International Journal of Bio-Technology

and Research (IJBTR)

ISSN 2249-6858

Vol. 3, Issue 1, Mar 2013, 81-90

© TJPRC Pvt. Ltd.

A GENOTYPE-INDEPENDENT AGROBACTERIUM MEDIATED TRANSFORMATION OF

GERMINATED EMBRYO OF COTTON (GOSSYPIUM HIRSUTUM L.)

MANOJ KUMAR1, ANOOP KUMAR SHUKLA

2, HARPAL SINGH

3, PRAVEEN C VERMA

4 & PRADHYUMNA

K SINGH5

Plant Molecular Biology & Genetic Engineering Division

CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow, India

ABSTRACT

Explants transformation through Agrobacterium, followed by in vitro regeneration is only method used for

genetic improvement of cotton. The currently used method are based on somatic embryogenesis, therefore time consuming,

genotype-dependent and expensive. We report here a genotype-independent method for genetic transformation of cotton.

Embryonic axes of 48 hours of germinating cotton seeds were incised at the cotyledonary node and co-cultivated with A.

tumefaciens. Transfected embryos were transferred into test tubes and grown for 10 days at 28 + 2oC. Later, the seedlings

were transferred to pots in glass house. T1 seeds obtained from the putative T0 transgenics were screened for the promising

transformation events on the basis of formation of root laterals in Hoagland liquid medium containing hygromycin B. The

transgenic nature of the positive T1 seedlings was confirmed by PCR and PCR-Southern analysis. Histochemical assay

showed the presence of the GUS reporter protein in T0 and T1 transgenics. This method gives 2-3 % transformation

efficiency and provides convenient method for transformation of cotton. The strategy, in principle, should be applicable to

all the cultivars and genotypes of cotton which are susceptible to Agrobacterium tumefaciens infection.

KEYWORDS: Agrobacterium-Mediated Transformation, Embryo Transformation, δ-endostoxin, Transgenic Cotton

ABBREVIATION

CaMV35S: Cauliflower mosaic virus 35S promoter; hptII:Hygromycin Phosphotransferase II; GUS: β-

Glucuronidase

INTRODUCTION

Cotton (white gold) is a major fiber crop of India and accounting for about 16 per cent of India’s export earnings

(Anonymous, 2007). Therefore, there is a dire need to produce maximum and best quality cotton. There are many desired

traits that can be improved through genetic engineering for commercial advantage. Genes conferring tolerance to various

biotic and abiotic stresses, or improving the yield and fiber quality have already been isolated and characterized in our

laboratory. The introduction of these genes into the cotton genome cotton is by no means an easy task. Most elite cotton

varieties remain recalcitrant and not amenable to genetic manipulation to protocols so far developed. Genetic

transformation of cotton by A. tumefaciens requires tissue culture dependent regeneration process, which undergo

prolonged tissue culture, genotype-dependency and expensive regeneration method. Efficient in vitro techniques for

regeneration and somatic embryogenesis from cotton are limited when compared to other major commercial crops. Only a

limited numbers of cultivars can be induced to produce somatic embryos and regenerative plants and the most responsive

lines are coker varieties which are no longer under cultivation (Trolinder and Goodin 1987, 1988 a, b; Davidonis and

Hamilton 1983; Kumaria et al. 2003).

82 Manoj Kumar, Anoop Kumar Shukla, Harpal Singh, Praveen C Verma & Pradhyumna K Singh

In the past decade, extensive research efforts have been focused for genetic improvement and a number of genes

with potential to confer agronomic advantages have been introduced by A. tumefaciens (Perlak et al. 1990, Bayley et al.

1992 and Lyon et al. 1993, Leelavathi et al. 2004) and particle bombardment (Thomas et al. 1995, Finer and McMullen

1990). Improvement of tissue culture methods to induce efficient transformation in a genotype independent manner is

desirable (Trolinder and Goodin 1989). The transformation efficiency is quite different in different cotton genotypes (Zhao

et al 2006, Katageri et al 2007). Although different laboratories have their own favourite genotypes for cotton

biotechnology research, few genotypes such as Coker remained choice for genetic transformation studies (Hu et al., 2011).

