001) reported that green fluorescent protein (GFP) proved to be a valuable tool in elucidating the timing and localization of transient gene expression in cotton. In visualizing conversion of transient events to stable transformation events, the effects of Agrobacterium strains, acetosyringone and temperature on stable transformation in cotton were also evaluated. Strain LBA4404 proved to be significantly better than EHA-105.

Addition of acetosyringone just prior to cocultivation to the culture at a final concentration of 100 M increased significantly the stable transformation efficiency in cotton. Cocultivation at 21C, compared to 25C, consistently resulted in higher transformation frequencies. Leelavati et al. (2003) presented a protocol for efficient transformation and regeneration of cotton. Embryogenic calli cocultivated with Agrobacterium carrying cryIIa5 gene were cultivated under dehydration stress and antibiotic selection for 3-5 weeks to generate several transgenic embryos, 75 globular embryo clusters were observed on selection plates and these embryo clusters were cultured on multiplication medium followed by development of cotyledonary embryos on embryo maturation medium to obtain on an average of 12 plants per Petriplate of cocultivated callus, 83 per cent of these plants have been confirmed to be transgenic by southern blot analysis and ten kanamycin resistant plants per plate were obtained. Shuangxia et al. (2005) developed a reliable and high efficiency system of transforming embryogenic callus (EC) mediated by Agrobacterium tumefaciens in cotton. Various aspects of transformation were examined in efforts to improve the efficacy of producing transformants. The effects of Agrobacterium strains, aceto syringone (AS), cocultivation temperature, cocultivation duration, Agrobacterium concentration and physical status of EC on transformation efficiency were evaluated. Strain LBA4404 proved significantly 8 -1 better than C58C3. Agrobacterium at a concentration of 0.5 x 10 cells ml (OD600=0.5) improved the efficacy of transformation. Relatively cocultivation temperature (19 C) and short cocultivation duration (48 hours) were optimal for developing a highly efficient method of -1 transforming EC. Concentration of AS at 50 mg l during cocultivation significantly increased transformation efficiency. EC growing 15 days after subculture was the best physiological status for transformation. An overall scheme for producing transgenic cotton is presented, through which an average transformation rate of 15 per cent was obtained. Wu et al. (2005) developed a simple protocol of transformation of cotton at a high frequency via Agrobacterium mediation, coupled with the use of embryogenic calli as explants. Embryogenic calli derived from only one to two somatic embryogenics calli lines of two Chinese cotton cultivars Ekang 9 and Jihe - 321 which have low embryogenic potency were first inoculated with a A. tumerfaciens strain LBA-4404 harbouring binary vector. pBin 438 carrying a synthetic bacillus thurengensis active cry1AC and API-B chimeric gene. Infected embryogenic calli were cocultivated for 48 h and were then moved on to the selection r medium with kanamycin (100 mg/l) for 7-8 weeks. Then, the kanamycin resistant calli (km ) subcultured in proliferation medium would re-differentiate to form somatic embryos in 30 days. Putative transformants were confirmed by polymerase chain reaction and southern blot analysis. Forty-five regenerated plants were successfully transferred to soil, of which 12 proved to have the active cry1AC and API-B chimeric gene. Insect bioassay showed that plants were highly resistant to cotton bollworm larvae, with mortality ranging from 75.8 to 100 per cent. Malathi et al. (2006) produced semi-looper resistant transgenic castor plants through Agrobacterium mediated genetic transformation method using super binary vector, pTOK233 carrying gus A and hpt gene. Furthermore, pSB111 vector carrying a synthetic delta endotoxin gene cry1Ab and herbicide resistance gene bar both driven by CaMV 35S promoter were used. PCR analysis revealed the presence of both bar and cry1Ab gene in the basta tolerant primary transformation. The primary transformants subjected to ELISA disclosed varied levels of cry protein. These transgenes expressing cry1Ab when bioassayed against freshly hatched semilooper larvae induced substantial (>88%) insect mortality. Southern analysis of 2T1 plants revealed the presence of cry1Ab gene. Transgenes, expressing cry1Ab exhibited ample resistance against the castor semi-loopper. Guo et al. (2007) transferred three constructs harbouring novel Bacillus thuringiensis cry1c, cry2A, cry9c and bar gene into four upland cotton cultivars via Agrobaterium mediated transformation, 84.8 per cent resistant calli were confirmed positive by PCR tests and total 50 transgenic plants were regenerated. Bioassay showed 80 per cent of the transgenic plantlets generated resistance to both insect and herbicide. This result showed that bar gene can replace antibiotic marker genes.

Katageri et al. (2007) achieved Agrobacterium mediated genetic transformation of an elite Inidan genotype (Bikaneri Nerma) of cotton (Gossypium hirsutum L.) using shoot apical meristems isolated from seedlings as explants and a synthetic genes encoding cry1Ac endotoxin of Bacillus thuringiensisi with the transformation efficiency of 0.2 per cent. Regeneration of shoots was carried out in selection medium containing kanamycin (100 mg/l) after cocutlivation of the explants with Agrobacterium tumefacines. Rooting was accomplished on a medium containing naphthalene acetic acid and kanamycin. Progeny obtained by selfing T0 plants were screened for the presence of neomycin transferase (npt II) and cry1AC genes by polymerase chain reaction (PCR) and southern hybridization. Expression of cry1Ac in the leaves of transgenic plants was detected by Xpress strips and quantified by Quan-T ELISA kits. Results of insect bioassay and field tests showed considerable potential of the transgenic cotton for resistance against cotton bollworm.

2.1.4 Estimation of Agrobacterium tumefaciens

One of the major problems encountered during plant tissue transformation with A. tumefaciens is the effective elimination of the Agrobacterium after transfer of genetic information has taken place. If A. tumefaciens is not elminated, it will overgrow on the tissue and affect the further growth of explants. Antibiotics are used for the elimination, but the higher levels of antibiotics needed to kill the organism may have deleterious effects on the regeneration. Hence, the level of antibiotic requirement is to be selected for individual crops. Although many antibiotics have been described for the effective elimination of Agrobacterium and cefotaxime have minimal toxicity on most of the tissues and efficiently eliminate Agrobacterium cells (Pollock et al., 1983). However, this antibiotic showed plant hormone like activity on plant tissues, especially when the plant tissues were grown in a medium containing low concentration of carbenicillin or cefotaxime.

2.1.5 Factors affecting Agrobacterium mediated transformation

The frequency of Agrobacterium mediated transformation is very low. For optimization of the transformation protocol, interaction between Agrobacterium tumefaciens and plant is probably the most important aspect to be considered. Many factors influencing Agrobacterium mediated transformation of dicotyledonous plants such as preculture of the explants, colonization period, wounding methods like, scalpel wounding, vacuum infiltration, blot drying, chilling injury, sand injury, scalpel wounding followed by colonization with Agrobacterium culture have been elucidated. 2.1.5.1 Preculture studies Preculturing of the explants on the regenerating medium prior to inoculation of Agrobacterium increased the frequency of transformants in pigeonepa to 62 per cent from cotyledonary node and 27 per cent from shoot tip (Geetha et al., 1999), 28.8 per cent from callus and 1.2 per cent from shoots regenerated (Lawrence and Koundal, 2001). 2.1.5.2 Duration of vacuum infiltration Applying a vacuum to plant organs in the presence of Agrobacterium removes intercellular fluids and air, which are replaced by bacteria when the vacuum is released (Hewezi et al., 2002).Vacuum infiltration assisted Agrobacterium mediated transformation was reported for the first time in Arabidopsis thaliana (Bechold et al., 1993). This method involves application of Agrobacterium to whole plants via vacuum infiltration of flowering stage. Mehrazad et al. (2002) reported a rapid protocol based on vacuum infiltration of Agrobacterium into lentil cotyledonary nodes. Vacuum infiltrated tissues had significantly (P<0.05) higher transient GUS expression than non-filtrated tissues. Under optimal conditions (infiltration at 200 mmHg for 20 minutes), about 95 per cent of Agrobacterium infiltrated explants exhibited an average of 16 blue foci. An Agrobacterium mediated gene transfer system was developed for cotton (Gossypium hirsutum L.) cv. Cocker-312 (Ikram, 2004). In this study, two month old hypocotyls derived embryogenic calli were treated for 10 minutes at 27 psi in a suspension of

Agrobacterium culture. In four independent experiments, upto 28.23 per cent transformation efficiency was achieved. 2.1.5.3 Duration of cocultivation period Length of cocultivation time employed varies widely from a few hours to a few days depending upon the species, explant and culture condition. Liu et al. (1990) studied influence of different concentration periods, tissue types and th bacterial strains for transformation in Capsicum annuum L. In general, 4 cocultivation period gave rise to slightly but significantly greater number of larger tumours. However, differences were not significant for leaf tissue per cent tumour formation in cultivars ECW, YWL and JPT, for tumor size in ECN and LBB. Because of 24 hours cocultivation period produced tumors on approximately 85 per cent of explants within 4 weeks and nopaline was detected in tumors with strain, C58. They concluded that 24 hours cocultivation would be adequate for transformation of pepper. Shuangxia et al. (2005) examined various aspects of transformation to improve the efficacy of producing transformants in cotton and found that shoot cocultivation duration of 48 hours was optimal for developing a highly efficient method of transforming EC. 2.1.5.4 Chilling (or) dehydration and rehydration of explants Hewezi et al. (2002) reported that split embryogenic axis of 21 days old immature sunflower embryos were bombarded, vacuum infiltrated or dehydrated before being infected by Agrobacterium tumefaciens strain EHA-105, bearing a binary vector carrying the selectable marker npt II and the scorable marker uid A gene. Of the 3 methods used, only dehydration and rehydration with Agrobacterium give rise to transient GUS expression in shoot apices and stably transformed plants as confirmed by Southern blotting analysis. Dehydration/rehydration of RHA 266 immature embryos of sunflower allowed bacteria to reach the innermost meristematic cell layers. T-DNA was successfully transferred and integrated and transgenes were expressed in shoot apices with the recovery of one transgenic plant in the progeny with transformation efficiency of 0.22 per cent.

2.2 SELECTION OF TRANSFORMED TISSUES/PLANTS AND CONFIRMATION

2.2.1 Selectable marker gene
A selectable marker gene is added to the gene construct in order to identify cells or tissues that have successfully integrated the transgene. This is necessary because, achieving incorporation and expression of transgenes in plant cells is an event, occurring in just a few per cent of the targeted tissues or cells. Selectable marker genes encode proteins that provide resistance to agents that are non-toxic to plants, such as an antibiotics or herbicides. A suitable marker gene allows the preferential growth of transformed cells in the presence of the corresponding selective agent. Many antibiotics and herbicide genes are used as selectable markers. Selection efficiency depends on the size and developmental state of the plant cells, regeneration response and concentration of the selective agent. Kanamycin resistance is the most widely used selection for higher plant transformation. The gene npt II conferring resistance to the amino glycoside antibiotics, such as kanamycin, was first established as useful dominant selectable marker for higher plants in 1983 (Bevans et al., 1983; Fraley et al., 1983; Herrera-Estrella et al. (1983). Since then, its usefulness has been demonstrated with a broad group of plants (Steinbiss and Davidson, 1989). Kanamycin resistance is conferred by transgenic expression of neomycin phosphotransferase, the product of the npt-II gene from the bacterial transposon Tn5. The enzyme neomycin phosphotransferase transfers a phosphate from ATP to the amino glycoside and thereby inactivates it. The neomycin phosphotransferase type-II (npt-II) gene has been used in transformation of more plant species than any other selectable marker. The original coding

sequence of npt-II was from bacterial transposon, Tn5 Npt5-II which provides resistance to certain amino glycoside antibiotics such as kanamycin, paramomycin and geneticin, but kanamycin is the most widely used. NPT -II coding sequence has been fused to constitutively regulated promoters such as nopaline synthase (nos) and cauliflower mosaic virus (CaMV) 35 S regulatory sequence (Fraley et al., 1983) and later cloned into plant transformation and expression vectors by many researchers (An et al., 1985, Bevan, 1984, Klee and Rogers, 1987). Kanamycin has proven to be the most widely applicable selective agent but the concentration is species specific. Species like Lycopersicum esculeutum (Fillatti et al.,1987) and Brassica napus (Moloney et al., 1989) are selected at low concentration of kanamycin (15-100 mg/l) whereas Beta vulgaris needs relatively higher concentration of kanamycin (400 mg/l). Neomycin phospho transferase-II, which is known to confer kanamycin resistance and a gene of bacterial origin of betaglucuronidase, which is commonly used as a reporter gene have been transferred to tobacco by leaf disc transformation using strain LBA 4404 of A. tumefaciens, which carries a binary vector (Oktem et al., 1994). Ming Cheng et al. (1996) have reported that selection on kanamycin (150 mg/l) facilitated the screening of regenerants and resulted in a relatively smaller population of putative transformants for further evaluation. Further they indicated that all GUS-positive shoots rooted in rooting medium containing 150 mg/l kanamycin and the frequency of transformed fertile plants was 0.2-0.3 per cent of the explants inoculated. They have further reported that a major obstacle to cotton transformation has been the optimization of methods for using a selectable marker such as kanamycin resistance. In tobacco, transformed tissue at any stage of development can be selected on high levels of kanamycin (up to 1000 mg/l) while in cotton such levels are toxic. De Neve et al. (1997) have reported that host factors are required for T-DNA integration. These authors have employed the root explant transformation procedure, which involves the treatment of sterilized Arabidopsis root section for transformed calli induced by transfer of a T-DNA carrying a selectable marker such as kanamycin resistance. Stable transformation was measured as the stable integration and somatic inheritance of neomycin phosphotransferase gene present on the engineered T -DNA. They further reported that the protocol has an inherently high rate of variation from one explant to another. Hemphill et al. (1998) have excised shoot apices from 1 day-old seedlings and demonstrated three different phenotypes during the kanamycin selection procedure. The 'green' phenotype showed early kanamycin resistance the leaves and stems were green and the shoots were cut or trimmed while growing throughout the selection phase. Following kanamycin selection, the green phenotypic shoots were placed on Ms3AC medium and then transferred to soil. The remaining shoots showed different degrees of stress. Some showed "mottled green" sectors on their cotyledonary leaves and their green primary leaves showed white vascular tissue and leaf edges. The non responsive apices phenotype elongated to approximately 2 cm and eventually became necrotic and died. When the 'mottled green' phenotypes were transferred from kanamycin selection medium I to kanamycin selection medium 2, these shoots developed green leaves from the growing apical meristem. These shoots were routinely cut or trimmed to keep them under continual antibiotic selection pressure; many died at the higher kanamycin level. However, several of the mottled green phenotypes were transferred to soil and tested positive for histochemical localization of gusactivity. Momtaz et al. (1998) following cocultivation with A. tumefaciens, callus induction media containing 500 mg/l carbenicillin and 25 mg/l kanamycin allowed transformed tissues to form kanamycin resistant micro calli at wound sites after 12-15 days. No callus from control tissue initiated at this level of kanamycin. At the same time one to two kanamycin resistant calli per hypocotyl segment were observed. Further they have tested a total of 499 putative transformed calli for npt II production. On lower levels of kanamycin (15 mg/l), some non transformed calli proliferated very slowly but eventually turned brown and died. The percentage of kanamycin resistant and npt-II positive calli was higher when higher levels of

kanamycin (25 or 35 mg/I) were used for selection. Kanamycin selection has been proven to be the most widely applicable selective agent but the concentration is species specific. Species like Brassica napus (Moloney et al., 1989) and Lycopersicum esculentum (Fillatti et al., 1987) are selected at low concentration of kanamycin (15-100 mg/l), where as Beta-vulgaris and cotton (Gossypium spp) needs relatively higher concentration of kanamycin (400 mg/l). Zapata et al (1999), reported that transgenic cotton (Gossypium hirsutum L.) plants of Texas cultivar CUBOHPRIS were obtained using Agrobacterium mediated transformation coupled with the use of shoot apex explants. After inoculation with Agro strain LBA 4404 containing the PBI-121 plasmid, regeneration of primary plants was carried out in a medium containing kanamycin (100 mg/l). Progeny obtained by selfing were germinated in the green house and selected for expression of the T-DNA marker gene encoding npt-II by painting kanamycin 2 per cent on the leaves. Plants'-that survived leaf painting were analyzed by DNA blots. Evidence for integration of the GUS was observed in two successive generations from the regenerants (To). The transformed plants appeared to have more than one copy of the TDNA. The use of shoot apex circumvents the problem of genotype independent regeneration of cotton. Zapata et al. (1999) have tested the kanamycin resistant plants for the presence of the GUS gene by DNA blot analysis. Hybridization of undigested DNA indicated that the introduced T-DNA was integrated into high molecular weight DNA. Digestion of genomic DNA with EcoR-I produced three hybridizing bands that were different in mobility from that of the digestion with Hind-III, which again indicates genomic integration of the T-DNA. Furthermore, since the T-DNA of the PBI-121 has single EcoR-I and Hind-III sites, the presence of these hybridizing fragments suggests three independent insertions of the transforming T-DNA into the genome of this plant. Seeds (T2) from TI plant were grown, and a DNA blot analysis of some of the progenies was carried out. All these plants had the same hybridization pattern of DNA as in the T1 plant. The fact that kanamycin resistant plants could be recovered and that most of the plants tested from resistant plants were GUS positive in the DNA blot, strongly suggested that the stable integration of the npt-II gene and gus gene. Xiang et al. (1999) have carried out the selection for transformants resistant to kanamycin. They have collected the seeds in bulk from plants vacuum infiltrated with Agrobacterium harboring PB 1101.2 gshl. Dry seeds were germinated either directly in soil (3000 seeds/flat) or on 150 mm agar plates (3000 seeds/plate). The medium in the plates was half-strength MS. Salts and 85 vitamins solidified with 2.0 g/l phytogel, supplemented with 50 mg/1 kanamycin. Seeds germinated in solium were kept in growth chamber. Plants grown at higher light intensities seemed to be more kanamycin resistant. Seeds germinated on plates and in soil received a 2 day cold treatment at 40 C to promote uniform germination. For in solium selection, seedlings at the stage of their first pair of true leaves were sprayed with 0.1 per cent Triton X-1OO solution with kanamycin concentrations ranging from 100 to 500 mg/l using a spray bottle. After spraying, the seedlings were covered for 16 h with a plastic dome to prevent excessive dehydration due to application of the Triton X-1OO. The kanamycin spray was applied daily in sufficient quantity to just wet the surface of the leaves. The plants were for 2 days with 100 mg/l kanamycin, followed by with 200 mg/l kanamycin, followed by 1 day with 500 mg/l kan. There was a slight amount of burn on the edges of leaves following the final spray treatment. Although selectable marker genes, that confer resistance to antibiotics or herbicides, are generally incorporated along with the gene of interest in a transformation process, so as to allow the recognition of the transformed cells from the background of untransformed once, subsequent to the generation of transgenic plants, the presence of these marker genes becomes no more of practical utility and thus arguably a matter of public euphoria, speculating the risk they can pose to the environment and health. Since the recurrent transformations to pyramid desirable genes are practically difficult with the same selectable markers used, novel strategies are evolved to essentially eliminate these marker genes or to modulate their spatial/temporal expression. Use of environmentally safe selectable markers

and transplastomic plants also serves the purpose.

2.2.2 Safer marker genes

The green fluorescent protein (gfp) gene is an ideal selectable marker and reporter for gene expression analysis and plant transformation. In nature, GFP is made by the jellyfish Aequorea victoria, and has been used as a convenient marker in heterologous systems. The first example of its expression in graminaceous plant was reported by Sheen et al. (1995) where bright GFP expression was detected in maize mesophyll protoplasts. These results have since been extended and applied to other cereals and grasses (Pang el al., 1996). In the traditional selection system, using antibiotics or herbicides, the transgenic cells convert the selective agent to a detoxified compound that may still exert a negative influence on plant cells. Further, the release of toxic metabolites by dying adjacent cells may also inhibit the growth of transformed cells. In contrast, a new set of markers known as positive selection markers are being developed, which can overcome some of the limitations encountered by the traditional selection system. Joersbo and Okkels (1996) have reported a selection system, which uses the glucuronidase gene from E.coli as a selectable gene and a glucuronide derivative of the cytokinins, benzyladenine as a selective agent, Benzyladenine N-3 glucuronide is inactive as cytokinin but upon hydrolysis by GUS becomes active cytokinin, stimulating growth and regeneration of cells in vitro. The gus gene served as both selectable as well as screenable marker. The efficiency of the transformation was reported to be two-fold higher than with kanamycin. Likewise the other selection systems, which originated from the concept of favouring the transgenic cells while starving rather than killing the non transgenic cells, is mannose selection system, where in Pmi gene (Phosphomannose isomerase) is used as selectable gene and mannose as a selective agent. The authors have observed that the use of mannose selection in sugar beet resulted in a tenfold increase in transformation frequency when compared to traditional kanamycin selection. Daniell et al. (2001) engineered chloroplast genome without the use of antibiotic selection. The Betaine aldehyde dehydrogenase (BADH) gene from Spinach was used as a selectable marker. The selection process involves the conversion of the toxic BA by the chloroplast BADH enzyme to a nontoxic glycin betaine, which also serves as an osmoprotectant for enhancing drought and stress tolerance in plants. Komari et al. (1996) have reported that one of the strategies to produce transgenic plants free from selectable marker is that the marker and gene of interest are placed on two separate T-DNAs in a single plasmid or on separate plasmids, which are contained in one or more agrostrains. The transgene and marker gene are thus inserted at the loci, which should recombine at reasonably high frequencies; the gene of interest can be segregated from the selectable marker gene in the next generation. Using this technique, transgenic plants free of a selectable marker gene were developed in rice, Brassica napus and Nicotiana tabacum. Over the past decade, several approaches have been developed either for excising marker genes from transgenic plants or their modulation and partial or temporal expression. Further, alternative environmentally safe selectable markers or containment of the transgenes through transplastomic plants have given a new dimension (Pawan et al., 2002).

2.2.3 Polymerase Chain Reaction

Kary Mullis invented the polymerase chain reaction (PCR) in 1985 (Mullis, 1990). One of the major applications of this outstanding invention is to know the presence of a transgene. The polymerase chain reaction results in the selective amplification of a transgene, if an appropriate primer is provided. Any region of transgene can be chosen, so long as the sequences at the borders of the region are known. The border sequences must be known because in order to carry out a PCR, two short oligonucleotides, must hybridize to the DNA molecule, one to each strand of the double helix. These oligonucleotides, which act as

primers for the DNA synthesis reactions, delimit the region that will be amplified. Amplification is usually carried out by the DNA polymerase-I enzyme from Thermus aquaticus, which lives in hot springs, and many of its enzymes, including Taq polymerase, are thermostable, meaning that they are resistant to denaturation by heat treatment. Thermostability of Taq polymerae is an essential requirement in PCR methodology along with dNTPs, MgCI2, reaction buffer, sterile water. To begin PCR amplification, the enzyme is added to the primed template DNA and incubated so that it synthesizes new complimentary strands. The reaction mixture is then heated to 940 C so that the newly synthesized strands detach from the template, and then cooled, enabling more primers to hybridize at their respective positions, including positions on the newly synthesized strands. Taq polymerase, which unlike most types of DNA polymerase is not inactivated by the heat treatment, now carries out a second round of DNA synthesis. The cycle of denaturation hybridization - synthesis is repeated, usually 25-30 times, resulting in eventual synthesis of several hundred million copies of the amplified DNA fragment. At the end of a PCR, a sample of the reaction mixture is usually analyzed by agarose gel electrophoresis, sufficient DNA having been produced for the amplified fragment to be visible as a discrete band after staining with ethidium bromide. This may by itself provide useful information about the presence of transgene that has been amplified. Gould et al. (1991) have subjected Maize DNA from six F1 of C56 and from one F2 of C1 to PCR amplification of 250 bp fragment within the GUS coding region. In a separate amplification, primers for a 1000 bp fragment within the NOS/NPT Ii gene were used. Amplification of the expected fragments were achieved from the DNA of four C56F1 as well as from the DNA of an F2 of the C1 family. These results indicated the presence of both GUS and NPT-II in the progeny of two original transformants. Manoharan et al. (1998) have used to demonstrate the presence of T -DNA in the transgenic plants. Two specific primers derived from npt-II gene sequences were used to detect a 0.7 kb fragment. The amplified DNA samples were electrophoresed- on 0.8 per cent agarose gel. 0.7 kb fragment co-running with the amplified product from PBI-121 could be detected from the transgenic plants but not from non transformed plant. Zhang et al. (1998) have reported that the resistance to the bollworm was observed in the R2-progeny derived by selfing the five R1 plants, which was evident from the high mortality rate of bollworm. On the basis of resistance analysis some resistant plants were sampled for the presence of the Bt-transgene with PCR. The amplified band on the Bt-transgene appeared in the samples of R1plants S 545 and S 591. The PCR products were subjected to southern blot analysis with the labelled transgene as probe. All the amplified bands could hybridize with the probe, proved that the PCR bands were not amplified from contaminating DNA, but from the Bt-transgene, thus establishing that transgene was heritable through selfing. The putatively transformed plantlets were subjected to PCR analysis to detect the presence of uid A and npt-II genes. In the transformants with GUS-activity, an expected fragment of 500 nucleotide uid A was amplified. An additional confirmation was also done using npt-II primers and PCR analysis revealed amplification of a 700 base-pair fragment. No amplification was found in non transgenic plants, pigeonpea (Geetha et al., 1999). For the detection of the npt-II coding sequence in the progeny, genomic DNA was subjected to PCR using the following primers and conditions: forward 5'TCATCTCACTTGCTCCTG-3' and reverse 5'AGCCAACGCTATGTCCTG-3' primers at a concentration of 50 pM were used to amplify a 363-bp region (position 1860 2222 of Tn5) of the npt-II gene (pHP 23 served as a positive control) with 0.5 g genomic DNA of each sample, 2 mM MgCI2, and 2 units of a (native) Taq Polymerase (and the recommended 0 buffer) from Gibco in a 50 l assay. Cycling conditions were denaturation, 1 min at 94 C; 0 0 0 annealing, 1 min at 50 C, elongation 2 min at 72 C, annealing, 1 min at 50 C, elongation 2 0 min at 72 C (30 cycles) and a final elongation step of 10 min. For detection of 363 bp fragment DNA was separated on 1 per cent agarose gels. All the 4 plants indeed showed the

presence of the expected 363 bp fragment, which is part of the coding region of the npt-II gene. In one of the putatively transformed lines, an additional fragment of approximately 2.5 kbp was amplified, probably due to rearrangement of two or more incomplete copies (Krishnamurthy et al., 2000). Prasad et al. (2000) have carried out PCR analysis of the npt-II gene for initial screening of putative transformants of Brassica juncea for the presence of npt-II gene. About 85per cent of the putative transformants gave the 0.7 kb full length npt-II gene. These results clearly indicated the successful integration of the gene cassette in these putative transformed lines of Brassica juncea. Weir et al. (2001) have performed PCR in 10 l of reaction solution using 25-40 ng of wheat genomic DNA 100 M each dNTP, 10 pmol of each primer and I unit of Taq DNA polymerase. The cycling program was 1 cycle of 95 C for 5 min, 35 cycles of 95 C for 30s, 56C for 30s and 72C for 2 min. Primers to amplify the npt gene were 511 1 1 AAAAGCCTGAACTCACCG-3 and 5 -TCGTCCATCACAGTITGCC-3 . The PCR products were visualized by running the completed reaction in a one per cent agarose gel containing ethidium bromide and then photographed. PCR analysis of lines transformed with PBGxI and selected with hygromycin B showed that npt gene was present in callus lines showing different intensities of GFP expression, with the production of a well defined product of 250 bp. PCR amplification of the npt-II gene using specific primers was carried out to check the presence of transgene in the plant genome. Plant DNA was isolated from immature leaves by cetyl trimethyl ammonium bromide (CTAB) method as described by Peterson et al. (1993) DNA concentration was estimated spectrophotometrically. The npt-II specific primer sequences (51-31) GAG GCT ATT CGG CTA TGA CTG and ATC GGG AGG GGC, GAT ACC GTA were used. Each PCR reaction was performed in 25 l (total volume) of reaction mixture consisting of 10 x reaction buffer, 150 ng DNA, 200 mM dNTPs, 25 mM MgCh, 100 ng of each primer DNA and 1 U of. Taq DNA polymerase. The PCR reaction buffer consisted of 10 mM Tris HCI (pH 8.3), 25 mM KCI and 0.01 per cent gelatin. PCR was carried out in a thermal cycler under the following conditions, 94 C for 3 min as preheating, then 35 cycles of 94 C denaturing for 30 S, 55 C annealing for 45 S 72C synthesis for 1 min and 7 min at 72C as final extension. Amplified DNA fragments were elctrophoresed on 1.0 per cent agarose gel and detected by ethidium bromide staining and photographed under ultraviolet light (Sathyavathi et al., 2002).

2.2.4 Dot blots and Southern Blots

This technique is used to detect the presence of a given sequence of DNA or RNA in the non-fractionated DNA, where sample DNAs from several individuals can be tested in a single test-run. Dot blots are useful in detecting presence of the sequence being transferred in a number of suspected transgenic individuals and the presence of specific mRNA following cDNA synthesis in different tissues of a single individual. This technique has been used in tobacco to confirm the transfer of glucanase and chitinase gene (Dinesh, 1998). Southern blotting was used to demonstrate the presence of the gene in question in transgenic, where detection of DNA fragments that are complementary to given DNA is critical. This is common method to confirm the stable integration of DNA in the genome and for determining the copy number. Southern blotting is infact required as a final confirmation and it is routinely used to know the presence of transgene in pigeonpea (Geetha et al., 1999; Lawrence and Koundal, 2001; Kumar et al., 2003; Singh et al., 2004) with radioactive probes.

2.2.5 Expression of transgene

Unlike southern hybridization, northern hybridization detects transcription of DNA sequence of the gene of interest. This technique was used to know the expression of cowpea protease inhibitor at mRNA level in transgenic pigeon pea (Lawrence and Koundal, 2001). Bhattacharya et al. (2002) confirmed cabbage transgenics with cryIAb from B. thuringiensis by northern blotting and reported differences in the level of transgene expression. RT-PCR is another technique that can be employed to detect transcription quantitatively. Smith et al. (1988) have reported the inhibition of expression of the endogenous, developmentally

regulated gene for polygalactouronase in stably transformed tomato expressing antisense RNA. Five cauliflower transgenic plants showed the expected 1 kb amplified fragment in RTPCR indicating transcriptionally active crylAb gene in the plant genome. The 2.2kb mRNA transcript obtained after splicing of a 0.19kb intron from the Bt-gene further supported RT PCR results (Chakrabarthy et al., 2002). By immuno blotting protein expressed by the transgene can be detected and quantified based on antigen-antibody reaction. In this method total protein from plants is fractionated on SDS-PAGE (l0% poly acrylamide) and transferred to PVDF and detected by antiserum of specific antigen. Bhattacharya et al. (2002) detected B. thuringiensis cryIAb proteins using the rabbit anti cryIAb serum and goat. anti rabbit IgG coupled to alkaline phosphatase as secondary antibody. They, could detect 81.3 kDa Cry protein by this method, which was further tested to know viability on second instars larvae of Plutella xylostella. They observed reduction in growth rate and high mortality.

2.2.6 Reporter gene

In all transformation protocols, invaribily some or at times, many non-transformed plants (escapes) are noticed along with transgenic plants. The transformed plants can be identified by Southern blot analysis of the DNA isolated from them, enzymatic assay, antibody assay and detecting the selectable marker gene products. But, these assays are laborious, time consuming and require radioactive substances, antibodies etc. These drawbacks can be overcome by including an independent reporter gene in the gene transfer cassette, whose presence can be measured. A reporter enzyme should be stable or tolerate amino terminal fusions with simple and versatile assay procedures and no intrinsic background activity in the organisms (Jeffenson, 1987). Genes commonly employed are -glucuronidase (GUS) and luciferase (LUC), anthocyanins green fluorescent protein gene (gfp), chloromphenicol acetyl transferase (cat), etc. GUS activity can be quantified using either flurometric or spectrometric assays, both of which are reasonably simple. Localization of GUS expression is also detected by histochemical assay. However, the histochemical assay is expensive and procedure is destructive to plant cells (Jefferson, 1987). To know the efficiency of Agrobacterium mediated transformation uid A gene has been transferred to pigeon pea (Arundhati, 1999; Geetha et al., 1999), groundnut (McKently et al., 1995; Venkatachalam et al., 2000), chickpea (Kar et al., 1996; Ramana et al., 1996), pea (Pounti-Kaerlas et al., 1989) and chilli (Manoharan et al., 1998). The reporter gene gus ++ has certain limitation like its activity is inhibited by some divalent metal ions like Cu and ++ Zn .GUS is reasonably resistant to thermal inactivation but the assay is destructive and cost of the substrate is very high. The drawbacks of reporter genes are thought to be replaced by gfp, where the protein itself fluoresces spontaneously in the heterologous cells. This has been already used in sugarcane (Elliot et al., 1998), wheat (Weir et al., 2001) and Australian barley (Wang et al., 2001) gfp is expressed independent of cell type, location with small molecular size and no adverse effect on cell metabolism (Sonia et al., 1998).

3. MATERIAL AND METHODS

A pre-requisite for transferring genes into plants is the availability of efficient transformation protocol. Although successful Agrobacterium mediated transformation have been reported earlier, the transformation efficacy was found to vary within and between plant species. It is therefore very important to identify conditions suitable to both high rates of transformation and genotype independent regeneration methods. The present investigations were carried out at Agricultural Research Station, Dharwad farm, University of Agricultural Sciences, Dharwad during 2006-2008.

3.1 EXPERIMENTAL MATERIALS

3.1.1 Genotypes
Two varieties viz., Surabhi (Gossypium hirsutum, L) and Jayadhar (Gossypium herbaceum L.) were used in the present investigation. Surabhi is considered as superior fibre quality hirsutum cotton suitable for 50s count. Due to its wider adaptability and stability, Jayadhar has been popular herbaceum cotton suitable for 20s count which is under cultivation in rainfed condition since 50 years.

3.2 EXPERIMENTAL CONDITIONS

In vitro transformation studies were carried out under aseptic conditions in the laminar flow cabinet. The bench of laminar flow chamber was exposed to UV-light for 10-15 minutes. The working surface was sterilized by swabbing with 70 per cent alcohol. The walls of the hood were also sprayed with 70 per cent ethanol to ensure total sterile conditions. Any material being used from outside was flamed before and after use. Before starting the work, hands were also swabbed well with ethanol. The experiments were conducted under well defined conditions of the culture room maintained at 252C uniform light (ca 1000 lux) provided by fluorescent tubes (7200 over a light/dark cycles of 16/8 hour. K)

3.2.1 Glassware and chemicals

Glassware like bottles, culture tubes, conical flasks petriplates, beakers, pipettes etc. were of Borosil make. Before washing, glassware were rinsed in water and then soaked in labolin (labogel) overnight. The labolin was drained out and the glassware were rinsed in distilled water and dried in a hot air oven. The forceps, scalpels etc. were also cleaned and dried. Cleaned glassware were sealed with aluminium foil, petriplates (with blotting/filter papers inside) wrapped in polythene covers and were autoclaved at 121C at 15 lbs pressure for 15 minutes. All chemicals and plant growth regulators used were of analytical grade from standard chemical manufacturing companies.

3.2.2 Nutrient medium

Analytical grade Murashige and Skoog (1962) medium with 3 per cent sucrose, vitamins, supplied by HI-media laboratories was used in this study. This medium was supplemented with different growth regulators at varying concentrations depending upon the purpose. The pH of the medium was adjusted to 5.6-5.8 with 0.1 N HCl (or) 1N NaOH. Distilled water was added to make up to the final volume, agar was added (8 g/l) and melted by gently heating, and then it was poured in culture tubes (150 x 25 mm diameter) or bottles (300 ml capacity). The containers were then plugged with non-absorbent cotton plugs or glass bottles with suitable caps. The container having the medium was autoclaved along with bottles, tubes and petriplates used for culture at 121C for 15 minutes. Medium where, thermolabile compounds had to be added, filter sterilized solutions were added to warm (50-60C) autoclaved medium and then dispensed in pre-autoclaved containers

3.2.3 Preparation of antibiotics

Kanamycin (Hi-media) stock of 50 mg/ml prepared in double distilled sterile water was o stored at 4 C. Cefotaxime (Hi-media) stock of 200 mg/ml was also prepared in double distilled o sterile water, filter sterilized and was stored at 4 C. The required amount of antibiotic from stock solutions was added using sterile pipettes or syringe.

3.2.4 Preparation of Growth regulators

1) Filter sterilized 6-Benzyle amino purine (Sigma) stock of 10 mg/ml prepared in double distilled water was stored at 4oC. 2) Filter sterilized NAA (Sigma) stock of 10 mg/ml prepared in double distilled water was stored at 4oC.