Most of the desirable genes are introduced initially into Coker varieties and then back-crossed with the desired genotype.

Several years of backcrossing and selection are required to identify agronomically suitable lines for commercialization

(Satyavathi et al., 2002).

However, most of the plants SE regenerated transformed plants, show abnormalities (Trolinder and Goodin 1988).

The low embryogenic potential, maturation and conversion of putative transgenic embryos into plantlets also remain a

problem. Alternate strategies to obtain transgenic cotton by direct methods which are genotype independent and do not

intervene lengthy tissue culture methods are desirable. Transgenic plants have also been generated by regeneration from

shoot apical tissue (Gould and Magallanes-Cedeno 1998, Gould et al 1991).

Another method for raising cotton transgenic is by pollen tube pathway transformation (Zhou et al 1983, Huang et

al 1999). Earlier report of transformation of embryonal segment of pigeon pea is reported by our group (Surekha et al.

2005). Several other groups also reported embryo transformation of several crops including safflower (Rohini and Rao

2000), sunflower (Schoneberg et al. 1994; Sankara Rao and Rohini 1996), peanut (Sankara Rao 2000) and maize (Wang et

al., 2007).

The generation of transgenic cotton plants is a routine practice in our laboratory for the validation of various

biotic (insect), abiotic stress (drought) and fiber related genes. Genotype limitation, abnormal somatic embryos, callus

induced genetic damage are commonly observed among regenerated plants (unpublished data). We are able to regenerate

limited number of plants from callus cultures by somatic embryogenesis due to phenotypic abnormalities (Aydin et al.,

2010), and cytogenetic changes (Stelly et al 1989).

Even in high embryogenic potential Coker cultivars, maturation and conversion of putative transgenic embryos

into plantlets still possess a problem. Further, insect resistant genes used for transformation have some detrimental effect

on somatic embryo development (Rawat et al., 2011). All these observations led us to develop an efficient transformation

method in a genotype independent manner.

In planta transformation experiments having high transformation efficiency is the prerequisite for large scale

functional analysis of the genes in cotton. Therefore, an efficient method for direct Agrobacterium-mediated

transformation of different cotton varieties using germinated embryos is developed and transformed with gus reporter gene

under CaMV35s promoter.

MATERIALS AND METHODS

Seed Sterilization and Germination

Sterilization of seeds of Khandwa-2, Anjali and Coker 310 was carried out as described earlier (Kumar and Tuli

2004). Seeds were grown aseptically in 250 ml flask containing 50 ml autoclaved distilled water. After 24 hours of

germination, seeds were de-coated to expose the embryos. Embryos were incised with scalpel in such a way that shoot

apical meristem became exposed (Fig. 1).

A Genotype-Independent Agrobacterium Mediated Transformation of 83 Germinated Embryo of Cotton (Gossypium hirsutum L.)

Construction of Binary Vector

Plasmid construction was carried out in plant transformation vector as described by Sambrook et al. 1989.

Construct in binary vector containing CaMV35S promoter for expression in plant of GUS protein was utilized for

Agrobacterium-mediated transformation (Fig 2).

Agrobacterium tumefaciens Infection and Co-Cultivation

Agrobacterium tumefaciens (LBA4404) harbouring binary vector was streaked on YEB medium plate, a single

isolated colony was inoculated in 5 ml YEP medium containing rifampcin 50 mgL-1

, streptomycin 200 mgL-1

and

kanamycin 50 mgL-1

and grown at 28oC and 200 rpm as primary culture. 50 µl of primary culture was inoculated in 50 ml

of YEP medium and grown in similar condition as secondary culture till OD600 reached to 1.4.

Cells were harvested by centrifugation (6,000Xg, 4oC, 5 min). Pellet was resuspended in 100 ml Induction

Medium (IM) (containing 1gL-1

NH4Cl2, 0.3 gL-1

MgSO4.7H2O, 0.15 gL-1

KCl, 0.01 gL-1

CaCl2, 0.0025 gL-1

FeSO4.7H2O,

0.272 gL-1

KH2PO4, 0.390 gL-1

MES, 100 µM acetosyringone and 5.0 gL-1

glucose) at pH 6.0 and incubated for 4 hours

at 175 rpm and 26 o

C in incubator shaker. Cells were again harvested, resuspended in 100 ml MSO medium (MS salts, B5

vitamins, MES 1.95 mgL-1

, 100 µM acetosyringone and glucose 20 gL

-1 at pH 5.65) and incubated for 2 hours at 150 rpm

and 250C.