3.2.5 Preparation of acetosyringone

10 mM Acetosyringone stock was prepared by dissolving 196.2 mg of acetosyringone in 1 ml of ethyl alcohol, made up volume upto 100 ml with sterile double distilled water, and stored at 4C.

3.3 METHODOLOGY
3.3.1 Preparation of Explants
3.3.1.1 Delinting of seeds Hundred ml of concentrated commercial H2SO4 was mixed with a kg of seed and continuously stirred with glass rod for 10-15 min or until the surface of seeds become shiny. Seeds were washed with tap water to remove the acid completely from the surface. Seeds floating on the surface of water were discarded as they are known to show poor germination. 3.3.1.2 Surface sterilization of seeds Delinted seeds were dipped in 0.2% HgCl2 for 20 minutes with constant stirring followed by the repeated washes with sterile distilled water under laminar airflow. Seeds were allowed to germinate aseptically at 26C. 3.3.1.3 Plant explants From germinating seeds, shoot apex with apical meristems were excised using sterilized scalpel blades and used in transformation studies. 3.3.1.4 Establishment of regenerated plants Small regenerated seedlings were transferred to plastic packets containing sterilized soil and peat in equal proportion, and placed in growth chamber for hardening. Seedlings were covered with plastic bags to maintain higher humidity. They were watered twice a week. Seedlings were then transformed to earthen pots containing soil and vermicompost in equal proportion and pots were placed in transgenic green house for flowering. 3.3.2 Agrobacterium strain and binary vectors The disarmed Agrobacterium strain EHA-105 harbouring binary vector pBINAR, carrying cry2Aa gene linked to the CaMV35S promoter, OCS terminator II and npt II gene under the control of nopaline synthase (nos) promoter and terminator was used as a selectable marker. This construct was obtained from National Research Centre on Plant Biotechnology, IARI, New Delhi. Its genetic map is presented in Fig.1.

3.3.2.1 Maintenance of Agrobacterium The Agrobacterium EHA-105 containing Cry2Aa was maintained on solid Yeast Extract Mannitol Agar (YEMA) medium (Appendix I) containing kanamycin @ 50mg/ml and 50mg/ml rifampicin by subculturing once in every 30-40 days on fresh medium and incubated at 26 C temperature for 48 hours. 3.3.2.2 Preparation of Agrobacterium culture for co cultivation Solid culture: one loopful of Agrobacterium was picked from a streak culture using a sterile inoculation loop and was streaked on a fresh petridish having solid YEMA medium with 50 g/ml of Kanamycin. The dish was kept at 28C for 48 hours in dark. After 48 hours the growth was carefully collected into a 2 ml new autoclaved eppendorf tube and 400 M Acetosyringone was mixed before 30 minutes of its use by vortexing for about 2-3 minutes. For developing liquid culture, one loopful of Agrobacterium was picked using a sterile inoculation loop and transferred to 150ml YEM liquid medium with 50 g/ml of kanamycin, which helps in applying selection pressure and allows only Agrobacterium to grow in the medium. Culture was grown at 28C temperature for 48 hours under orbital shaker with 150rpm. Culture that read 0.6 OD at 600 nm was used to get the pellet of bacterium culture by giving rotation of 8000 rpm for 5 minutes. Six ml MS broth and 400 l of acetosyringone (10 mM) was added to the Agrobacterium culture before 30 minutes of its use.

3.4 TRANSFORMATION AND PLANT REGENERATION

Shoot tips with shoot apical meristem (SAM) were used as explants. Following different methods of colonization, the explants were gently shaken in the bacterial suspension for 10 minutes and blotted dry on a sterile filter paper. They were transferred to the MS o medium (Appendix II) and co-cultivated under dark condition for two days at 262 C. After cocultivation explants were washed with sterile water containing 400 mg/l cefotaxime, blotted dry with sterile blotting paper and transferred to the regeneration medium containing MS with 400 mg/l cefotaxime. Standardization of protocol For standardization of in vitro regeneration and transformation protocol different factors such as, preculture, colonization period, cocultivation period, wounding methods like scalpel wounding, vacuum infiltration, blot drying or desiccation, chilling injury, sand injury and removal of apical dominance to induce axillary shoots are considered. The methods followed under in vivo treatments such as, scalpel wounding followed by colonization with Agrobacterium, induction of axillary shoots by removing apical dominance are described below.

3.4.1 In vitro transformation studies

3.4.1.1 Effect of explant preculture on regeneration after colonization and co-cultivation Shoot apices were grown on MS medium with different concentrations of BAP viz., 1 mg/l, 2 mg/l, 4 mg/l, 8 mg/l, 16 mg/l for 48 hours. They were then colonized for 10 minutes and co-cultivated with liquid Agrobacterium for 48 hours followed by washing explants with cefotaxime before transferring to shoot regeneration medium. 3.4.1.2 Effect of Agrobacterium colonization period on regeneration Shoot apices isolated from aseptically germinated seeds were subjected to 10 min, 30min, 60 min, and 24 hrs of colonization with liquid Agrobacterium tumefaciens EHA105 carrying cry2Aa gene and co-cultivated for 48 hours in murashige and skoog medium. After washing with sterile distilled water containing 400 mg/l of cefotaxime, explants were transferred to MS medium with 400mg/l cefotaxime.

Fig 1. Map of the cry2Aa gene construct in Ti plasmid

3.4.1.3 Effect of cocultivation duration on regeneration The standardization of cocultivation period was done by incubating explants with Agrobacterium for different duration viz., 48, 72, and 96 hours. After washing with cefotaxime, explants were transferred to the standardized regeneration medium (MS) containing 400 mg/l cefotaxime 3.4.1.4 Wounding methods 3.4.1.4.1 Effect of scalpel wounding and colonization duration on regeneration One fourth slant cut was given by using scalpel near the apical meristem region of shoot apex isolated from aseptically germinated seeds. Those wounded explants were suspended in Agrobacterium suspension for different periods of colonization viz., 10 min, 30min, 60 min, and 24 hrs 3.4.1.4.2 Influence of vacuum infiltration duration on regeneration. Different explants type viz., shoot apices and shoot apices with th slant cut at apical meristem region were placed in culture bottle and to this Agrobacterium suspension was added just to immerse explants. The culture bottle containing explants with Agrobacterium suspension was placed into a bell jar. Vacuum was created for 10, 20, 30, 60 minutes. Such explants were cocultivated for 2 days on MS medium containing 400 mg/l cefotaxime. 3.4.1.4.3 Effect of blot drying or dessication on the regeneration Dessication wounding was done by placing the embryos/shoot apices on Whatman No. 1 filter paper in a petridish. The dishes were then left in horizontal sterile laminar air hood for 15 and 30 minutes to dry. The explants were then removed from the dry filter paper and placed directly into another petri dish containing an Agrobacterium suspension for inoculation, after inoculating for 10 min, the embryos were cocultured for 2 days. 3.4.1.4.4 Effect of chilling on regeneration Shoot, apices were chilled at 4C for different periods viz., 10 min, 30 min, 60 min, 24 hours, and 48 hours. Later, the explants were rehydrated with Agrobacterium inoculum, blot dried and incubated in MS medium for 48 hours in dark at 22C. 3.4.1.4.5 Effect of Sand injury on regeneration. Dissected shoot apices were suspended in Agrobacterium culture and sterilized sands to induce wounds, then kept on shaker with vigorous shaking at 200 rpm at 25C for different durations (10 min, 30 min, 60 min, and 24 hours). Following this, explants were blotted on sterile Whatman No. 3 paper and transferred to cocultivation medium for 2 days.

3.4.2 In vivo transformation studies

3.4.2.1 Effect of scalpel wounding at the shoot apex region and colonization of Agrobacterium on regeneration of plants in pots Wounding was done to the seedlings grown in greenhouse. Several small incisions were done at the apical meristem region by using scalpel. Vertical cut was also given at the apical meristem region. Liquid and solid Agrobacterium culture was applied to the Shoot apical meristem region where cut was given. 3.4.2.2 Effect of scalpel wounding and colonization of Agrobacterium on plant proliferation in MS media Wound injury was done by using scalpel near the shoot apical meristem region of aseptically grown seedlings in semi solid MS medium and that wounded region was infected with Agrobacterium culture. In another treatment, shoot apex region was halved by using scalpel and infected with liquid agroculture.

3.4.3 Selection of putative transformants

Kanamycin was used for selecting the transformants. Lethal dose of the antibiotic was determined by culturing the untreated (control) shoot apices on shoot induction medium containing different concentrations (0, 50, 100, 150, 200 mg/l) of kanamycin. Based on this study 150 mg/l kanamycin was used for selecting the transformants. Sixty days old putative transformants were screened using shoot apices for kanamycin resistance at 150 mg/l concentration.

3.5 CONFIRMATION OF PRESENCE OF TRANSGENE

To confirm the presence of transgene, the control and those were positive for Kanamycin screening were also tested for the presence of npt II and cry2Aa gene by PCR with specific primers.

3.5.1 Extraction of genomic DNA for PCR analysis

Tissue was collected using 1.5 ml Eppendorf tube lid to ensure uniform size. The collected tissue was macerated in pestle and mortar at room temperature without buffer for 15 sec. The extraction buffer (400 l)s was added and kept in hot water bath at 65C for 45 minutes. The solution was centrifuged at 8000 rpm for 10 min and 400 l of supernatant was transferred to fresh Eppendorf tube. The supernatant was mixed with 400 l of chloroform and phenol (1:1) and invert gently and centrifuged at 8000 rpm for 10 min and the supernatant was removed. The supernatant was again mixed with 400 l of chloroform and centrifuged at 8000 rpm for 10 min and supernatant was removed and mixed with 800 l of isopropanol and incubated at -20C for 1 hour. It was centrifuged at 8000 rpm for 10 min, the supernatant was discarded by retaining only pellet. To that pellet, 70% alcohol was added and centrifuged for 5,000 rpm for 5 min. after centrifuge, alcohol was removed without disturbing to the pellet dried and dissolved in 80 l T10E1 and stored at 4C until further use.

3.5.2 DNA quantity and quality estimation

The concentration of DNA was assessed spectrophotometrically by recording the absorbance at 260 nm and 280 nm and also by gel electrophoresis using 0.8 per cent agarose with known concentration of uncut DNA. In spectrophotometric analysis, 5 l of DNA sample diluted with TE buffer and volume made up to 3000 l was subjected to spectrophotometer reading at absorbance of 230nm, 260nm and 280nm. A good DNA preparation generally exhibits the following spectral properties. A230 < 0.10, A230/A260 < 0.45, A280/A260 < 0.55, or A260/A280 > 1.80 DNA concentration was calculated using O.D at 260 nm with following formula. Concentration of DNA (g/ml) = O.D. at 260 x 50 To test the quality of DNA, samples were run on 0.80 per cent agarose in 1x TAE (Tris Acetic acid EDTA) buffer and stained with ethidium bromide and checked for

contamination by RNA and the DNA was evaluated by comparing it with a standard undigested DNA sample.

3.5.3 PCR amplification

The chromosomal DNA of plants obtained from cocultivated explants, control plants and plasmid DNA were used as template for PCR confirmation of the targeted cry2Aa specific gene. Gene specific primers were used since this is a part of cry2Aa gene cassette within the T-DNA region. The nucleotide sequences of cry2Aa specific primer are as follows. Forward: 5l GGG CAC TGT GTC CTC CTT CCT CCTC-31 Reverse: 5 GGG GAG ATG GTG AAG CCG GTG TAG-3 PCR Reaction mix The PCR mix was made fresh in bulk depending on the number of samples each time. Each 20l contained SDDW Mgcl2 dNTPs Taq Buffer Forward primer Reverse primer Taq polymerase Template Total : 12.67l : 0.5l : 1l : 2-5l : 1l : 1l : 0.33l : 1l : 20l
l l

The PCR amplification steps were as follows Stage I II Step 1.Initial Denaturation 1.Denaturation 2.Annealing 3.Extension III 1.Final Extension 2.Dump Temperature (oC) 94 94 49 72 72 4 Duration (min) 5 1 1 2 20 1 o

No. of cycle 1

37

After the completion of required cycles of amplification the samples were stored at 4 C in a refrigerator until further use.

3.5.4 Agarose Gel Electrophoresis of DNA

Procedure: 1. Sufficient 1x electrophoresis buffer was prepared from 50x stock.

2. Agarose powder was added (1%) to TAE buffer (1x) and was dissolved by o o melting at 100 C. The solution cooled to 50 C and ethidium bromide was added (0.5 g/ml) and positioned the comb 0.5-1.0mm above the plate. Then agarose solution was poured into the gel frame and allowed to set. The gel tank was filled with TAE buffer (1x) just enough to cover the surface of the gel to a depth of 1 mm. 3. The DNA sample was mixed with gel loading buffer and it was slowly loaded into the wells of the submerged gel using a disposable micropipette. 4. The lid was connected to the power supply and electrophoresis was carried out at 100 volts for 30-45 min. 5. It was examined by gel documentation system or ultra violet illumination

3.6 STUDIES ON INDUCTION OF AXILLARY SHOOTS

3.6.1 Induction of axillary shoots by removing shoot apical meristem
The apical dominance of embryo axes isolated from aseptically germinated seeds were removed by giving different types of horizontal cut viz, removal of leaf primordia / SAM, 1/4th of 1mm of shoot apex, 1/3rd portion of shoot apex to induce axillary shoots and incubated in MS medium for regeneration.

3.6.2 Induction of axillary shoots by removing apical meristem of seedlings

The apical meristamatic tissues of aseptically grown 8 days old seedlings were removed by using scalpel and grown in MS broth with varied concentrations of BAP (5, 10, 20, 40, 100 mg/l), TDZ (5, 10, 20, 40, 100 mg/l ) and combination of NAA (0.1mg/l)+BAP ( 5, 10, 20, 40 mg/l) to induce axillary shoots.

3.7 STATISTICAL PROCEDURES AND ANALYSIS

The data obtained from the in vitro experiments that were conducted in the laboratory, under uniform culture conditions were analysed under completely randomized design in MS Excel employing the formula given by Gomez and Gomez (1983).

3.8 HISTOLOGICAL STUDIES

The following explants were used for histological studies. 1) Apical meristamatic tissue of aseptically grown seedlings of 8-10 days old seedlings were removed to induce axillary buds. After 6 days of removal of apical meristems, explants were observed under microscope for the presence of axillary buds. Cotyledonary leaves were detached from the seedlings and approximately 5 to 10 mm length of shoot apex region along with axillary buds were fixed for histological analysis. 2) Shoot apices isolated from aseptically germinated seeds with different types of cut viz., removal of leaf primordia, 1/4th portion of 1mm of shoot apex, 1/3 rd portion of shoot apex were used for histological analysis. Histological analysis The tissues can be sliced into very thin sections provided they are first processed to prevent cell damage. The processing involves a series of steps; fixation, dehydration, embedment and subsequent sectioning with a microtome. Fixation Fixing cells with formaldehyde, will preserve the general organelle structure of the cell, but may destroy enzymes and antigens, which are located in the cell. Since cellular decomposition begins immediately after the death of an organism, one must fix the cells to

prevent alterations in their structure through decomposition. Routine fixation involves the chemical cross-linking of proteins (to prevent enzyme action and digestion) and the removal of water to further denature the proteins of the cell. Samples were fixed in fixative. FAA (Formalin, Acetic acid, Alcohol) Mix: EtOH 95% Acetic acid Formalin (37% Formaldehyde) Dehydration Fixatives, such as formaldehyde, have the potential to further react with any staining procedure which may be used later in the process. Consequently, any remaining fixative is washed out by placing the blocks in running water overnight or by successive changes of water and/or a buffer. If the tissues are to be embedded in paraffin, all traces of water must be removed: water and paraffin are immiscible. The removal of water is dehydration. The dehydration process is accomplished by passing the tissue through a series of increasing alcohol concentrations. The blocks of tissue were transferred sequentially to 50%, 60%, 70%, 80%, 90%, and 100% alcohols for about two hours each. The samples were then placed in a second 100% ethanol solution to ensure that all water was removed. It is important to distinguish between dehydration and drying. Tissues should never be allowed to air dry. Dehydration involves slow substitution of the water in the tissue with an organic solvent Infiltration After dehydration, the tissues can be embedded in paraffin. For paraffin embedding, first clear the tissues. Clearing refers to the use of an intermediate fluid that is miscible with ethanol and paraffin, since these two compounds are immiscible. The most often used clearing agent is xylene. Incubate samples for 1 hour up to 1 day, depending on toughness and size: EtOH 100 % 50 ml 5 ml 10 ml

EtOH:Xylene 3:1 EtOH:Xylene 1:1 EtOH:Xylene 1:3 Xylene Xylene 100 % 100 % (samples can be stored) (samples can be stored)

Embedding Infiltrated samples were embedded on pre-warmed bench and wax blocks were stored at 4C until further processing. Sectioning Sections of 10 m thick were cut on a microtone, affixed to microscope slides by using 0.5 per cent gelatin as a adhesive. Staining

Deparatinize the slides in xylene or butanol using coplin jars for 4 hours to overnight. a. 1:3 (alcohol:xylene) for 10 mins b. 1:1 (alcohol:xylene) for 10 mins c. 3:1 (alcohol:xylene) for 10 mins

d. 100% alcohol 5 mins e. 90% alcohol 5 mins f. 80% alcohol 5 mins

g. 70% alcohol 5 mins h. 50% alcohol 5 mins i. Water for mins Then staining for 5-10 mins with saffranin and slides were air-dried. These were mounted with a xylene-based mountant DPX after dehydration through ethanol and histoclear. Microscope slides were examined under bright field with an Olympus light microscope and images were recorded using camera attached to an Olympus imaging system.

4. EXPERIMENTAL RESULTS
Tissue culture step is highly genotype dependent and Indian cultivars of cotton are shy for regeneration, so standardizing genotype independent regeneration methods are of great importance, because such system would also avoid problems encountered in other methods such as prolonged culture period of somatic embryogenesis, low frequency of embryo maturation high frequency of abnormal embryo development, low conversion rate of somatic embryo into plantlets and lack of shoot elongation. Hence the current investigations were carried out to standardize regeneration and transformation protocol in cotton through both in vitro and in vivo methods. The results obtained are presented in this chapter. Standardization of regeneration and transformation protocol

4.1 IN VITRO TRANSFORMATION STUDIES

4.1.1 Effect of explant preculture on regeneration after colonization and cocultivation
The shoot apices isolated from the aseptically grown seedlings of Jayahdar and Surabhi were inoculated in MS media supplemented with varied concentration of BAP before colonization and co-cultivation for 48 hours at 22C in dark to know the effect of BAP on the regeneration of plants. Then they were washed with cefotaxime (400 mg/l) and incubated in MS media supplemented with cefotaxime. All observations on plant survivability, regeneration, and shoot growth were recorded 35 days of culture and shown in Table 1. In Jayadhar, regeneration response was significantly higher when explants were supplemented with BAP of 1 mg/l (49), 2 mg/l(48) and 4 mg/l (44) than 0 mg/l (42) BAP. There was no significant difference between two media viz., 8 mg/l BAP (42) and 16 mg/l (42.5) and 0 mg/l (42). Highest response of 80 and 81.7 per cent was observed in media supplemented with 2 mg/l and 1 mg/l respectively. Which were on par with each other. Significantly higher regeneration response was observed in 1 mg/l (48.5), 2 mg/l (47.5), and 4 mg/l (47.5), 8 mg/l (44.5) BAP supplemented media in Surabhi than 0 mg/l (42) BAP. The higher concentration of BAP, 16 mg/l (43) showed on par with 0 mg/l (42).