To inoculate A. tumefaciens on to the embryonic apical meristem in the incised embryos, they were dipped in to

bacterial suspension (MSO medium) and cultivated for 1 h at 125 rpm, 250C in incubator shaker in dark. Then embryos

were collected, blot dried on sterile Whatman filter paper (Cat. Log no. 3030917) and transferred to co-cultivation medium

(MS salts, B5 vitamins, myoinositol 100 mgL-1, glucose 30 gL-1 and agar 0.8%) for 3 days at 280C in diffused light. Co-

cultivated embryos were washed with cefotaxime (250 mgL-1

) for 5 min followed by autoclaved distilled water. These

embryos were then transferred aseptically to test-tubes containing Hoagland medium on paper bridges.

After 10 days seedlings were transferred to pots containing potting mix (sandy loam soil, sand, vermiculite and

peat moss in 2:1:1:1 ratio). The plants were covered with polythene bags to maintain high humidity and irrigated with

sterile water for 14-18 days of hardening. During this period, the plants were incubated at 28±2 oC, 60 µmol m-2s-1 light

intensity and 16 hour of photoperiod in glass house. The polythene bags were removed and plants were then transferred to

net-house after 4-6 weeks.

ANALYSIS OF TRANSGENIC PLANTS

PCR Analysis

To establish transformed nature of the putative transgenic plants hpt II gene was used as targets for PCR analyses.

Genomic DNA was isolated from one month old plant leaves by CTAB method (Murray and Thompson, 1980) and used

for PCR amplification of hpt II gene.

The primers used were forward primer HPF 5’-TCCACTATCGGCGAGTACTTCTA-3’ and reverse primer

HPR-5’ACGCGGATTTGCGCTCCAACAAT-3’ with 500- bp amplicon size.

The vir C gene was used as control to detect the presence of contaminating Agrobacterium in the plant tissue. The

730-bp fragment of Agrobacterium-borne vir C gene was detected by using 5’-ATCATTTGTAGCGACT-3’ (forward) and

5’-AGCTCAAACCTGCTTC-3’ (reverse) primers. The amplicons were visualized after electrophoresis in 0.8% agarose

gels.

84 Manoj Kumar, Anoop Kumar Shukla, Harpal Singh, Praveen C Verma & Pradhyumna K Singh

Southern Analysis of PCR Product

The identity of hptII gene fragment in the PCR product was confirmed by Southern hybridization technique. The

PCR product for hptII gene from putative transgenic plants after gel separation were blotted onto nylon membranes

(Hybond N+; Amersham-Pharmacia The Probe was synthesized from 5’ site hpt II gene and was radioactively labeled with

α-[32 P]dCTP using the Random primer Kit from BRIT (India) according to the manufacturer’s instructions, and used for

the hybridization. The blotting and subsequent hybridization were carried out as described (Samboork et al., 1989).

RNA Isolation and RT PCR

RNA was isolated by total RNA isolation kit (Sigma, USA). cDNA was synthesized from 2 µg of RNA using first

strand cDNA synthesis kit (invitrogen, USA). cDNA was used for PCR amplification using gene specific primers of hpt II

(HPF and HPR).

Hygromycin Selection of Transgenic Plants

Putative transgenic cotton seeds (T1 and T2) were screened by growing them on hygromycin B (35 mgL-1 (Sigma,

USA) for the presence of transgene. Seeds obtained from T0 and subsequent generation of transgenic cotton plants were

evaluated on the basis of presence of root-laterals in selection medium (Hoagland with hygromycin B). After 7 days, plants

with root-laterals were considered positive while without root-laterals were considered as negative.

RESULTS

Agrobacterium mediated transformation of germinated shoot apical meristem is rapid and an efficient method of

transformation as after co-cultivation embryos were directly transferred to Hoagland medium for further growth. Genetic

transformation of total of 304 cotton seeds of three different cultivars yielded 91 transformed T0 plants. Almost similar

transformation frequency (27%-31 %) was observed on A. tumefaciens-mediated transformation of incised apical meristem

of germinated embryos of three different cultivars. DNA was isolated from all plants and PCR amplification results

showed presence of insert in nearly 29% of plantlets. The results are shown in Table1 and PCR amplification of some

plants is shown in Fig 3a. virC specific primers did not show any amplification from the genomic DNA of transgenic

plants, indicated the absence of residual bacteria (Fig 3b). RT-PCR confirmed the presence of Hpt II gene in transgenic T1

generation (Fig 4). It was observed that all plants were chimeric as GUS staining of T0 plant leaves showed mosaic pattern

(Fig 5A).

A total of 91 PCR positive T0 putative transgenic plants were transplanted in to net house after 4-6 weeks of

screening and hardening. Most of the plants grow normally without showing any phenotypic variation. Transformed T0

plants were allowed to pollinate naturally. Seeds obtained from T0 plants were grown on selection medium (Hoagland

medium containing 35 mgL-1 Hygromycin B). T1 Seedlings showing presence of root-lateral on 7th day were considered

positive and were transferred to pots in glass house.

A total of 5045 seeds were harvested at maturity and treated for Hygromicin selection and lateral root formation.

Individually, 85 hygromycin resistant plants out of 2827 in case of Khandwa -II, 22 out of 965 in case of Coker-310 and

Anjali var. yields 42 out of 1253 seeds on the basis of root lateral formation on hygromycin selection (Table 1). PCR

amplification and GUS assay of T1 transgenics confirmed the presence of the gus gene and GUS protein. Further, T1 plants

were not chimeric in nature as GUS staining was uniform from different leaves of the same plant (Fig 5B).

A Genotype-Independent Agrobacterium Mediated Transformation of 85 Germinated Embryo of Cotton (Gossypium hirsutum L.)

The confirmed positive T1 plants were assayed again using PCR-Southern blot hybridization. Genomic DNA

isolated from T1 generation transgenic cotton plants confirmed integration of the uid A gene in the transgenic cotton lines.

Genomic DNA isolated from non-transgenic plants did not hybridize with the Hpt II probe (Fig 6). Plants (T1) were

selected and seeds from these plants were analyzed for segregation ratio analysis. The Mendelian inheritance could not be

deduced due to small number of seeds obtained. Homozygous T2 plants were obtained by growing seeds from a single boll

on selection medium. Boll giving all seeds positive indicates that its mother plant (T1) was homozygous. The method

presented here showed not only the integration of both marker gene as well as antibiotic resistant gene, but also confirmed

the transmission of the introduced genes.

DISCUSSIONS

Development of a transformation method, which is independent of cultivars or tissue culture barriers, would

represent a major achievement in the area of transgenic development. Shoot apex transformation is carried out in different

cultivars of cotton, in which bisected shoot apex have been used for transformation cotton (Satyavathi et al. 2002; Gould et

al. 1998). A practical approach of embryo transformation is a short-term process of transformation of any genotype of

cotton. The advantage of using the embryo as an explant is that it allows genotype independent transformation and the

relatively rapid recovery of transgenic progeny (Christou, 1996; John 1997). This method of transformation avoids

somaclonal variations induced by long-term exposure of tissue/cells to culture media before they mature to give

regenerants with varying degree of somaclonal variation.

The feasibility of this transformation strategy was initially evaluated on the basis of number of seedlings

germinated after wounding and infection with A.tumefaciens. In embryo transformation, we utilized incised embryonal axis

for transformation. After co-cultivation embryos were directly transferred to Hoagland medium, almost all the embryos

transferred to test tubes, survived and were healthy. After 7-10 days, seedlings were transferred to pots in glass house for

further development.