4.1.2 Effect of Agrobacterium colonization period on regeneration

For the optimization of infection period before cocultivation, shoot apices isolated from aseptically grown seedlings subjected for 10 min, 30min, 60 min and 24 hrs of colonization with Agrobacterium tumefaciens EHA105 carrying cry2Aa gene and cocultivation for 48 hours in Murashige and Skoog medium containing (400mg/l) cefotaxime. Observations on number of plants obtained after 35 days of culture was recorded and data is presented in Table 2. The Analysis of variance showed significant effect due to colonization or infection period. Highest regeneration rate of 78.75 per cent in Jayadhar and 80 per cent in Surabhi was observed, when explants were not infected with Agrobacterium. In Jayadhar, maximum number of plants were obtained when explants were infected with Agrobacterium for 10 min (72.5%) followed by 30 min (67.5%) of colonization. The per cent regeneration was significantly on par (29 plants) when explants were colonized for lowest duration (10 min) in the experiment with no colonization (31.5 plants). The drastic reduction in regeneration was recorded in 24 hours of colonization (37.5%) response. In Surabhi, regeneration of explants were maximum, when explants were infected with Agrobacterium for 10 min (73.75%) followed by 30 min (67.5%) of colonization.

4.1.3 Effect of cocultivation duration on regeneration

For the optimization of cocultivation period, shoot apices isolated from germinated seeds were dipped in Agrobacterium suspension EHA105 harboring cry2Aa gene for 10 minutes, blot dried and cocultivated for 48, 72 and 96 hours. After washing, the co-cultivated

Table 1. Effect of explant preculture on regeneration after colonization and cocultivation

Sl. No 1 2 3 4 5 6 7 8 9 10 11 12

Genotype Jayadhar

Composition of media (BAP) 0 mg/l 1 mg/l 2 mg/l 4 mg/l 8 mg/l 16 mg/l

No. of explants responded 42.0 49.0 48.0 44.0 42.0 42.5 43.0 48.5 47.7 47.5 44.5 43.0 45.1 0.29 0.81 1.5

Per cent response 70.0 81.7 80.0 73.3 70.0 70.8 71.7 80.8 79.4 79.2 74.2 71.7

Surabhi

0 mg/l 1 mg/l 2 mg/l 4 mg/l 8 mg/l 16 mg/l Mean S.Em CD (1%) CV (%)

Note : A total of 60 explants were cultured under each treatment in three replications

Table 2. Effect of Agrobacterium colonization period on the regeneration of two cotton genotypes Sl. No. 1 2 3 4 5 6 7 8 9 10 Surabhi Duration of colonization 0 min 10 min 30 min 60 min 24 hrs 0 min 10 min 30 min 60 min 24 hrs Mean S.Em CD (1%) CV (%) No. of explants responded 31.5 29.0 27.0 25.0 15.0 32.0 29.5 27.0 26.5 16.0 25.85 0.86 2.4 5

Genotype Jayadhar

Per cent response 78.75 72.5 67.5 62.5 37.5 80.0 73.75 67.5 66.25 40.0

Note : A total of 40 explants were cultured under each treatment in three replication

Table 3. Effect of cocultivation duration on regeneration of two genotypes of cotton Sl. No. 1 2 3 4 5 6 Surabhi Jayadhar Duration of colonization 48 hours 72 hours 96 hours 48 hours 72 hours 96 hours Mean S.Em CD (1%) CV (%) No. of explants responded 14.3 8.3 5.7 15.0 8.7 6.3 9.71 0.15 0.43 2.76

Genotype

Per cent response 71.5 41.5 28.5 75.0 43.5 31.5

Note : A total of 20 explants were cultured under each treatment in four replication

Table 4. Effect of scalpel wounding and colonization duration on regeneration of two genotypes of cotton

Sl. No. 1 2 3 4 5 6 7 8 9 10

Genotype Jayadhar

Duration of colonization 0 min 10 min 30 min 60 min 24 hrs

No. of explants responded 31.5 24.0 23.0 22.3 13.5 32.0 24.5 23.0 22.5 15.5 23.18 0.2 0.58 1.63

Per cent response 78.75 60.00 57.50 55.75 33.75 80.00 61.25 57.50 56.25 38.75

Surabhi

0 min 10 min 30 min 60 min 24 hrs Mean S.Em CD (1%) CV (%)

Note : A total of 40 explants were cultured under each treatment in three replication

shoot apices were cultured on plain MS medium containing 400 mg/l cefotaxime. Observations on regeneration was recorded and presented in Table 3. Analysis of Variance showed significant difference between duration of co-cultivation in both the genotypes. Highest plant survivability was observed in 48 hours of co-cultivation that is 71.5 per cent in Jayadhar and 75 per cent in Surabhi. 48 hours of co-cultivation recorded significantly higher regeneration than 72 and 96 hours of co-cultivation in both the genotypes. There was no significant difference in regeneration response between two genotypes in 48 hours of co-cultivation.

4.1.4 Wounding methods

Transformation efficiency can be increased in Agrobacterium transformation by wounding the explants prior to, during or after incubation with the Agrobacterium. 4.1.4.1 Effect of scalpel wounding and colonization duration on regeneration Wound injury was made by using scalpel near the shoot apical meristem region of th shoot apex with slant cut and then explants were suspended in Agrobacterium culture for 0 min, 10 min, 30 min, 60 min and 24 hours of colonization period. Cocultivated for 48 hours in dark at 24C, followed by washing with cefotaxime and incubated in MS medium with cefotaxime 400 mg/l. Observations on regeneration was recorded and presented in Table 4. There was a significant effect due to scalpel wounding and colonization. Highest regeneration rate of (31.5) of 78.75 per cent in Jayadhar and 80 per cent in Surabhi (32) was observed, when explants were neither trimmed, nor infected with Agrobacterium. In Jayadhar maximum number of plants were obtained when trimmed explants were infected with Agrobacterium for 10 min (60%) followed by 30 min (57.5%). In Surabhi regeneration of explants were maximum for 10 min (61.25%) followed by 30 min (57.5%). 4.1.4.2 Influence of vacuum infiltration duration on regeneration Shoot apices isolated from aseptically grown seedlings with slant cut at SAM region and with no cut were vacuum infiltrated for 0 min, 10min, 20min, 30min, 60min with Agrobacterium tumefaciens EHA105 carrying cry2A gene before cocultivation for 48 hrs and cultured on Murashige and Skoog medium. The observations were recorded on total number of plants established on the regeneration medium and presented in Table 5. In Jayadhar, highest regeneration rate of (70%) was observed, when explants were not subjected for vacuum infiltration. The per cent regeneration was significantly on par (27.5), when explants were vacuum infiltrated for lowest duration (10 min) than no vacuum infiltration (28 plants). In Surabhi, highest regeneration rate of (65%) was observed, when explants were not subjected for vacuum infiltration. The per cent regeneration was significantly on par (24.5), when explants were vacuum infiltrated for lowest duration (10 min) than no vacuum infiltration (26 plants). There was a reduction in per cent survivability with trimming or slant cut in both the genotypes. Per cent regeneration varies from 0 min (47.5%), 10 min (41.25%), 20 min (45%), 30 min (48.75%) and 60 min (46.25%) in Jayadhar. In Surabhi maximum regeneration with trimmed explants accounted for (51.25%) with 0 minutes of vacuum infiltration and lowest accounted for 35 per cent with 60 minutes of vacuum infiltration. 4.1.4.3 Effect of blot drying or dessication on the regeneration Shoot apical meristem explants were subjected for drying for 0 min, 15 min, and 30 min and immersed in Agrobacterum suspension and cocultivated for 48 hours. After washing with cefotaxime explants were transferred to regeneration medium. The observation on regeneration was recorded and presented in Table 6.
th

Table 5. Influence of vacuum infiltration duration on regeneration of two cotton genotypes Sl. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 CUT at sam region Surabhi No cut at sam region Cut at sam region Duration of vacuum infiltration No cut at sam region 0 min 10 min 20 min 30 min 60 min 0 min 10 min 20 min 30 min 60 min 0 min 10 min 20 min 30 min 60 min 0 min 10 min 20 min 30 min 60 min Mean S.Em CD (1%) No. of explants responded 28 27.5 27 24.4 19 20 18 18 17 16.5 27 25 24.5 23.6 20 20.5 19 18.5 15.5 14 21.15 0.69 2.1 Per cent response 70 68.75 67.5 61 47.5 47.5 41.25 45 48.75 46.25 65 61.25 66.25 59 50 51.25 52.5 46.25 38.75 35

Genotype Jayadhar

Cv % 3.6 Note : A total of 40 explants were cultured under each treatment in two replications

Table 6. Effect of blot drying or dessication on the regeneration of cotton genotypes

Sl. No. 1 2 3 4 5 6

Genotype Jayadhar

Duration of blot drying 0 min 15 min 30 min

No. of explants responded 16.75 13.0 11.5 16.0 14.75 11.75 13.96 0.288 0.83 4.461

Per cent response 83.75 65.0 57.5 80.0 73.8 58.75

Surabhi

0 min 15 min 30 min Mean S.Em CD (1%) CV (%)

Note : A total of 20 explants were cultured under each treatment in four replications

Higher plant regeneration of 83.8 per cent in Jayadhar 80 per cent in Surabhi was observed, where explants were not subjected for desiccation / air drying. Lower survivability was recorded with explants which were dehydrated for 30min that is 57.5 per cent in Jayadhar and 60 per cent in surabhi than 15min survivability of 65 per cent in Jayadhar and 73.8 per cent in Surabhi. 4.1.4.4 Effect of chilling on regeneration Shoot apices of both the genotypes were subjected for chilling at 40c for different periods viz., 10 min, 30 min, 60 min, 24 hours, and 48 hours. Then explants were suspended in Agrobacterium culture, blot dried and incubated in MS medium for 48 hours in dark at 22 The observation on number of explants regenerated were taken after 35 days of culture C. and presented in Table 7. There was 78 per cent and 76 per cent survivability in Jayadhar and Surabi genotypes respectively, when explants were not subjected for chilling. In Jayadhar regeneration from explants chilled for 10 min (58%), 30 min (60%), 60 min (58%) and 24 hours (56%) were significantly higher than 48 hours (44%) of chilling. Significant differential response of regeneration was not observed between 10, 30 and 60 minutes of chilling injuries in Jayadhar. Regeneration from explants chilled for 10 min (52%), 30 min (60%), 60 min (54%) and 24 hours (48%) were significantly higher than 48 hours (40%) of chilling in Surabhi. There was no significant differential response of regeneration between 10, 30 and 60 minutes of chilling injuries in Surabhi. 4.1.4.5 Effect of Sand injury on regeneration Shoot apices isolated from germinated seeds were mixed with sterilized sand during colonization period before co-cultivation to make wounding on the explants. After 48 hours of co-cultivation, explants were washed with cefotaxime 400 mg/l and transferred to MS medium containing 400 mg/l cefotaxime. Observations on plant regeneration was recorded and data is presented in Table 8. Regeneration was more with 10 minutes of sand injury in both genotypes that is 68 per cent in Jayadhar and 60 per cent in Surabi than 30 min (54%), 60 min (48%) and 24 hours (32%) in Jayadhar and 30 min (50%), 60 min (40%), and 24 hours (28%) in Surabhi. Plant regeneration was significantly lower in all durations of sand injury (28-68%) than no injury in both genotypes 82 and 78 per cent in Jayadhar and Surabhi, respectively.

4.2 IN VIVO TRANSFORMATION STUDIES

4.2.1 Effect of scalpel wounding and colonization of Agrobacterium on plant proliferation of seedlings grown in pots
Wounding was done to the 8 days old seedlings grown in greenhouse. Several small incisions were done at the apical meristem region by using scalpel. Vertical cut was also given at the apical meristem region. Liquid and solid Agrobacterium culture was applied on the Shoot apical meristem region where cut was given. Observations on number of plants produced axillary shoots were recorded and shown in Table 9. Plant proliferation was significantly reduced in wound and vertical cut seedlings than control (no wounding and no colonization). However, as much as 65 per cent and 64 per cent plant proliferation was recorded in Jayadhar and Surabhi respectively when explants were wounded before colonization. Vertical cut followed by colonization resulted in reduction of plant proliferation in both the genotypes that is 41.5- 51 per cent in Surabhi and 42-50 per cent in Jayadhar.

Table 7. Effect of chilling on regeneration of two cotton genotypes Sl. No. 1 2 3 4 5 6 7 8 9 10 11 12 Surabhi No. of explants responded 19.5 14.5 15.0 14.5 14.0 11.0 19.0 13.0 15.0 13.5 12.0 10.0 14.25 0.135 0.37 1.26

Genotype Jayadhar

Duration of chilling 0 min 10 min 30 min 60 min 24 hrs 48 hrs 0 min 10 min 30 min 60 min 24 hrs 48 hrs Mean S.Em CD (1%) CV (%)

Per cent response 78 58 60 58 56 44 76 52 60 54 48 40

Note : A total of 25 explants were cultured under each treatment in three replications

Table 8. Effect of Sand injury on regeneration of cotton genotypes Sl. No. 1 2 3 4 5 6 7 8 9 10 Surabhi Genotype Jayadhar Duration of injury 0 min 10 min 30 min 60 min 24 hrs 0 min 10 min 30 min 60 min 24 hrs Mean S.Em CD (1%) CV (%) No. of explants responded 20.5 17.0 13.3 12.0 8.0 19.5 15.0 12.5 10.0 7.0 13.48 0.972 2.768 2.295 Per cent response 82.0 68.0 53.2 48.0 32.0 78.0 60.0 50.0 40.0 28.0

Note : A total of 25 explants were cultured under each treatment in three replications.

Table 9. Effect of scalpel wounding and colonization of Agrobacterium on plant proliferation of Jayadhar genotype in MS media

Sl. No.

Treatment

No of explants responded

Per cent response

No wounding

50

100

Wound at SAM+liquid culture

36

72

Vertical cut at SAM+liquid culture

22.5

45

Vertical cut at SAM+solid culture

19.2

38.4

Mean

31.92

S.Em

0.17

CD (1%)

0.498

CV %

2.38

Note : A total of 50 explants were cultured under each treatment in five replications.

Table 10. Effect of scalpel wounding at the shoot apex region and colonization of Agrobacterium on regeneration of two cotton genotypes in pots

Sl. No.

Genotype

Treatments

No. of explants responded

Per cent response

Jayadhar

No wounding

50

100

Wound injury

32.5

65

Vertical cut+liquid culture

25

50

Vertical cut+solid culture

21

42

Surabhi

No wounding

50

100

Wound injury

32

64

Vertical cut+liquid culture

25.5

51

Vertical cut+solid culture

20.75

41.5

Mean

32.09

S.Em

0.29

CD (1%)

0.79

Cv %

2.5

Note : A total of 50 explants were cultured under each treatment in four replications

4.2.2 Effect of scalpel wounding at the shoot apex region and colonization of Agrobacterium on regeneration of seedlings grown in MS media
Apical meristem or leaf primordial region of 10 days old seedlings of Jayadhar genotype grown in greenhouse was injured by doing small incisions and also leaf primordial region was halved followed by application of Agrobacterium culture to the Shoot apical meristem region. The observation on number of plants produced axillary shoots were recorded after 35 days and shown in Table 10. In Jayadhar, there was significant reduction in regeneration response of wound and vertical cut seedlings followed by colonization than control (no wounding and no colonization). When explants were wounded before colonization, plant proliferation of 72 per cent was observed. There was a reduction in plant proliferation (38.4-45%), when plants were given vertical cut followed by colonization of Agrobacterium.

4.3 INDUCTION OF AXILLARY SHOOTS

Though meristem based transformation systems are advantageous in that they are faster, but the plants are chimeras and it is difficult to identify germline transform ants. If axillary or multiple shoots could be produced from the transformed meristem cells, then it improves the probability of germline transformation of recalcitrant cultivars. This experiment involves regeneration of plant and induction of axillary shoots by removing shoot apical meristem or primordia.

4.3.1 Induction of axillary shoots by removing shoot apical meristem of shoot apex
Apical meristem of embryo axes isolated from aseptically germinated seeds were removed by giving different types of horizontal cut viz, removal of leaf primordia, 1/4th of 1mm rd of shoot apex, 1/3 portion of shoot apex. Observation on number of explants produced shoots were recorded and shown in Table 11. Plant regeneration was significantly lower in trimmed explants (6-16% in Jayadhar, and 14-28% in Surabhi), than control (70% & 60% in Jayadhar and Surabhi, respectively).

4.3.2 Effect of removal of apical dominance of seedlings which were grown in MS broth supplemented with PGRs on induction of axillary shoots
Leaf primordia were removed from the aseptically grown 8 days old seedlings. and grown in MS broth with varied concentration of BAP (5 mg/l, 10 mg/l, 20 mg/l, 40 mg/l, 100 mg/l) TDZ (5 mg/l, 10 mg/l, 20 mg/l, 40 mg/l, 100 mg/l) and combination of NAA (0.1 mg)+BAP (5 mg/l, 10 mg/l, 20 mg/l, 40 mg/l) for 5 to 6 days to identify the effect of growth regulators on induction of axillary shoots. Plants were observed under microscope and observation on number of plants produced axillary shoots were recorded and presented in Table 12 and 13. In Jayadhar, there was no induction of axillary or cotyledonary shoots from the explants where apical dominance was not suppressed. 71.7 per cent of plants could produce axillary shoots in MS medium and it was on par with plants which were grown in MS broth with 5 mg/l BAP. Induction of axillary shoots by growing seedlings in MS broth with 10 mg/l (64.2%), 20 mg/l (65%), 40 mg/l (59.2%), 100 mg/l (58.3%) BAP significantly were lower than MS broth (71.7%). Similarly TDZ at 5 mg/l (70.8%) was on par with MS broth 71.7 per cent and rate of axillary shoot induction in MS with 10 mg/l (61.7%), 20 mg/l (60.8%), 40 mg /l (58.3%), 100 mg/l (59.2%) BAP were on par. Combination of NAA (0.1 mg/g) and BAP at 5 mg/l (65%), 10 mg/l (65.8%), 20mg/l (65.8%), and 40 mg/l (64.20%) were on par. In Surabhi, there was no induction of axillary or cotyledonary shoots from the explants where apical dominance was not suppressed. Less number of plants produced axillary shoots when they were grown in MS broth containing 10 mg/l (65%), 20 mg/l (58.2%), 40 mg/l (58.75%) and 100 mg/l (55%) than MS broth (70%).