Transformation efficiency measured as percentage of confirmed transgenic plants out of total number of plants

raised. The T0 generation transformation percentage is based on PCR analysis using hpt II gene primers, which was about

29% and is in accordance with earlier reports of embryo transformation in Safflower (Rohini and Rao, 2000) and higher

than in case of Cajanus cajan, which was found to be 15% in T1 generation, where embryonal segment was used for the

transformation (Surekha et al., 2005). The percentage of confirmed transgenic plants appeared to be lower (approximately

4-10% less) when compared to the previous reports of wheat (Supartana et al., 2006), kennaf (Kojima et al., 2004) and

maiz (Chumakov et al., 2006; Wang et al., 2007), where in planta transformation was carried on either on pistil filaments,

or meristem.

The difference in the transformation efficiency of three different cultivars may be attributed to the appropriate

incision of the embryos to expose apical meristem. An extra slicing of meristem was resulted into death of seedling and

similarly less incision lowered the chance of expose to Agrobacterium.

In this transformation method, as the Agrobacterium tumefaciens transfers the gene in to the meristmatic cells that

are still to be differentiated. Therefore, the T0 plants are chimeric in nature i.e. the parts of the T0 plants, originating from

inoculated meristems, could be transformed, while the other remained untransformed. Due to this, no step involved for

Agrobacterium killing in the T0 stage plants and has been inferred that A. tumefaciens could be eliminated by the self

defense action of plants. Indeed, we have verified the absence of A. tumefaciens in the transformants by two ways. Firstly

86 Manoj Kumar, Anoop Kumar Shukla, Harpal Singh, Praveen C Verma & Pradhyumna K Singh

by inoculation of leaf homogenate on LB plates (data not presented), no colonies appeared on inoculated plates and

secondly by confirming the absence of Vir C gene in genomic DNA.

In cases of T1 generation plants, however, the entire plant tissue should be of transformed nature and for

confirmation and screening of positive plants, we give 7-days antibiotic selection to T1 seeds to check the presence of root-

laterals. Assumption that T-DNA integration at heterochromatin regions will not be eliminated in T0 generation, as we are

not giving selection of hygromycin to eliminate them which we do in tissue culture by selecting them in medium

containing antibiotic for four to five cycle, doesn’t hold good.

Another assumption that T-DNA may preferentially target euchromatic regions of the genome and avoid

heterochromatin came from transgene expression studies by Koncz et al. 1989. He positioned a promoterless nptII gene

into T-DNA near a T-DNA border, transformed tobacco and Arabidopsis plants (selecting for kanamycin resistance

encoded by nptII gene within the T-DNA), and recorded the frequency of NPTII-positive plants. NPTII activity could

result only from T-DNA integration into transcriptionally active regions of the genome, allowing the promoterless nptII

gene to be transcribed by an active promoter. The results of these experiments indicated that in both Arabidopsis and

tobacco, approximately 30% of the T-DNA insertions resulted in transgenic plants expressing NPTII activity. Because the

genome size of Nicotiana is more than 10 times that of Arabidopsis, in part resulting from increased levels of repetitive

and heterochromatic DNA, thus author suggests that T-DNA specifically targeted transcriptionally active regions of the

genome. In our study also, integrations of the gene in transcriptional active region may resulted in high level of expression.

Embryo transformation of cotton was easy and inexpensive as it did not require lengthy tissue culture procedure

i.e. regeneration of transformant via somatic embryogenesis. Another advantage of having genotype independency of this

method, as it can be used to transform those cotton varieties that are recalcitrant to regeneration. It is highly efficient

technique for cotton as number of genes can be stably introduced in the genome in a short span of time.

The possibility of some of the progeny resulting in fully transformed plants for the introduced gene has been

demonstrated in this study. The procedure was applied to different species of cotton also for the validation of different

genes. Transformants were obtained with other crops also (Tomato, Tagetes and Pyrethrum) with different transformation

efficiencies (Lab data). The Agrobacterium susceptibility of different crops may be one of the reasons for this difference

In conclusion, the embryo transformation method of cotton transformation described in present study provides

highly efficient and frequent method of transgenic cotton production via Agrobacterium transformation. Using this method

approximately 27%-30% of gus positive plants were obtained which gives up to 3.3% of transformation efficiency in T1

generation.

REFERENCES

1. Anonymous.: Indian Textile industry - important from perspective of overall economy (2007)

2. Ministry of Textiles, Annual Report, Industry Research

3. Aydin, Y, Talas-Ogras, T., Alunkut, A., Insailoglu, I. (1992) Cytohistological studies during cotton somatic

embryogenesis with Brassinosteroid application. - IUFS Journal of Biology. 69: 33-39

4. Bayley, C., Trolinder, N., Ray, C., Morgan, M., Quisenberry, J.E., Ow, D.W. (2010) Engineering 2,4-D resistance

into cotton.- Theor Appl Genet. 83: 645–662

5. Christou, P. (1996) Transformation technology. - Trends Plant Sci. 1: 423-431

A Genotype-Independent Agrobacterium Mediated Transformation of 87 Germinated Embryo of Cotton (Gossypium hirsutum L.)

6. Crane, Y.M., Gelvin, S.B. (2007) RNAi-mediated gene silencing reveals involvement of Arabidopsis chromatin-

related genes in Agrobacterium-mediated root transformation. - Proced Nat Acad Sci. 104: 15156–15161

7. Gould, F. (1998) Sustainability of transgenic insecticidal cultivars: integrating pest genetics and ecology. - Annu

Rev Entomol. 43: 701-726

8. Gasser, C.S., Fraley, R.T. (1992) Transgenic crops. - Sci Am. 266: 62–69

9. Finer, J., Mc, Mullen, M. (1990) Transformation of cotton (Gossypium hirsutum L.) via particle bombardment. -

Plant Cell Rep. 8: 586–589

10. HuL, Yang, X., Yuan, D., Zeng, F., Zhang, X. (2011) GhHmgB3 deficiency deregulates proliferation and

differentiation of cells during somatic embryogenesis in cotton. - Plant Biotec J. 9:1038-48

11. Thomas, J.C., Brown, J.K., Adams, D.G., Keppenne, V.D., Wasmann, C.C., Kanost, M.R., Bohnert, H.J. (1995)

Protease inhibitors of Maduca sexta expressed in transgenic cotton. - Plant Cell Rep. 14: 758–762

12. Keller, G., Spatola, L., McCable, D., Martinell, B., Swain, W., John, M.E. (1997) Transgenic cotton resistant to

herbicide bialaphos. – Transgen Res. 6: 385-392

13. Koncz, C., Martini, N., Mayerhofer, R., Koncz-Kalman, Z., Körber, H., Redei, G.P., Schell, J. (1989) High-

frequency T-DNA-mediated gene tagging in plants. - Proc Natl Acad Sci. USA. 86: 8467–8471

14. Lyon, B.R., Cousins, Y., Llewellyn, D.J., Dennis, E.S. (1993) Cotton plants transformed with a bacterial

degradation gene are protected from accidental spray drift damage by the herbicide 2, 4-dichlorophenoxyacetic

acid. - Transgene Res. 2: 162–169

15. Perlak, F.J., Deaton, R., Armstrong, T., Fuchs, R.L., Sims, S.R., Greenplate, J.T., Fischhoff, D.A. (1990) Insect

resistant cotton plants. – Biotechnology. 8: 939–943

16. Rawat, P., Singh, A.K., Ray, K., Chaudhary, B., Kumar, S., Gautam, T., Kanoria, S., Kaur, G., Kumar, P.,

Pental, D., Burma, P.K. (2011) The expression of Bt endotoxin Cry1Ac has detrimental effect on the in vitro

regeneration as well as in vivo growth and development of tobacco and cotton transgenics. - Journal of

Bioscience. 36(2): 363-376

17. Rohini, V.K., Rao, K.S. (2000) Embryo transformation, a practical approach for realizing transgenic plants of

Safflower (Carthamus tinctorius L.). - Annals of Botany. 86: 1043-1049

18. Rohini, V.K., Sankara, Rao. K. (2000) Transformation of peanut (Arachis hypogaea L.): a non tissue culture

based approach for generating transgenic plants. - Plant Science. 150: 41–49

19. Sambrook, J., Fritsch, E.F., Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, second ed., CSHL

Press, Cold Spring Harbor, NY

20. Sankara, Rao., K., Rohini, V.K. (1999) Agrobacterium-mediated transformation of sunflower (Helianthus annuus

L.): a simple protocol. - Annals of Botany. 83: 347–354

21. Satyavathi, V. V., Prasad, V., Gita, Lakshmi. B., Lakshmi, Sita. G. (2002) High efficiency transformation

protocol for three Indian cotton varieties via Agrobacterium tumefaciens. - Plant Science. 162: 215-223