Table 11. Effect of removal of apical meristem of shoot apex by giving different types of cuts on regeneration

Sl. No. 1 2 3 4 5 6 7 8

Genotype Jayadhar

Types of cut No removal Removed leaf primordia Removed 1/4th of 1mm of SAM Removed 1/3 rd of SAM

No. of explants responded 24.3 4 2 1.5 24 7 4.5 3.5 8.85 0.187 0.548 2.73

Per cent response 97.2 16 8 6 96 28 18 14

Surabhi

No removal Removed leaf primordia Removed 1/4th of 1mm of SAM Removed 1/3 rd of SAM Mean S.Em CD (1%) CV %

Note : A total of 25 explants were cultured under each treatment in three replications

Table 12. Effect of removal of apical dominance of seedlings and grown in MS broth supplemented with PGRs on induction of axillary in Jayadhar genotype

Sl No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1/2MS

Composition of media

No. of plants produce axillary shoots 43 42 38.5 39 35.5 35 42.5 37 36.5 35 35.5 39 39.5 39.5 38.5 38.4 0.683 2.013 2.845

Per cent response 71.7 70 64.2 65 59.2 58.3 70.8 61.7 60.8 58.3 59.2 65 65.8 65.8 64.2

1/2 MS+BAP(5mg/l) 1/2 MS+BAP(10mg/l) 1/2 MS+BAP(20mg/l) 1/2 MS+BAP(40mg/l) 1/2 MS+BAP(100mg/l) 1/2 MS+TDZ(5mg/l) 1/2 MS+TDZ(10mg/l) 1/2 MS+TDZ(20mg/l) 1/2 MS+TDZ(40mg/l) 1/2 MS+TDZ(100mg/l) 1/2 MS+NAA(0.1mg/l)+BAP(5mg/l) 1/2 MS+NAA(0.1mg/l)+BAP(10mg/l) 1/2 MS+NAA(0.1mg/l)+BAP(20mg/l) 1/2 MS+NAA(0.1mg/l)+BAP(40mg/l) Mean S.Em CD (1%) CV %

Note : A total of 60 explants were cultured under each treatment in two replications

Table 13. Effect of removal of apical meristem of seedlings and grown in MS broth supplemented with PGRs on induction of axillary shoots in Surabhi genotype No. of plants produce axillary shoots 28 27.25 26 23.25 23.5 22 27.5 24.75 24.75 22.75 22.5 26.75 25.5 24 23.5 24.8 0.300 0.808 2.203

Sl No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1/2MS

Composition of media

Per cent response 70 68.125 65 58.125 58.75 55 68.75 61.875 61.875 56.875 56.25 66.875 63.75 60 58.75

1/2 MS+BAP(5mg/l) 1/2 MS+BAP(10mg/l) 1/2 MS+BAP(20mg/l) 1/2 MS+BAP(40mg/l) 1/2 MS+BAP(100mg/l) 1/2 MS+TDZ(5mg/l) 1/2 MS+TDZ(10mg/l) 1/2 MS+TDZ(20mg/l) 1/2 MS+TDZ(40mg/l) 1/2 MS+TDZ(100mg/l) 1/2 MS+NAA(0.1mg/l)+BAP(5mg/l) 1/2 MS+NAA(0.1mg/l)+BAP(10mg/l) 1/2 MS+NAA(0.1mg/l)+BAP(20mg/l) 1/2 MS+NAA(0.1mg/l)+BAP(40mg/l) Mean S.Em CD (1%) CV %

Note : A total of 40 explants were cultured under each treatment in four replications

Table 14. Effect of different concentration of Kanamycin on the explants of cotton

Sl. No. 1 2 3 4 5 + -

Kanamycin Treatment (mg/l) Control (0) 50 100 150 200 = Survival = Death

Explants after two weeks + + + -

Plate 1. Kanamycin sensitivity test A- Kanamycin susceptible plant B- Kanmycin resistant plant

Similarly, 5 mg/l of TDZ (27.5) was on par with MS broth in producing axillary shoots . Axillary shoot induction in MS with 10 mg/l (61.8%), 20 mg/l (61.9%) TDZ were on par and 40 mg/l (56.9%), 100 mg/l (56.25%) TDZ were on par. Per cent regeneration for different combination of NAA (0.1 mg/l) and BAP (5 mg/l, 10 mg/l, 20 mg/l, 40 mg/l) varied from 58.7566.9 per cent.

4.4 DETERMINATION OF KANAMYCIN SENSITIVITY

In this experiment various levels of kanamycin were used viz., 50 mg/l, 100 mg/l, 150 mg/l, 200 mg/l. Shoot apex along with 2 to 3 top leaves of control plants were transferred to MS medium containing 50 mg/l, 100 mg/l, 150 mg/l and 200 mg/l of kanamycin. Among these, kanamycin concentration at 150 mg/l and 200 mg/l resulted in immediate death of control plants (Table 14). Hence shoot apex position along with top 2 to 3 leaves of all treated plants were transferred to MS medium containing 150 mg/l of kanamycin.

4.4.1 Selection of putative transformants

Out of 11220 successfully cocultivated and regenerated plants established in vitro. Only 26 of them showed kanamycin resistance (Plate 1).

4.4.2 Confirmation of cry2Aa gene integration in Jayadhar and Surabhi cotton genotypes
PCR Amplification To look for the presence of the transgenes, amplification of npt-II and cry2Aa gene was done through PCR. The genomic DNA from the putatively transformed plants as well as the control plants and plasmid DNA from the Agrobacterium was isolated. Plasmid DNA served as a positive control. DNA from control untransformed plant was used as a negative control. The primer specific for the npt-II and cry2Aa gene was used in the PCR analysis. The PCR products were electrophoresed on a 1.2 per cent agarose gel using 1kb ladder as the marker. An approximately 700 base-pair band of the npt-II gene and 1.2 kb of cry2Aa was obtained. The untransformed control plants naturally did not produce any bands (Plate 2). Although 14 plants belonging to Jayadhar cultivar were resistant to kanamycin, only three were PCR positive, one in scalpel wounding, two in chilling method and similarly, in Surabhi out of 12 kanamycin resistant plants only one was PCR positive in preculture treatment at 1 mg/l of BAP (Table 15, 16 and 17).

4.5 HISTOLOGICAL STUDIES

The type of explants used for histological analysis are 1) Apical dominance of aseptically grown seedlings of 8-10 days old seedlings were removed to induce axillary buds. After 6 days of removal of apical dominance, explants were observed under microscope for the presence of axillary buds and were photographed (Plate 3). Cotyledonary leaves were detached from the seedlings and approximately 5 to 10 mm length of shoot apex region along with axillary buds were fixed for histological analysis. 2) Shoot apices isolated from aseptically germinated seeds with different types of cut viz., removal of leaf primordia, 1/4th portion of 1mm of shoot apex, 1/3 rd portion of shoot apex were used for histological analysis (Plate 4).

Plate 2a. Amplification of cry 2Aa transformed plants of Jayadhar and surabhi genotypes M Marker + Plasmid DNA - Untransformed Plants Lane 2,4,5 and 7 Shows positive amplification for cry 2Aa primers

Plate 2b. nptll amplification of cry 2Aa transformed plants of Jayadhar and Surabhi genotypes M Marker + Plasmid DNA - Untransformed Plants Lane 2,4,5 and 7 Shows positive amplification for cry 2Aa primers

Plate 3. Explant preparation for induction of axillary shoots in Jayadhar and Surabhi ABCDEControl Primordial Leaves removed Removal of 1/4th of 1 mm SAM th Removal of 1/3 of SAM to H Histological view of SAM were different types of horizontral cut was given

D Plate 4. Axillary bud induction in Surabhi and Jayadhar A- Control (no removal of apical meristem) B- Removal of apical meristem C- Hisological view of explant where apical meristem was not removed D- Histological view of explant apical meristem was removed

Table 15. Transformation studies in Jayadhar genotype

Sl. No.

Treatments

No. of plants cocultivated

No of plants regenerated

No of kanr plants 3 0

No of PCR positive plants 0 0

1 2

Preculture studies Effect of colonization duration Effect of cocultivation duration Scalpel wounding on apical meristem of shoot apex Vaccum infiltration Chilling injury Blot drying Sand injury Effect of scalpel wounding on SAM of established seedlings grown in MS media Effect of scalpel wounding on SAM of established seedlings grown in pots Total

1080 600

494 382

240

85

600

367

5 6 7 8 9

800 450 240 375 1000

215 362 261 294 638

1 3 1 0 0

0 2 0 0 0

10

800

417

6110

3515

12

Table 16. Transformation studies in Surabhi genotype No of PCR positive plants 1 0

Sl. No.

Treatments

No. of plants cocultivated

No of plants regenerated

No of r kan plants 2 0

1 2

Preculture studies Effect of colonization duration Effect of cocultivation duration Scalpel wounding on apical meristem of shoot apex Vaccum infiltration Chilling injury Blot drying Sand injury Effect of scalpel wounding on SAM of established seedlings grown in pots Total

1080 600

495 393

240

90

600

369

5 6 7 8 9

800 450 240 375 800

208 362 482 296 470

3 2 1 0 0

0 0 0 0 0

5110

3165

14

Table 17. Gene integration studies in T0 (putative) transgenic plants

Cultivar

No. of cocultivated plants

No. of well established plants

No. of r plants kan

PCR positive

Transformation frequency (%)

Jayadhar

6110

3166

12

0.049

Surabhi

5110

3515

14

0.019

Total

11220

6681

26

0.036

5. DISCUSSION
Conventional means of genetic manipulation of economically important crop plants has significantly increased the production in several crops. However, it has several limitations: The transfer of genes is possible only between sexually compatible species and there is a possibility of transfer of undesirable genes along with desirable genes and others. With the advent of molecular biology and genetic engineering techniques, it is now possible to overcome the difficulties associated with conventional plant breeding. One of such method is the transformation of plants with an alien gene. The transformation of cotton can be attempted, once an efficient and reproducible regeneration system is established. The two basic methods employed for the introduction of the genes into cotton are direct gene transfer via particle bombardment and Agrobacterium mediated transformation. The regeneration of a plant from the transformed cell is dependent on the nature of the explants as well as the genotype transformed (Kumaria et al., 2003). The prolonged culture period and the genotype dependent response can be overcome by biolistic delivery of the transgene of interest. Transformation of embryogenic suspension cultures of cotton by particle bombardment was first reported by Finer and McMullen (1990); they recovered transgenic plants within five-month period. The method was adopted to produce herbicide resistant Acala and Coker cotton (Rajasekaran et al., 1996). Further refinement of this technique increased the recovery rate of 30 per cent from cryopreserved culture. Refinement consisted of subjecting rapidly growing suspension cultures to multiple bombardments followed by gradually increasing the selection pressure. This procedure promotes the development of stable transformed lines (Rajasekaran et al., 2000). The bombardment of meristems has been reported to completely bypass the somatic embryogenesis step. However, the protocol is extremely labour intensive, as it requires the removal of leaf primordial tissues around the meristems and their excision from seeds or seedlings. Very low percentages (0.001-0.01) of the bombarded meristems were transformed. This is because stable transformation occurs, only when the germline cells in the L2 and L3 layers of the meristematic tissue are transformed. The penetration of particles in the epidermal layer is higher but they dont transmit the gene to advance generation (McCabe and Martinell 1993; Chlan et al. 1995). Splitting the meristem to expose the L2 layer has been found to promote reorganization of the meristem layers, thereby increasing the selection pressure. Wilkins et al. (2000) tried this method to improve germ-line transformation events. Pruning of meristems has been found to induce the development of lateral buds, giving rise to germline transformants. John (1997) reported that by this method the frequency of transformation improved (0.09%). The induction of multiple shoots from the meristem also increased the number of shoot- producing transformed meristematic cells (Agarwal et al. 1997, Gupta et al. 1997, Morre et al. 1998). Even though the germline transformation is genotype independent, it does not involve any tissue culture phase and takes less time than somatic embryogenesis- based protocols. Bombardment leads to multiple insertions of the transgene, of which a few could be truncated with or without re-arranged sequences. Thus such an event leads to gene silencing in subsequent generations, resulting in the loss of trait. The procedure is highly skilled, labour intensive and the trasgenics can be obtained only in the T1 generation. Furthermore, chimaeras are frequently observed (Wilkins et al. 2000). Direct gene transfer to cotton protoplasts has not been demonstrated as the isolation and culture of protoplasts is a difficult procedure with only a single report of regeneration (Peeters et al. 1994). The transformation of cotton via Agrobacterium is a simple and efficient method of choice. Cotton transformation via Agrobacterium was first reported by Firoozabady et al. (1987) and Umbeck et al. (1987). Since then eighteen US patents have been granted on cotton transformation and regeneration. Besides transformation of seedling explants and embryogenic suspensions, meristems have also been transformed via Agrobacterium (Gould and Magallanes-Cedeno 1998, Zapata et al., 1999). The transformed meristems were selected on media supplemented with Kn or BA and NAA to obtain well rooted plants or plants that were grafted to a rootstock (Luo et al., 1999; Satyavathi et al., 2002). Since the meristems did not de-differentiate, the interventing callus phase and somatic variations were

also absent. This method has the advantage of being genotype independent; it is rapid and also possesses a fewer number of transgene insertions. The main disadvantage is that it is labour intensive and the transformants are chimaeric. For optimization of the transformation protocol, interaction between Agrobacterium tumefaciens and plant is probably the most important aspect to be considered. Regardless of the method of delivery of the new genetic material, it is necessary to regenerate whole fertile plants from the transformed cells. The production of stably transformed transgenic plants involves two processes: transformation of plant cells and regeneration of those transformed cells to whole plants. Because neither the transformation nor the regeneration is 100% effective, the chance of obtaining a transformed plant depends on these two processes occurring consecutively in the same cell. In many cases, the production of transgenic plants is prevented due to the inability to regenerate plants from those tissues amenable to transformation. In view of the above, current investigations were aimed at developing a protocol to increase the regeneration and transformation efficiency by using both in vitro and in vivo methods and the findings are discussed under the following heads. Many factors influencing Agrobacterium mediated transformation of dicotyledonous plants such as preculture of the explants, colonization period, cocultivation period, wounding methods like scalpel wounding, vacuum infiltration, chilling injury, blot drying, sand injury and also removal of apical dominance are considered critical.

5.1 IN VITRO TRANSFORMATION STUDIES

5.1.1 Effect of preculture studies on regeneration
To transform most plants using Agrobacterium, a single plant cell that has received the new DNA from the bacteria has to be regenerated into a whole plant. This process involves the culturing of the transformed cell to provide replication of that cell. Levels of plant hormones can be manipulated to cause this mass of cells to form roots and shoots of a regenerated plant. Use of precultured shoots in the inoculation step is to ensure activation of cell division in apical meristematic tissues. In this study, explants were precultured for 48 hours at different concentration of BAP before co-cultivation with Agrobacterium tumefaciens. The results show that regeneration response was significantly higher when explants were supplemented with 1 mg/l (49), 2 mg/l (48) and 4 mg/l (44) than 0 mg/l (42) in Jayadhar. Significantly higher regeneration response was observed in 1 mg/l (48.5), 2 mg/l (47.5) and 4 mg/l (47.5) BAP supplemented media in Surabhi than 0 mg/l (42) BAP. Preculturing of the explants on the regenerating medium prior to incubation of Agrobacterium increased the frequency of transformants in pigeon pea to 62 per cent from cotyledonary node and 27 per cent from shoot tip (Geetha et al., 1999), 28.8 per cent from callus and 1.2 per cent from shoots regenerated (Lawrence and Koundal, 2001). Satyavathi et al. (2002) have reported consistent production of transgenic cotton plants in three Indian varieties. Among the different combinations of BAP and NAA tested, 0.1 mg/l of BAP and NAA in the medium influenced efficient regeneration of shoots by organogenesis. Shoot bud proliferation and elongation were achieved in 3-4 weeks time on medium supplemented with GA3. Moralejo et al. (1998) have described a procedure for genetic transformation of Eucalyptus globulus (Labill) and they have studied the influence of explant pre-cultivation and reported that when seedlings were precultured for 4-6 days, the level of GUS transient expression was significantly greater than that of control (i.e. without pre-culture) and that the seedlings precultured for 6 days seemed to be more suitable for stable integration of transgenes. They further reported that the improvement of DNA uptake could be due to stimulation of cell division by the hormones in the pre-cultivation medium, since mitotic cells would be more susceptible to Agrobacterium (or) would have a higher level of transcription. However, they also opined that the physiological status optimal for DNA uptake (transient expression) is not necessarily optimal for integration for foreign DNA in the host genome.

Infact, division of cells would not be sufficient for transformation and integration may primarily depend on the availability of DNA repair enzymes. These proteins, although not yet identified are thought to play a key role in the last steps of T-DNA integration (Tinland, 1996). Gould et al. (1998) also opine that presence of actively dividing cells (transformation competant cells) in the shoot apex is important in Agrobacterium mediated transformation.

5.1.2 Effect of duration of colonization on regeneration

Krishnan et al. (2008), reported effective transformation both in 15 and 20 minutes infection period, but the transformation efficiency was more when more callus was used for co-cultivation with infection carried out for 20 minutes in Centella asiatica. In the present investigation regeneration response of 72.5 per cent and 73.75 per cent was accounted in 10 minutes of colonization in Jayadhar and Surabhi respectively. Regeneration rate decreased with increase in colonization period.

5.1.3 Effect of duration of co-cultivation on regeneration

The length of cocultivation time employed varies widely from a few hours to a few days depending upon the species, explants and culture condition. Longer periods of cocultivation seem to be effective for efficient transfer of the Ti plasmid to plant cells. Cao et al. (1998) suggested that low efficiency of transformation in earlier work on apples was due to insufficient length of cocultivation. The prolonged period of cocultivation from 2 to 4 days increased GUS expression by almost 100 fold. In the current study, the optimum time period for cocultivation was determined to be 48 hrs. Co-cultivation beyond this period resulted in softening, browning and death of the explants and the survival was very poor. Muthukumar et al. (1996) have observed overgrowth of bacterium beyond three days of cocultivation among different days to co-cultivation based on resistant shoot production on media containing hygromycin. Liu et al. (1990) studied the influence of different concentration periods, tissue types and bacterial strains for transformation in Capsicum annuum L. In general, 24 hours cocultivation period gave rise to slightly but significantly greater number of larger tumours. However, differences were not significant, for leaf tissue, per cent tumour formation in cultivars ECW, YWL and JPT, for tumour size in ECN and LBB. Because, 24 h cocultivation period, produced tumours on approximately 85 per cent of explants within 4 weeks and nopaline was detected in tumours with strain C58. They concluded that 24 hours cocultivation would be adequate for transformation of pepper. Shauangxia et al. (2005) found that shoot cocultivation duration of 48 hours were optimal for developing a highly efficient method of transforming EC in cotton. Cervera et al. (1998) reported that the 5-day culture period resulted in over growth of Agrobacterium and a subsequent decrease in regeneration frequency of the transformed shoots. Therefore in our study, the effects of varying periods of co-cultivation was studied and it was found that 48 hours was optimum with regeneration response of 71.5 per cent and 75 per cent in Jayadhar and Surabhi, respectively. Co-cultivation beyond 48 hours resulted in overgrowth of the bacteria, resulting in softening and death of the tissue.

5.1.4 Effect of wounding methods on regeneration

Recently, it has been observed that the efficiency of transformation can be enhanced by supplementing with wounding methods. Wounding methods may include, but are not limited to vacuum infiltration, scalpel, needles, desiccation or blot drying, sand injury, chilling injury. 5.1.4.1 Scalpel wounding Certain plant species have proved to be more difficult to transform with Agrobacterium than others, particularly members of the monocotyledonous plant family. Transformation of the dicot cotton has also been particularly difficult. Since monocotyledonous plants generally do not form crown galls, it was initially assumed that the

host range of Agrobacterium was restricted to dicotyledonous plants. However, Stephen and Graves (2006) described a process for transforming plants, including the monocot corn, in US pat. Nos. 5,177,010 and 5,187,073. These methods involve making a wound in a seedling in an area containing rapidly dividing cells, then inoculating the wound with Agrobacterium. Explants which were trimmed followed by colonization of bacterium showed regeneration response of 60 per cent and 61.75 per cent respectively in Jayadhar and Surabhi as compared to control explants (no trimming, no colonization) accounted 78.75 per cent and 80 per cent of regeneration response in Jayadhar and Surabhi respectively. Bombardment of the plant tissues with micro-projectiles promoted Agrobacterium mediated transformation (14.9%) in tobacco (Bidney et al., 1992) and Eucalyptus (Moralejo et al., 1998). 5.1.4.2 Effect of vacuum infiltration Vacuum infiltration is one of the simple methods to improve the reach of Agrobacterium to different parts of the tissue. Physically vacuum creates a negative atmospheric pressure that causes the air spaces between the cells in the plant tissue to decrease. The longer the duration and the lower the pressure of the vacuum, the less air space there within the plant tissue. An increase in the pressure allows the infiltration medium, including the infective transformation vector, to relocate into the plant tissue. For plant transformation, vacuum is applied to a plant part in the presence of Agrobacterium for a certain period. The length of time that a plant part or tissue is exposed to vacuum is critical as prolonged exposure causes hyperhydricity. Shoot apices of both the genotypes Jayadhar and Surabhi were vacuum infiltrated for 10 min with Agrobacterium culture, gave maximum frequency of shoots on MS medium (68.75% in Jayadhar and 61.25% in Surabhi) as compared to control plants with no vacuum infiltration (70% in Jayadhar and 65% plants in Surabhi). Applying a vacuum to plant organs in the presence of Agrobacterium removes intercellular fluids and air, which are replaced by bacteria when the vacuum is released (Hewezi et al., 2002). Vacuum infiltration assisted Agrobacterium mediated transformation was reported for the first time in Arabidopsis thaliana (Bechold et al., 1993). This method involves application of Agrobacterium to whole plants via vacuum infiltration of flowering stage. During vacuum infiltration Agrobacterium infection is supposed to improve the reach and contact between tissue/cell surfaces and the bacteria (Effendi et al., 2000). Mehrazad et al. (2002) reported a rapid protocol based on vacuum infiltration of Agrobacterium into lentil cotyledonary nodes. Vacuum infiltrated tissues and significantly (P<0.05) higher transient GUS expression than non-filtrated tissues. Under optimal conditions (infiltration at 200 mmHg for 20 minutes), about 95 per cent of Agrobacterium infiltrated explants exhibited an average of 16 blue foci. An Agrobacterium mediated gene transfer system was developed for cotton (Gossypium hirsutum L.) cv. Cocker-312 (Ikram, 2004). In this study, two month old hypocotyls derived embryogenic calli were treated for 10 min at 27 psi in a suspension of Agrobacterium culture. In four independent experiments up to 28.23 per cent transformation efficiency was achieved. 5.1.4.3 Effect of blot drying Dehydration and rehydration of embryonic axes with Agrobacterium was aimed to mimic the effect of vacuum infiltration. Because the loss of turgor pressure would result in an increased uptake of Agrobacterium and improve transformation efficiencies. Blot dried explants showed lower regeneration response of 57.5 per cent in Jayadhar and 60 per cent in Surabhi for 30 min of desiccation than 15min survivability of 65 per cent in Jayadhar and 73.8 per cent in Surabhi.

Hewezi et al. (2002) reported dehydration or rehydration of RHA266 (sunflower) immature embryos allowed bacteria to reach the innermost meristematic cell layers. T-DNA was successfully transferred and integrated, and transgenes were expressed in the shoot apices with the recovery of one transgenic plant in the progeny. Although the stable transformation efficiency of this procedure is still low (0.22%). 5.1.4.4 Effect of chilling United states patent (7122722), reported that their invention relates to methods for transforming cotton using isolated shoot apical tips of seedlings. The apical shoot tips of cotton seedlings are isolated and chilled at a low temperature to slow the metabolic activity of the isolated cells, and then inoculated with a transforming agent carrying a desired gene. The invention also provides a method of regenerating transformed cotton plants with a desired trait from transformed apical shoot tips. Explants chilled for more than 48 hours showed lower regeneration response (44% in Jayadhar and 40% Surabhi) as compared to 10 min (58%), 30 min (60%), 60 min (58%), 24 hours (56%) of chilling in Jayadhar and 10 min (52%), 30 min (60%), 60 min (54%), 24 hours (48%) of chilling in Surabhi. 5.1.4.5 Effect of sand injury Though, Plant regeneration was significantly lower in all durations of sand injury (2868%) than no injury in both the genotypes (82 and 78%) respectively in Jayadhar and Surabhi, 10 minutes of sand injury in both genotypes gave maximum regeneration response ( 68 per cent in Jayadhar and 60 per cent in Surabhi) than 30 min (54%), 60 min(48%) and 24 hours (32%) in Jayadhar and 30 min(50%),60 min(40%), and 24 hours(28%) in Surabhi. Bidney et al. (1992) have reported that bombardment of plant tissues with microprojectiles is an effective method of wounding to promote Agrobacterium mediated transformation in sunflower. The leaves bombarded with particles suspended in TE buffer prior to Agrobacterium treatment produced at least 100 times more kanamycin resistant colonies than leaves treated by the standard particle gun transformation protocol. In addition, large sectors of GUS expression, indicative of meristem cell-transformation, were observed in plants recovered from sunflower apical explants only when the meristems were wounded first by particle bombardment prior to Agrobacterium treatment. Similar results in two different tissue types suggest that, particles may be used as a wounding mechanism to enhance Agrobacterium transformation frequencies, and Agrobacterium mediation of stable transformation is more efficient than the analogous particle/ plasmid protocol.

5.2 IN VIVO TRANSFORMATION STUDIES

Although Agrobacterium mediated transformation with regeneration via somatic embryogenesis or direct organogenesis is a viable approach, these methods are very labour intensive, not very efficient, and in some cases very slow. Hence, an Agrobacterium mediated in vivo transformation method has been carried out where in plants were exposed directly to Agrobacterium cells, eliminating the need for preparation of cell or callus cultures explants or other materials for treatment. Moreover, regeneration steps are eliminated; treated plants are allowed to mature and set seed. Wilkins et al. (2000) tried a method to improve germ-line transformation events. Pruning of meristems has been found to induce the development of lateral buds, giving rise to germline transformants.

5.2.1 Effect of scalpel wounding on SAM of established seedlings grown in pots followed by colonization with agroculture
Wounded explants showed as much as 65 per cent and 64 per cent plant proliferation respectively in Jayadhar and Surabhi than control plants with no wounding and colonization. Vertical cut followed by colonization resulted in reduction of plant proliferation in both the genotypes that is 41.5- 51% in Surabhi and 42-50 per cent in Jayadhar.

5.2.2 Effect of scalpel wounding on SAM of established seedlings grown in MS media followed by colonization with agroculture
Wound and vertical cut seedlings followed by colonization showed significant reduction in regeneration response than control (no wounding and no colonization) was observed. Plant proliferation of 72 per cent was observed with wounded explants. There was a reduction in plant proliferation (38.4-45%), when plants were given vertical cut followed by colonization of Agrobacterium.

5.3 STUDIES ON INDUCTION OF AXILLARY SHOOTS

Maximum number of apical dominance suppressed plants which were grown in MS broth produced axillary shoots (71.7% in Jayadhar and 70% in Surabhi) than plants grown in different combination of BAP, TDZ, and combination of NAA and BAP. None of the growth regulators concentration gave better response than MS broth in inducing axillary shoots. Lee (1987) reported that Plants of the tetraploid cotton species Gossypium hirsutum L. ordinarily produce secondary shoots only from axillary buds. Destruction of the primary shoot by excision below the cotyledonary axils occasionally leads to the production of adventitious shoots. Two plants of the cultivar Coker 201 produced adventitious shoots from what appeared to be callus tissue growing outward from stem cambium after 12 plants had been severed at the ground line. A plant of the "honseyard" stock Orinoco produced several shoots from globular masses of tissue that grew from the roots after the shoot was removed. Another plant of Orinoco, grown from seed produced on the first, produced shoots at two sites near the ground line of a stump from which the trunk had been removed. After removal of the shoots, the plant grew three additional groups of shoots, including one group from a root about 2 cm from the stump masses of tissue that grew from the roots after the shoot was removed. Another plant of Orinoco, grown from seed produced on the first, produced shoots at two sites near the ground line of a stump from which the trunk had been removed. After removal of the shoots, the plant grew three additional groups of shoots, including one group from a root about 2 cm from the stump. Potrykus (1990) has reported that in cereals the shoot apical meristem of plants is a very important tissue in the developmental biology of plant as it generates the whole green part of the plant body, including the flowers. In particular, the meristem is a tissue with a very high regeneration potential. Gene transfer to meristem cells could therefore, overcome many of the regeneration problems due to difficult tissue culture systems in many important crop species. Medford (1992) has reported that the meristem is often confused with the complete shoot apex, which also contains the leaf primordia and the young leaves. Furthermore, the meristem as a tissue may represent a complicated pattern of cells. Each of these cells may differ physiologically due to its unique position in the meristem. Hemphill et al. (1998) reported a clonal propagation system to regenerate mature cotton (G. hirsutum L.) plants from preexisting meristem from in vitro grown tissues. Shoot apices, lateral nodes and cotyledinary nodes were co cultivated with A. tumefaciens and grow to a two-leaf stage by this in vitro culture system. They further reported that this procedure resulted in 121 kanamycin selected shoots and 40 mature viable plants, which produced viable T1 seeds. Mature T1 plants expressed GUS activity in pollen grains that suggested that the transgene was inherited by progeny.

5.4 SCREENING OF TRANSFORMANTS

As meristem based transformation produces more chimeras, kanamycin screening during regeneration is not suitable, because it may reduces the probability of getting transformants. So kanamycin screening was done after 35 days of culture. Kanamycin was used as a selective agent to pick up the transformed plants invitro. In order to avoid the possibility of selecting the untransformed plants on medium containing kanamycin, the intrinsic kanamycin resistance of cotton shoots was determined.

Shoot apex along with top 2-3 leaves were tested for in vitro kanamycin sensitivity. Shoot apex were inoculated on MS medium with different concentrations of kanamycin and 150 mg/l was found to be lethal to all explants of cotton as this concentration resulted in browning and complete death of explants two weeks after incubation .However, Momtaz et al. (1998) chose 25 mg/l of kanamycin for selection of transformed callus from cotyledons and hypocotyl tissues. In a system such as tobacco, transformed tissue at any stage of development can be selected on high level of kanamycin (up to 1000 mg/l), while in cotton such levels were found to be toxic. One of the major disadvantages of obtaining plants through organogenesis is the involvement of more than one cell in the shoot initiation process. Similar observation was made by Vasil (1985). When organogenesis is used for transformation and subsequent transgenic plant regeneration, the risk of recovering chimeric plants exists. In this study although thirteen plants showed kanamycin resistance, none of them were PCR positive. The presence of kanamycin in the rooting media was inhibitory for root formation. Rooting was achieved on kanamycin free medium. Giovanna et al. (1993) made similar observation and the use of effective kanamycin selection, concentration, timing and absence of kanamycin in the root development phase were crucial in screening of chickpea transformants. However, Draper et al. (1988) feel that roots being more sensitive to antibiotics, the ability to root on selection medium containing high levels of selective agent is by itself a strong indication of transformed nature of plantlets. While escapes failed to root, but the transformant did root in kanamycin containing rooting medium. According to Geetha et al. (1998), the efficient uptake of kanamycin by shoots during rooting stage might provide more effective selection of positive transformants. During rooting stage, shoots which apparently escaped previous selection eventually died within 2 weeks. Putatively transformed shoots that survived on selection medium produced roots within 3 weeks. As the preliminary experiments showed that kanamycin at 150 mg/l was lethal to non transformed plants. This concentration was used to screen the putative transformants in vitro. Two types of shoots developed on the selection medium, shoots that were green and actively growing and those that bleached and died. Small shoot explants of cotton are sensitive to kanamycin. In the meristem based method, if the meristem is killed the procedure fails. One of the current problems in the shoot apex method is that the promoters used to drive npt-ll may not be efficiently expressed in meristem. Under such circumstances, the selection pressure used must be low. Then, the escapes and the transformed shoots are not killed. This is the reason, why there is a wide gap between the number of kanamycin resistant shoots and PCR positive transformants. Gould and Cedeno (1998) also opined that the use of a permissive selection protocol is important in the practice of this method because plant regeneration is dependent on vitality of the meristem. Further, they also explain that the promoter used with the selectable resistance gene such as npt II, must be active in the meristem for selection to work, as one would normally expect. If cells in the meristem die, the organization inherent in the meristem is destroyed and regeneration from meristem is abolished. The CaMv 35S promoter is not active in plant meristem or shoot apex and the transformation events in this region do not provide protection to this vital region. The amount of selection tolerated also depends on the size and preparation of shoot explant, which is skill dependent and highly individualistic. Although, earlier workers have recommended 10-50 mg/l of kanamycin for the selection passage, we have used 50 mg/l for initial screening and during the subsequent subcultures, this level was increased to 150 mg/l to avoid too many escapes. Ramana et al. (1996) have also used 50 mg/l of kanamycin in the selection medium. Despite the popular belief that meristematic tissues of plants cannot be used in Agrobacterium mediated transformation, there is no direct evidence to support this view. To date, meristem based methods have been successfully used in Petunia (Ulian et al., 1988), Pea (Hussey et al., 1989) and sunflower (Schrammeijer et al., 1990). Despite the recalcitrance of the Gossypium genus, shoot apical meristem has widely been used for cotton transformation. However, regenerants from cocultured shoot apices with A. tumefaciens carry the transgenes mainly in chimeric fashion since the transformation events giving rise to shoots occur in pre-existing multicellular meristems as de novo formation of shoots from single transformed cell is less probable.

Kar et al. (1996) also opine that in a organogenetic system, there is a risk of losing events of transformation due to chimera.. It was observed that repeated application of selection pressure during shoot formation largely inhibits shoot formation by eliminating untransformed cells. Repeated selection in rapidly multiplying shoots is thought to eliminate untransformed tissues. Lowe et al. (1995) adopted a similar strategy to enrich transgenic sectors and eliminate chimeras.

5.5 CONFIRMATION OF TRANSGENE

The transformants were selected on MS medium containing kanamycin. In all, 26 plants from selection medium were further checked for the presence of transgene using nptII and cry2Aa specific primers, but the amplification of desired fragment was observed only in four plants. In Jayadhar, out of 14 kanamycin resistant plants, one in scalpel wounding, two in chilling method were found to be PCR positive and similarly, in Surabhi out of 12 kanamycin resistant plants only one was PCR positive in preculture treatment at 1 mg/l of BAP. In most cases, survival of shoots/plants on selection medium (kanamycin) is not a proof of transformation. Yet, it is thought to narrow down the number of individuals to be tested for the presence of transgene. Differential exposure of the in vitro cultures to the selective agent, residual resistance of the plant tissue to the selective agent and the level applied might affect the outcome. The low frequency of PCR positive plants among the selected shoots from tissue culture selection regimes is dependent on several factors such as tissue type size of explant, chemical properties and concentration of selection agent and time of application (Bowen et al., 1998). In this study, different factors such as preculture studies, colonization duration, cocultivation duration, wounding methods like scalpel wounding, vacuum infiltration, blot injury, chilling injury, sand injury and scalpel wounding followed by colonization with Agrobacterium were tried for optimization of Agrobacterium mediated regeneration and transformation protocol in cotton. Definite inferences could not be drawn from these experiments, as there was very poor correlation between the plants, which survived on selection medium and their being positive for the presence of the transgene.