22. Schoneberg, J.M., Scelonge, C.J., Burrus, M., Bidney, D.L. (1994) Transformation of sunflower using embryonic

axis explants. - Plant Science 103: 199–207

88 Manoj Kumar, Anoop Kumar Shukla, Harpal Singh, Praveen C Verma & Pradhyumna K Singh

23. Singh, P.K., Kumar, M., Chaturvedi, C.P., Yadav, D., Tuli, R. (2004) Development of a hybrid δ-endotoxin and

its expression in tobacco and cotton for control of a polyphagous pest Spodoptera litura. - Transgenic Res 13:

397–410

24. Stelly, D.M., Altman, D.W., Kohel, R.J., Rangan, T.S., Commiskey, E. (1989) Cytogenetic abnormalities of

cotton somaclones from callus cultures. - Genome 2: 762–770

25. Trolinder, N.L., Goodin, J.R. (1988) Somatic embryogenesis in cotton (Gossypium) I. Effect of source of explant

and hormone regime. - Plant Cell Tiss Org Cult 12: 31-42

26. Trolinder, N.L., Xhixian, C. (1989) Genotype specificity of the somatic embryogenesis response in cotton. - Plant

Cell Rep 8: 133–136

27. Wang, J., Sun, Y., Li. Y. (2007) Maiz (Zea mays) genetic transformation by co-cultivating germinating seeds with

Agrobacterium tumefaciens. - Biotechnol Appl Biochem 46: 51-55

28. Zhao, F.Y., Li, Y.F., Xu, P. (2006) Agrobacterium mediated transformation of cotton (Gossypium hirsutum L. cv.

Zhongmian 35) using glyphosate as a selectable marker. - Biotechnol Lett 28: 1199-1207

APPENDICES

Table 1: Showing Transformation Efficiency of Agrobacterium-Mediated Transformation in T0 and T1 Generations

Genotypes

Number of Embryos for

Transformation

Number of PCR

Positive Embryos

Transformed (T0)

Number of Positive T1

Plants. Root Lateral

Presence

% Efficiency

Khandwa-2 104 33 85/2827 3

Coker-310 98 27 22/965 2.2

Anjali 102 31 42/1253 3.3

Figure Captions

Figure 1: Figure Showing Different Steps in Embryo Transformation Protocol for Cotton. (a) 24 H Germinated

Seeds, (b and c) Dissection of Embryo, (d and e) Co- Cultivation of Embryo, (f and g) Growing Seedlings in Test-

Tubes, (h) Transgenic Plant in Pot

A Genotype-Independent Agrobacterium Mediated Transformation of 89 Germinated Embryo of Cotton (Gossypium hirsutum L.)

Figure 2: Schematic Diagram of the T-DNA of PCAMBIA Binary Vector. LB, Left Border; CaMV35s, Cauliflower

Mosaic Virus 35S Promoter, RB, Right Border; HPTII, Hygromycin Phosphotransferase; uidA, GUS Reporter

Gene and Tnos, NOS Terminator

Figure 3: (a) PCR Amplification of hpt II Gene in T1 Positive (on the Basis of Presence of Root-Lateral) was

Showing Amplification of 500 bp. (b) Positive Plants Showing no Amplification of vir C Gene

Figure 4: Reverse Transcription PCR of T1 Transgenic for Presence of hpt II Transcript in Total RNA. Lane M

Shows Marker, Lane 1, 2, 3, 4 Shows 500 bp Band from Transgenic T1 cDNA, Lane 5 RT- and Lane 6 Amplification

from Positive Plasmid

Figure 5: Histochemical GUS Staining. Panel A: Sharing Chimeric GUS Staining in T0 Transgenic Cotton. Panel B:

Uniform GUS Staining in T1 Transgenic Cotton