5.6 FUTURE AREAS OF RESEARCH

Based on the results obtained in the present study the following points need to be considered as thrust areas of research. 1) To study induction of multiple shoots from cotyledonary nodal regions 2) Although shoot apical meristem has been widely used for cotton transformation, efficiency is low and the regenerants are likely to be chimeric. Attempts should be made to increase the efficiency of transformation by screening the leaves of each branch which produces bolls at flowering stage

6. SUMMARY AND CONCLUSIONS

Several techniques have been developed to introduce foreign genes into plants. Agrobacterium mediated gene transfer is a simple and the most reliable method of creating transformants in a wide variety of crop species. The present investigation was carried out to increase transformation efficiency, the protocol for Agrobacterium mediated transformation through both invitro and invivo methods in Jayadhar and Surabhi genotypes of cotton. Transformation experiments were carried out using Agrobacterium strain EHA-105 harbouring binary vector pBINAR, carrying cry2Aa gene linked to the CaMV35S promoter, OCS terminator II and nptII gene under the control of nopaline synthase (nos) promoter and terminator was used as a selectable marker. In vitro transformation studies For in vitro transformation, the protocol adopted uses transformation competent cells in the shoot apex of germinating seedling for Agrobacterium mediated transformation and for plant driven regeneration. The factors influencing transformation of cotton apical meristems such as the influence of explant pre-cultivation, colonization duration, cocultivation duration, wounding methods like scalpel wounding, vaccum infiltration, chilling duration, sand injury, blot drying were studied. Induction of axillary shoots by suppressing apical dominance was also studied. Use of pre-cultured shoots in the inoculation step is to ensure activation of cell division in the apical meristematic tissues and regulating morphogenesis in explants. Low concentration of cytokinin at 1 mg/l of BAP was found effective for shoot induction in both Jayadhar and Surabhi cotton genotypes and only one PCR positive plant was found in Surabhi with 1mg/l of BAP preculture treatment. Attempts to standardize colonization duration revealed that colonization duration of 10 min was optimum for maximum regeneration. Cocultivation duration of 48 hours was found to be better and cocultivation beyond this resulted in softening, browning and death of the explants. To increase the Agrobacterium mediated transformation efficiency, wounding methods were followed. Though wounding methods reduces regeneration response, i) Scalpel wounding followed by colonization with agroculture resulted in 60 per cent and 61.25 per cent regeneration in Jayadhar and Surabhi respectively. One plant was found to be PCR positive in Jayadhar genotype with scalpel wounding and colonization for 10 min. ii) The per cent regeneration was significantly on par, when explants were vaccum infiltrated for 10 mins (27.5) and 20 mins (27.0) in Jayadhar with no vaccum infiltration. In Surabhi also explants vaccum infiltrated for 10 mins (24.5) and 20 mins (26.5) were significantly on par with no vaccum infiltration (26.0). iii) Per cent regeneration reduced with blot drying. However, as much as 65 per cent in Jayadhar and 73.8 per cent in Surabhi accounted for 15 mins of blot drying as compared to 57.6 per cent and 58.75 per cent which accounted for 30 mins of blot drying in Jayadhar and Surabhi, respectively. iv) Explants chilled for more than 48 hours showed lower regeneration response (44% in Jayadhar and 40% in Surabhi). Two plants were found to be PCR positive with 30 min of chilling in Jayadhar genotype. v) Though sand injury reduces the regeneration response in both the genotypes (28 to 68%) than no injury in both the genotypes (82 and 78%) in Jayadhar and Surabhi, respectively, 10 mins of sand injury gave maximum regeneration response of 68 per cent in Jayadhar and 60 per cent in Surabhi. There was a poor correlation between kanamycin selected shoots and putative transformants.

In vivo transformation studies Vertical cut followed by colonization resulted in reduction of plant proliferation in both the genotypes which were grown in pots i.e. 41.5 to 51 per cent in Surabhi and 42 to 50 per cent in Jayadhar. Wound and vertical cut seedlings followed by colonization showed significant reduction in regeneration response than control (no wounding and no colonization). Induction of axillary shoots Suppression of apical dominance resulted in higher rate of axillary shoot induction (71.7% in Jayadhar and 70% in Surabhi) in MS broth than any other combination of growth regulator viz., BAP (1, 2, 4, 8, 16 mg/l), TDZ (1, 2, 4, 8, 16 mg/l) and NAA (0.1 mg/l) + BAP (1, 2, 4, 8, 16 mg/l).

REFERENCES
Agarwal, D.C., Banerjee, A.K., Kolala, R.R., Dhage, A.B., Kulkarni, A.V., Nalawade, S.M., Hazra, S and Krishnamurthy, K.V., 1997, In vitro induction of multiple shoots and plant regeneration in cotton (Gossypium hirsutum L.). Plant Cell Rep., 16: 647652. *Akiyoski, D.E., Klee, H., Amansino, R.M., Nester, E.W. and Angordon, M.P., 1984, T-DNA of Agrobacterium tumefaciens encodes an enzyme of cytokinin biosynthesis. In: Proceedings of National Academy of Science, 81: 5994-5998. An, G., Watson, B.D., Stachel, S., Gordon, M.P. and Nester, E.W., 1985, New cloning vehicles for transformation of higher plants. Embo J., 4: 277-284. Anonymous, 2008, http://www.cotcorp.gov.in/statistics.asp *Arundhati, A., 1999, Agrobacterium mediated transformation of pigeonpea [Cajanus cajan (L.) Mill sp.] in using leaf discs. International Chickpea and Pigeonpea Newsletter, 6: 62-64. *Bechold, N., Ellis, J. and Pelletier, G., 1993, In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. Sciences de la vie, 316: 11931194. Bevan, M., 1984, Binary Agrobacterium vectors for plant transformation. Nucleic Acid Res., 12: 52-55. Bevans, M.W., Flavell, R.B. and Chilton, M.D., 1983, A Chimaeric Antibiotic resistance gene as a selectable marker for plant cell transformation. Nature, 304: 184-187. Bhattacharya, R.C., Viswakarma, N., Bhat, S.R., Kirti, P.B. And Chopra, V.L., 2002, Development of insect resistant transgenic cabbage plants expressing a synthetic cry1a(b) gene from Bacillus thuringiensis. Curr Sci., 83(2): 146-150. Bidney, D., Scelonge, C., Martich, J., Burrus, M., Sims, L. and Huffman, G., 1992, Microprojectile bombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens. Plant Mol. Bio., 18: 301-313. Binns, A. N. and Thomashow, M. F., 1988, Cell biology of Agrobacterium infections and transformation of plants. Ann. Rev. Mic., 42: 575-606. Birch, R.G., 1997, Plant transformation: problems and strategies for practical application. Annual Review of Plant Physiology and Plant Molecular Biology, 48: 297-326. Bowen, D., Rocheleau, T., Blackgurm, M., Andreev, O., Golabeva, E., Bhartia, R. and French Constant, R.H., 1998, Insecticidal toxins from the bacterium. Photorhabdu luminescens. Science, 280: 2129-2132. Cao, X., Liu, Q., Rawland, L.J. and Hammerschlag, F.R., 1998, GUS expression in blueberry (Vaccinium spp.) factors influencing Agrobacterium mediated gene transfer efficiency. Plant Cell Rep., 18: 266-270. Cervera, M., Pina, J. A., Juarez, J., Navarro, L. and Pena L., 1998, Agrobacterium mediated transformation of citrange: factors affecting transformation and regeneration. Plant Cell Rep., 18: 271-278. Chakraborthy, R., Viswakarma, N., Bhat, S.R., Kirti, P.B., Singh, B.D. and Chopra, V.L., 2002, Agrobacterium mediated transformation of cauliflower: optimisation of protocol and development of transgenic cauliflower. J. Biosci., 27(5): 495-502.

Chee, P.P., Fober, K.A. and Slighton, J.L., 1989, Transformation of soybean (Glycine max) by infecting germinating seeds with Agrobacterium tumefaciens. Plant Physiol., 91: 1212-1218. Chilton, M.D., Drummond, M. H., Merlo, D. J., Siacky, D., Montoya, A.L., Gordan, M.P., and Nester , E. W., 1977, Stable incorporation of plasmid DNA into higher plant cell. The Molecular Basis of Crown Gall Tumuorigenesis Cell, 11: 263-271. Chlan, C.A., Lin, J., Cary, J. and Cleveland, T.E. 1995, A procedure for biolistic transformation and regeneration of transgenic cotton from meristematic tissue. Plant Mol.Biol. Rep., 13: 31-37. Christou, P., Mccabe, D. E., Martinell, R. J. and Swain , W. F., 1990, Soybean genetic engineering commercial production of transgenic plants. Trends In Biotech., 8: 145-151. Christou, P., Murphy, J.E. and Swain, W.F., 1987, Stable transformation of soybean by electroporation and root formation from transformed callus. Proceedings of National Academy of Science, USA, 84: 3962-3966. Daniell, H., Wiebbe, P.O. and Fernandez Son Milan, A., 2001, Selectable markers for genetic transformation of barley. Plant Sci., 6: 237-239. Davey, M. T., Rech, E. L. and Mulligan, B. J., 1989, Direct DNA transfer to plant cells. Plant Mol. Biol., 13: 273-285. De Neve, M., De bluck, S., Jacobs, A., Van Montagu, M. and Depicker, A., 1997, T-DNA integration patterns in co-transformed plant cells suggest that T-DNA reports originate from co-integration of separate T-DNAS. Plant J., 11: 15-29. Deng Xiang-Yang, Wei Zhi-Ming and An Hai Long, 2001, Transgenic peanut plants obtained by particle bombardment via somatic embryogenesis regeneration. Cell Res., 11: 156-160. Dillen, W., Declercq, J., Kapila, J., Zambre, M., Van Mantagu, M. and Angerm, G., 1997, The effect of temperature on Agrobacterium tumefacins mediated gene transfer to plants. The Plant J., 12: 1459-1463. Dinesh, M., 1998, Transformation of tobacco with glucanase chitinase encoding genes using Agrobacterium tumefaciens. M.Sc.(Agri) Thesis, Univ. Agric. Sci., Bangalore. Donaldson, P.A. and Simmonds, D.H., 2000, Susceptibility of Agrobacterium tumefaciens and cotyledonary node transformation in short season soybean. Plant Cell Rep., 19: 478-484. *Draper, J., Scott, R., Hamil, J., 1988, In draper, J., Scott, R., Armitage, P., Walden, R., Plant genetic transformation and gene expression. A Laboratory Manual, (eds.): Blackwell Scientific Publication, Oxford, Great Britain, p.103. Effendi, H.K., Akira, K. and Toshiaki, K., 2000, Transformation of soybean by infecting and that of soybean and kidney bean by injecting the bacteria germinating into seeds. Plant Biotechnology, 17(3): 187-194. Elliot, A. R., Campbell, J. A., Brettell, R. J. S. and Grof, C. P. L., 1998, Agrobacterium mediated transformation of sugarcane using GFP as a screenable marker. Aust. J. Plant Physiol., 25: 739-743. Fillatti, J.J., Kiser, J., Rose, R and Comai, L., 1987, Efficient transfer of a glyphosate tolerance gene into tomato using a binary Agrobacterium tumefaciens vector. Biotech, 5: 726-730. Finer, J.J. and McMullen, M.D., 1990, Transformation of cotton (Gossypium hirsutum L.) via particle bombardment. Plant Cell Rep., 8: 886-889.

Firoozabady, E., Deboer, D.l., Merlo, D.J., Halk, E.L., Amerson, L.N., Rashka, K.E. and Murray, E.E., 1987, Transformation of cotton (Gossypium hirsutum L.) of Agrobacterium tumefaciens and regeneration of transgenic plants. Plant Mol. Biol., 10: 105-116. *Fraley, R.J., Rogers, S. G., Horsch, R.B., Sanders, P.R., Flick, J. S., Adams, S. P., Bittner, M. L., Fink, C. L., Fry, J. S., Gallupi, G. R., Goldberg , S. B., Hoffman, N. L. and Woo, S.C., 1983, Expression of bacterial genes in plant cells. In: Proceedings of National Academy of Science, 80: 4803-4807. Fromm, M., Taylor, L. P., and Walbot, U., 1985, Expression of gene transferred into monocot and dicot plant cells by electroporation. In: Proceedings of National Academy of Science, 82: 5824-5828. Geeta, N., Venkatachalam, P. and Lakshmi Sita, 1999, Agrobacterium mediated genetic transformation of pigeonpea (Cajanus cajan L.) and development of transgenic plants via direct organogenesis. Plant Biotech., 16: 213-218. Geetha, N., Venkatachalam, P., Prakash, V. and Lakshmisita, G., 1998, High frequency induction of multiple shoots and plant regeneration from seedling explants of pigeonpea (Cajanus cajan). Current Science, 75: 1036-1041. Gio vanna, S., luigi, S., Sofia, C., Giovanna F. and Domenico , M., 1993, Genetic transformation in the grain legume cicer arietinum L (chickpea). Plant Cell Rep., 12: 194-198. Gomez, K.A. and Gomez, A.A., 1983, Statistical procedure for agricultural research, john wiley and sons, pp.91-95. Gould, J. and Cedeno, M. M., 1998, Adaptation of cotton shoot apex culture to Agrobacterium mediated transformation. Plant Mol. Reports, 16: 1-10. Gould, J., Devey, M., Hasegawa, Osamu, Ulian, E.C., Gregory petrson and Smith, R.H., 1991, Transformation of Zea mays L. using Agrobacterium tumefaciens and the shoot apex. Plant Physiol., 95: 426-434. *Gould, J. and Smith, R., 1988, Shoot tip culture as a potential transformation system. Beltwide cotton production conferences. National Cotton Council, Memphis, pp.113-114. Guo, X., Huang, C., Jin, S., Nie, Y. and Zhang, X., 2007, Agrobacterium mediated transformation of cry1c, cry2A and cry9c genes into Gossypitum hirsutum and plant regeneration. Biologia Plantarum, 51: 242-248. Gupta, S.K., Srivastava, A.K, Singh, P.K. and Tuli, R., 1997, In vitro proliferation of shoots and regeneration in cotton. Plant Cell Tiss. Org. Cult. 51: 149-152. Gynheung, 1985, High efficiency transformation of cultured tobacco cells. Plant Physiol., 79: 568-570. Hayashimoto, A.L.Z. and Murai, N., 1990, A poly ethylene glycol mediated protoplast transformation system for fertile transgenic rice plants. Plant Physiol., 93: 57-863. Hemphill, J.K., Hoang, C.V. and Chapman, K.D., 1998, Agrobacterium mediated gene transfer system for cotton (Gossypium hirsutum L.), communicated. Herrera Estrella, Depicker, A., Van Montagu, M. and Schulj, 1983, Chimeric genes as dominant selectable markers in plant cells. Embo, J., 2: 987-995. Hewezi, T., Perrault, A., Alibert, G. and Kallerhoff, J., 2002, Dehydrating immature embryo split apices and rehydrating with Agrobacterium tumefaciens: A new method for genetically transforming recalcitrant Sunflower. Plant Mol. Biol. Reports, 20: 335345.

Hoekema, A., Hirsch, P.R., Hooykaas, P.J.J. and Schilperoort, R.A., 1983, A binary plant vector strategy based on separation of vir and t-region of Agrobacterium tumefaciens t1 plasmid. Nature, 303: 179-180. Hood, E.E., Helmer, G.L., Fraley, R.T. and Chilton, M.D., 1986, The hypervirulence of Agrobacterium tumefaciens a281 is encoded in a region of ptibo542 outside of tDNA. J. Bacterial., 168: 1291-1301. Horsch, R., Fraley, R.T., Rogers, S.G., Sanders, P.R., Lloyd, A. and Hoffmann, N., 1984, Inheritance of functional foreign genes in plants. Science, 223: 496-498. Horsch, R., Fry, J., Hoffmann, N., Wallroth, M., Eichholtz, D., Rogers, S. and Fraley, R., 1985, A simple and general method for transferring genes into plants. Plant Sci., 227: 1229-1231. Hussey, G., Johnson, R.D. and Warren, S., 1989, Transformation of meristematic cells in the shoot apex of the cultured pea shoots by Agrobacterium tumefaciens and A. rhizogenes. Protoplasma, 148: 101-105. Ikram Ul-Haq, 2004, Agrobacterium mediated transformation of cotton (Gossypium hirsutum.)via vacuum infiltration. Plant Mol. Bio. Reporter, 22: 279-288. Jefferson, R.A., 1987, Assaying chimeric genes in plant - The GUS gene fusion system. Plant Mol. Bio. Reporter, 4: 387-405. Joersbo, M. and Brunstedt, J., 1990, Direct gene transfer to plant protoplast by mild sonication. Plant Cell Rep., 9: 207-210. Joersbo, M. and Okkels, F.T., 1996, Stimulating growth and regeneration of transformed cells in vitro. Zbid, 16: 219-221. John, M.E., 1997, Cotton improvement through genetic engineering. Critical Rev. Biotechnol., 17: 185-208. Kadapa, S.N., Thimmaiah, G. and Khadi, B.M., 1983, Breeding for resistance or tolerance to bollworms in cotton (G. Hirsutum L.). Presented in cotton challenges in 80s seminar organised by Karnataka Chamber of Commerce Hubli, 27-29 October 1983, pp.1-8. Kanthi, S.G., 2005, Transformation and analysis of transgenic pigeonpea for pod borer resistance. M.Sc.(Agri.) Thesis, Univ. Agric. Sci., Dharwad. Kar, S., Tony, M.J., Pritilata Nayak and Sen, S.K., 1996, Efficient transgenic plant regeneration through Agrobacterium mediated transformation of chickpea (cicer arietinum L.). Plant Cell Rep., 16: 32-37. Katageri, I.S., Vamadevaiah, H.M., Udikeri, S.S., Khadi, B.M. and Kumar, P.A., 2007, Genetic transformation of an elite Indian genotype of cotton (Gossypium hirsutum L.) for insect resistance. Curr. Sci., 93(12): 25. Klee, H.J. and Rogers, S.G., 1987, Plant gene vectors and genetic transformation: Plant transformation systems based on the use of Agrobacterium tumefaciens. In: Cell Culture and Somatic Cell Genetics of Plants, Academic Press, 6: 1-23. Klee, H.J. and Rogers, S.G., 1989, Plant gene vectors and genetic transformation : Plant transformation systems based on the use of Agrobacterium tumefaciens. In: Cell Culture and Somatic Cell Genetics of Plants, Academic Press, 6: 1-23. Klein, T.M., Sanford, J.C. and Wolf, E., 1985, Particle gun technology; A novel method for the introduction of DNA into living cells, SM Biotechnology in Plant Science: Relevance to Agriculture in the Eighties. Correll University, Poster, p.28.

Komari, T., Hiei, Y., Saito, Y., Murai, N. and Kumashiro, T., 1996, Strategies to produce transgenic plants free from selectable marker gene. Plant J., 10: 165-174. Krishnan, N.V., Soni, K.B., and Rajmohan, K, 2008, Agrobacterium tumefaciens mediated genetic transformation in Centella asiatica L. urban. Current Botica, 2: 1-8. Krishnamurthy, K.V., Suhasini, K., Sagare, M.M., Kathen, A. De., Pickard, T. and Schieder, 2000, Agrobacterium mediated transformation of chickpea (cicer arietinum L). Embryo axes. Plant Cell Rep., 2000, 19: 235-240. Kumar, M.S., Sharma, S.D. and Pratibha Devi, 2003, Protocol for efficient plant regeneration and Agrobacterium mediated genetic transformation of pigeonpea (Cajanus cajana). Indian J. Genet., 63: 289-294. Kumaria, R., Leelavathi, S., Bhatnagar, R.K. and Vanga, S.R., 2003, Regeneration and genetic transformation of cotton : Present status and future perspectives. Plant Tissue Cult., 13(2): 211-225. Lawrence, P.K. and Koundal, K.R., 2001, Agrobacterium tumefaciens mediated transformation of pigeonpea (Cajanus cajan L. Millsp) and molecular analysis of regenerated plants. Curr. Sci., 80: 1428-1432.

Lee, J.A., 1987, Induction of Adventitious Shoots in Cotton. Crop Sci , 27: 349-350 . Leelavathi, S, Sunnichan, V.G., Kumria, R., Vijaykanth, G.P., Bhatnagar, R.K. and Reddy, V.S., 2003, A simple and rapid Agrobacterium-mediated transformation protocol for cotton (Gossypium hirsutum L.): Embryogenic calli as a source to generate large number of transgenic plants. Plant Cell Rep., (in press). Liu, W., Parrott, W.A., Hildebrand, D.F., Collins, G.B., and Williams, E.G., 1990, Agrobacterium induced gall formation of shoots like pepper (Capcicum annuum L.) and formation of shoot like structures expressing introduced genes. Plant Cell Rep., 9: 360-364. Lowe, K., Bowen, B., Hoerster, G., Ross, M., Bond, D., Pierce, D. G. and Kamm, B., 1995, Regeneration and transformation of maize linesvia embryo culture and a biolistic particle delivery system. Biotech., 13: 677-682. Luo, J., Biswas, G.G. and Gould, J., 1999, Shoot apex transformation and regeneration of Texas cotton for disease resistance. In: Plant and Animal Genome VII Conference San Diego. *Mahalaxmi, A. and Khurana, P, 1997, Agrobacterium mediated cereal transformation: a critical appraisal. Indian J. Exp. Bio., 35: 416-426. Malathi, B., Ramesh, S., Rao, K.V. and Reddy, V. D., 2006, Agrobacterium- mediated genetic transformation and production of semilooper resistant transgenic castor (Ricinus communis L.). Euphytica. 147: 441 449. Manoharan, M., Sreevidya, C.S. and Lakshmisita, G., 1998, Agrobacterium mediated genetic transformation in hot chilli (Capsicum annuum L. Var Pusa Jwala). Plant Sci., 131: 77-83. McCabe, D.E. and Martinell, B.J., 1993, Transformation of elite cotton cultivars via particle bombardment of meristems. Biotech., 11: 596-598. Mckently, A, H., More, G. A., Doostdar, H. and Niedz , R.P.,1995, Agrobacterium mediated transformation of peanut (Arachis hypogaea L.) embryo axes and development of transgenic plants. Plant Cell Rep., 14: 699-703. Medford, J.I., 1992, Vegetative apical meristems. Plant Cell, 4: 1029-1039.

Mehrzad, M., Yucel, M. and Outem, H.V., 2002, Transformation of lentil (Lens culineris.) cotyledonary nodes by vacuum infiltration of Agrobacterium tumefaciens. Plant Mol. Bio. Reporter, 20: 251-257. Miki, U. B. L. A., Reich, T. J. and Iyer, V. N., 1987, Microinjection an experimental tool for studying and modifying plant cells. In: Plant DNA Infectious Agent. Ed. Hohn and J. Schell, Springer Verlag, New York, pp.349-365. Ming Cheng, Robert L., Jarret, Zhijian Li, Aiqui xing and James W. Demski, 1996, Production of fertile transgenic peanut (Arachis hypogea L.) Plants using Agrobacterium tumefaciens. Plant Cell Rep., 15: 653-657. Mogali, S.C., 2005, Studies on genetic transformation in cotton (Gossypium hirsutum L.). Ph.D. Thesis, Univ. Agric. Sci., Dharwad. Moloney, M.M., Walker, J.M. and Sharma, K.K., 1989, High efficiency transformation of brassica napus using Agrobacterium vectors. Plant Cell Rep., 8: 238-242. *Momtaz, O.A., Diab, A.A., Abushady, M.R. And Madkour, M.A., 1998, Transformation of Egyptian cotton tissue (Gossypium barbadense L) using Agrobacterium tumefaciens. Proceedings of World Cotton Research Conference 2. Athens, Greece, September 6-12, pp. 314-319. Moralejo, M., Rochange, F., Boudet, A.M. and Teulieres, C., 1998, Generation of transgenic Eucalyptus globulus plant lets through Agrobacterium tumefaciens mediated transformation. Aust. J. Plant Physiol., 25: 207-212. Morre, J.L, Permingeat, H.R, Romagnoli, M.V, Heisterborg, C.M and Vallejos, R.H., 1998, Multiple shoot induction and plant regeneration from embryonic axes of cotton. Plant Cell Tiss. Org. Cult., 54: 131-136 Mullis, K.B., 1990, The unusual origins of the polymerase chain reaction. Scientific American, 262: 56-65. Murashige, T. and Skoog, F., 1962, A revised medium for rapid growth and bioassays with tobacco cultures. Physiologia Plantarum, 15: 473-479. Muthukumar, Mariamma, M., Veluthambi, K. and Gnanam, A., 1996, Genetic transformation of cotyledon explants of cowpea (Vigna unguiculata L. Warp) using Agrobacterium tumefaciens. Plant Cell Rep., 15: 980-985. Negrutiu, I., Shillito, R., Potrykus, I., Biansini, G.and Sala, F., 1987, Hybrid genes in the analysis of transformation condition. Plant. Mol. Biol. 8: 363-373. *Oktem, H.A., Ozkan, F., Ozacp, V.C. and Yucel, M., 1994, Agrobacterium mediated gene transfer in tobacco. Turkish J. Bot., 18(5): 397-405. Pang, S., De Boer, D.L., Wna, Y., Ye, G., Layton, J.G., Neha, M.K., Armstrong, C.L., Fry, J.E., Hinchee, M.A. and Fromm, M.E., 1996, An improved green fluorescence protein gene as a vital marker in plants. Plant Physiol., 112: 893-900. Pawan, K., Jaiwal, Lingaraj Sahoo, N., Dolendro Singh and Rana P., Singh, 2002, Strategies to deal with the concern about marker genes in transgenic plants: some environment friendly approaches. Curr. Sci., 83(2): 128-136. Pawan, K.J., Sautter, C and Potrykus, I., 1998, Agrobacterium rhizogenes-mediated gene transfer in mungbean (Vigna radiata L. (Wilczek). Curr. Sci., 75(1): 41-45. Peeters, M.C, Willems, K. and Swenen, R., 1994, Protoplast-to-plant regeneration in cotton (Gossypium hirsutum L. cv. Coker 312) using feeder layers. Plant Cell Rep., 13: 208-211.

Peterson, A.H., Brubaker, C.L. and Wendel, J.F., 1993, A rapid method of extraction of cotton (Gossypium spp.) Genomic DNA suitable for rflp or PCR analysis. Plant Mol. Biol. Rep., 11: 122-127. Polock, K., Barfield, D.G. and Shields, R., 1983, The toxicity of antibiotics to plant cell cultures. Plant Cell Rep., 2: 36-39. *Potrykus, I., 1990, Gene transfer to cereals: an assessment. Biotech., 8: 535-542. Pount-Kaerlas, J., Stable, P. and Eriksson, T., 1989, Transformation of pea (Pisum sativum L.) by Agrobacterium tumefaciens. Plant Cell Rep., 8: 321-324. Prasad, K.V.S.K., Sharmila, P., Kumar, P.A. and Pardha Saradhi, P., 2000, Transformation of Brassica juncea (L.) Czern with bacterial cod a gene enhancer its tolerance to salt-stress. Molecular Breed., 6: 489-499. Rajasekaran, K., Grula, J.W., Hudspeth, R.L., Pofelis, S. and Anderson, D.M., 1996, Herbicide resistant Acala and Coker cottons transformed with a native gene encoding mutant forms of acetohydroxy acid synthase. Mol. Breed., 2: 307-319. Rajasekaran, K., Hudspeth, R.L., Cary, J.W., Anderson, D.M. and cleveland, T.E., 2000, High frequency stable transformation of cotton (Gossypium hirsutum L.) by particle bombardment of embryogenic cell suspensions cultures. Plant Cell Rep., 9: 539545. Ramana, R.V., Venu, C.H., Jayashree, T. and Sadananda, M.A., 1996, Direct somatic embryogenesis and transformation. J. Biotech., 34: 716-718. Rhodora, R.A and Thomas, K.H., 1996, Agrobacterium tumefaciens-mediated transsformation japonica and indica rice varieties. Planta, 199: 612-617. Satyavathi, V.V., Prasad, V. and Lakshmi, B.G. and Sita, G.L., 2002, High efficiency transformation protocol for three Indian cotton varieties via Agrobacterium tumefaciens. Plant Sci., 162: 215-222. Schrammeijer, B., Sijimons, P.J.M. and Hoekema, 1990, Meristem transformation of sunflower via Agrobacterium. Plant Cell Reports, 9: 55-60. Sheen, J., Hwang, S., Niwa, Y., Koboyashi, H. and Galbraith, D.W., 1995, Green fluorescent protein as new vital marker in plant cells. The Plant J., 8: 777-784. Shillito, T. D., Saul, M. W., Paskowshi, J., Mullr M. and potrykus, I., 1985, High frequency gene transfer in plants. Biotechnology, 3: 1099-1103. Shroeder, G., Wafeenschmidt, S., Weiler, E.W. and Schroder, J., 1984, The T-region of Ti plasmid codes for an enzyme synthesizing indole 3-acetic acid. European J. Biochem., 138: 387-391. Shuangxia, J., Xianlong, Z., Shaoguang, L., Yichun, N., Xiaoping, G. and Chao, H., 2005, Factors affecting transformation efficiency of embryogenic callus of upland cotton (Gossypium hirsutum) with Agrobacterium tumefaciens. Plant Cell, Tissue and Organ Cult., 81: 229-237. Singh, D., N, Lingaraj Sahoo, Raman Saini, Neera, B. S, and Pawan K., 2004, In vitro regeneration and recovery of primary transformants from shoot apices of pigeonpea using Agrobacterium tumefaciens. Physiol. Mol. Biol., 10:65-74. Smith, C. J. S., Watson, C. F., Bir, C. R., Ray, J., Bird, C. R., Morris, P. C., Schuch, W. and Grierson, D., 1988, Antisense RNA inhibition of polygalacturonase gene expression in reansgenic tomatoes. Nature, 334: 724-726. Sonia, P. K., Jaiswal, A. A. and Sahoo, L., 1998, Green fluorescent protein : a novel reporter gene . Curr. Sci., 74: 402-405.

Steinbiss, H.H. and Davidson, A., 1989, Genetic manipulation of plants: agronomic applications. Sci. Prog. Oxford, 73: 147-168.

from tools to

Stephen and Graves, 2006, A rapid in vitro regeneration scheme of cotton plants compatible with Agrobacterium mediated transformation, US Pat. Nos. 5, 177, 016 and 5,187, 073. Sunilkumar, G. and Rathore, K.S., 2001, Transgenic cotton: Factors influencing suspension and callus cultures of cotton (Gossypium hirsutum L.). Plant Cell Rep., 15: 859864. Tidke, P.M. and Sane, S.N., 1962, Jassid resistance and morphological characters of cotton leaf. Indian Cotton Grower Review, 16: 324-327. Tinland, B., 1996, The integration on T-DNA into plant genomes. Trends in plant Science, 1: 178-184. Tomes, D. T., Weissinger, A. K., Ross, M., Higgins, R., Drummond, B. J., Schaaf, S., MaloneSchoneberg and Howard, J., 1990,Transgenic tobacco plants and their progeny derived by microprojectile bombardment of tobacco leaves. Plant Mol. Bio., 14: 261-268. *Toyoda, H., Matsuda, Y., Utsumi, R., and Oushi. S., 1988, Introduction microinjection for transformation of tomato callus cells. Plant Cell Rep., 7: 293-296. Ulian, E.C., Smith, R.H., Gould, J.H. and Mcknight, T.D., 1988, Transformation of plantsvia the shoot apex. In vitro Cellular and Developmental Biology, 24: 951-954. Umbeck, P., Johnson, G., Barton, K. and Swain, W., 1987, Genetically transformed cotton (gossypium hirsutum l.) plants. Biotech., 5: 263-265. United States Patent, http: //www.freepatentsonline.com/7122722.html Vasil, I.K., 1985, In Tissue Culture in Forestry and Agriculture. Plenum Publishing Corporation, pp.50-56. Venkatachalam, P., Geetha, N., Khandewal, A., Shaila, M.S. and Akshmisita, G., 2000, Agrobacterium mediated genetic transformation and regeneration of transgenic plants from cotyledon explants of groundnut (Arachis hypogea L.) via somatic embryogenesis. Curr. Sci., 78: 1130-1136. Wang, J.H., R.J. and Donaldson, B.J., 1996, Agrobacterium mediated transformation and regeneration Rose and expression of foreign genes in medicago trancatula. Australian J. Plant Physiol., 23: 265-270. Wang, W., Chen, W., Gao, Y. and Zhu, Z., 1998, Obtaining insect resistant cotton by transformation with Agrobacterium. Proceedings of the World Cotton Research Conference-2, Athens, Greece, September 6-12, pp.350-353. Weir, B., Gu, X., Wang, M., Upadhyaya, N., Elliot, A.R. and Brettel, R.J.S., 2001, Agrobacterium tumefaciens mediated Transformation of wheat using suspension cells as a model system and green fluorescent protein as a visual marker. Aust. J. Plant Physiol., 28: 819-823. Wilkins, T.A., Rajasekaran, K. and Anderson, D.M. 2000, Cotton Biotechnology. Crit. Rev. Plant Sci., 19: 511-550. Wu, J., Zhang, X., Nie, Y. and Luo, 2005, High-efficiency transformation of Gossypium hirsutum embryogenic calli mediated by Agrobacterium tumefaciens and regeneration of insect resistant plants. Plant Breeding,124: 142-146. Xiang, C., Peng Han and David J. Oliver, 1999, In solium selection for arabidopsis transformants resistant to kanamycin. Plant Mol. Bio. Reporter, 17: 59-65.

Yadav, H.N., Mittal, R.K. and Singh, H.G., 1967, Correlation studies between leaf midrib structure and resistance to jassids in cotton. Indian J. Agric. Sci., 37: 495-497. Zambrysky, P. C., 1992, Chronicles from the Agrobacterium plant cell DNA transfer stor. Ann. Rev. Plant Physio. Plant Mol. Biol., 43: 465-490. Zambrysky, P.C., Joos, H., Genetello, C., Leemans, J., Vanmontagu, M. and Schell, J., 1983, Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity. Embo J., 2: 2143-2150. Zapata C., Park, S.H., El-Zik, K.M. and Smith, R.H., 1999, Transformation of a texas cotton cultivar by using Agrobacterium and the shoot apex. Theor. Appl. Genet., 98: 252-256. Zhang, W.N., Zhang, B., Xu, Y. and Guo, S., 1998, Development of transgenic insect protected cotton plants. Proceedings of World Cotton Research Conference-2, Athens, Greece, September 6-12, pp.210-212.

*Originals not seen

APPENDIX I
YEMA medium (Yeast Extract Mannitol Agar)
Mannitol Yeast extract NaCl K2HPO4(2%) MgSO4.7H2O (1M) Agar Water 10 g 400 mg 100 mg 500 mg 200 mg 8g 1000 ml

APPENDIX II
Murashige and Skoog medium
MS powder 4.04 g CaCl2 440 mg Sucrose 30 g Gelrite 2.5 g Water 1000 ml

APPENDIX III
Loading dye (6x) 0.25% Bromophenol blue 40% (w/v) sucrose in water Stored at 4C TAE tris Acetate (Sambrook et al., 1989) 50x Tri Base 242 g Glacial acetic acid 57.1 ml 0.57 m EDTA (pH 8.0) 100 ml Distilled water 1000 ml

APPENDIX IV
DNA extraction buffer (Edwards et al., 1991) Tris HCl (pH (7.5) 200 mM NaCl 250 mM EDTA 25 mM SDS (w/v) 0.5% PCR Requirements SDDW 12.67 l MgCl2 0.5 l dNTPs 1 l Taq Buffer 2-5 l Forward primer 1 l Reverse primer 1 l Taq polymerase 0.33 l Template 1 l Total 20 l

GENETIC TRANSFORMATION STUDIES IN COTTON

SUMITHRA S. 2008
ABSTRACT
An investigation was carried out to obtain genotype independent regeneration protocol to enhance regeneration and genetic transformation efficiency in cotton. cry2Aa gene and cotton genotypes: Jayadhar (Gossypium herbaceaum) and Surabhi (G. hirsutum) were used in the investigation. Both in vitro and in vivo Agrobacterium mediated transformation methods using strain EHA-105 were tried. Shoot apex of a germinating seedling was used as explants for in vitro transformation studies. Out of 49 plants one was found to be PCR positive (2% transformation efficiency) in 1 mg/l of BAP preculture treatment. Colonization duration of 10 min followed by 48 hours of co-cultivation was optimum for maximum regeneration (71.575%). Co cultivation beyond 48 hours resulted in browning and death of the explants. Among wounding methods, out of 24 plants one plant was found to be PCR positive (4%) with scalpel wounding and colonization for 10 min with a regeneration response of 60 per cent as compared to scalpel wounded non colonized plants (78.75%). The per cent regeneration was significantly on par, when explants were vacuum infiltrated for 10 min and 20 min (61.368.8%) as compared to non vacuum infiltrated plants (65-70%). Per cent regeneration reduced with blot drying (57.5-73.8%) as compared to control explants (80-83.8%). Explants chilled for more than 48 hours showed lower regeneration response (40-44%) as compared to control explants (76-78%). Out of 15 plants two were found to be PCR positive (6.7%) with 30 min of chilling. Sand injury reduced the regeneration response (28-68%) than no injury (7882%). In vivo transformation method has been carried out where in plant cells were directly exposed to Agrobacterium cells by eliminating the regeneration steps. Explants which were wounded and given vertical cut showed lower regeneration response (38.4%-72%) as compared to non wounded in vivo plants (100%). However none of the plant showed transformation. Removal of leaf primordia and meristamatic region induced axillary shoots and drastically reduced the regeneration (6-28%) as compared to non removal of primordia from shoot apex (96-97%).

Dr. I. S. KATAGERI MAJOR ADVISOR