M. Guru Prasad et al.

, IJSID, 2011, 1 (3), 428-432

ISSN:2249-5347

IJSID
International Journal of Science Innovations and Discoveries
Research Article
An International peer Review Journal for Science

Available online through www.ijsidonline.info

STANDARDIZATION OF PROTOCOL FOR IN VITRO REGENERATION OF THE BRINJAL (SOLANUM MELONGENE) CVS-69
M.Guru Prasad 1, SK.Jaffar 2* K.L.N. Mallikharjuna rao3, V.C. Harika4, D.Venkateshwarlu Naik5
1Regional

Agriculture Research station Tirupathi, 517 502. A.P, India; 2Department of Biochemistry, Acharya Nagarjuna University, Guntur-522 510, A.P, India; 3Dept of Bichemistry, Acharya Nagarjuna University, A.P, India; 4Dept of Food &Nutritional Science, Acharya Nagarjuna University, Guntur-522 510, A.P, India; 5Department of Biochemistry, Acharya Nagarjuna University, Guntur-522510, A.P, India.

Received: 06.09.2011

ABSTRACT
Modified: 19.10.2011 Published: 29.12.2011
*Corresponding Author

A procedure for in vitro regeneration was developed using cotyledonary explants of a commercially grown brinjal cultivar. Shoot regeneration from inoculated cotyledons (40-60%) was obtained on MS medium containing zeatin riboside 1mg/l.Numerous shoots were developed from the established explants on the same medium. profuse rooting was achieved on strength MS salts, IBA 1mg/l and sucrose 60mg/l within 10 days. This regeneration procedure was used to facilitate gene transfer through agro bacterium tumefaciens in egg plant var s69. Using cry 1Ab gene present in the T-DNA of the binary vector, PCAMBIA which also contains gene that encodes neomycin phosphotransferase -11 (npt 11). The

Name: SK. Jaffar Place: Guntur, AP, India E-mail: Shaikjaffar2008@yahoo.com

explants were co cultivated with Agro bacterium tumefaciens and shoots were regenerated on medium as per the protocol standardized. The selection was done on a medium containing 75mg/l kanamycin and 500mg/l cefotaxime. The T0 brinjal plants were normal and tested positive for npt 11 through PCR with npt 11

INTRODUCTION

specific primer. The transformed progenesis are being multiplied for further evaluation and bioassay.

Key words: Brinjal, Transformation, Tissue culture, Zeatin Riboside, Indole acetic acid

INTRODUCTION
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INTRODUCTION Solanum melongene.l. (Brinjal) is an important vegetable crop grown in India. It is an integral part of the diet as the population is mostly vegetarian in nature. Conventional breeding as led to the improvement of few traits like drought tolerant and increase in yield. Man of the important agronomic and stress resistance traits has at to be improved. Despite the fact that some of the wild relatives were identified as resistant sources to several biotic and a biotic stress (Among the biotic stresses the shoot and fruit born leucinodes orbonalis causes sever damage and reducing the yield up to 70%). Even though some degree of insect protection is providing b the insecticides, management of shoot and fruit borer b safe and effective alternatives is necessary). The transfer of the crystal protein gene b bioassay) with the event of the biotechnological modern approaches several successful attempts have made to transfer Bt resistant crystal protein genes (Kumar.et al.1999) to the plants in order to have sustainable degree of the stress resistance. But the success of genetic transformation in plants upon the availability of efficient (Nayudu.et al) and reproducible tissue culture protocols for regeneration of whole plants from various explants. Among the brinjal genotypes cultivated in A.P.S-69 is a high yield and greater adaptability but susceptible (Sharma, et al) to several biotic stresses. Since large number of gene construct for biotic stresses were available in public do, genetic transformation is a potential option to improve the traits in the variety. Tissue culture techniques have been used to produce somatic embryo and haploid plant in brinjal (Gledd et .at 1986). In brinjal the using of agro bacterium transformation was first report b the guru and sink (1988) b using the nos neo marker. Agro bacterium mediated transformation using root explants was also developed (Franklin and lakshmisita 2003). In this article we describe a regeneration protocol in brinjal, using which the genetic transformation of brinjal was done to introduce a cry1ab gene. MATERIALS AND METHOD: Plant material and its tissue culture Cotyledonary explants of the S-69 were cultured and multiple shoots were induced on ms medium containing 1mg/l zeatin riboside .Regenerated plants were rooted on MS medium containing 1mg/l of Abate certified seeds of cultivated were washed in running tap water for few minutes .Later on washed seeds were treated with bavastin 1% for 20 minutes and then the seeds were surface sterilized with 70% ethanol for 5 min utes, 0.1% sodium hypochlorite for 20 minutes. Then treated seeds were rinsed 3-4 times with distilled water and germinated on half strength MS medium solidified with 0.8% agar. Agro bacterium strain and culture condition Agro bacterium strain LBA 4404 contain binary vector PCAMBIA was used for transformation .The binary vector (Rotino G L & Gleddie S et al) carrying the neomycin phosphotransferase (npt11) gene driven b nopaline synthase (nos) promoter which confers resistance to antibiotic kanamycin as a plant selection medium . the vector also contains CR1Ab gene driven b cauliflower mosaic virus 35s (CAMV 35S) promoter .A colony from a freshly
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prepared culture is inoculated in LB medium containing 50mg/l kanamycin and 15 mg/l rifampicin at 28c on a rotating shaker (200rpm) and grown for overnight. Agro bacterium mediated transformation: Cotyledon explants from 10 days old seedlings were pre-incubated on hormone free MS medium for 2 days and used for co- cultivation. 5ml of agro bacterium liquid culture (1 OD at 600nm) which was grown for overnight is taken and added to the pre-incubated explants which were taken and added to the pre-incubated explants which were taken in Petri plates and swirled gently for 5 min. infected explants were blot dried on sterile filter paper and co-cultivated on hormone free MS medium for 2 days. These segments were transferred to the selection medium (shooting medium containing 50 mg/l kanamycin) after 2 DAS of co- cultivation. PCR Assay for transgenic plants From the fresh leaves of putative transformants genomic DNA is isolated using CTAB method and subjected to pcr using npt11 specific primer the primers and PCR conditions were used as earlier described by (chakrabarti et al., 1999) RESULTS AND DISCUSSION The regenerated studies( Chadha M L et al .,)which were done before agro bacterium mediated transformation (Magioli C et al.,) this shoot buds developed from the cut ends of the explants on MS medium containing 1mg/l Zeatin riboside with in 15-20 days of culture initiation. Multiplication and elongation of shoots was obtained on the same. the roots were induced on half strength MS medium supplemented with 1 mg/l IBA.The results during regeneration suggest that gelatin plays a major role in shoot differentiation as no regeneration obtained in the absence of this growth regulator the regenerated plants were acclimatized under high humidity and transfer to field and the showed normal performance.

1.Cotyldons, 2.shoot tips and 3.regeneration of shoot lets (fig 1,2 and 3) In the transformation experiment (Andrasfalvy A et al, 1995) after 15 days after incubation shoot induction was observed from the cut ends of cotyledons in selection medium (fig 1,2 and 3). In contrast the untransformed
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explants turned brown and die on selection medium containing 50 mg/l kanamycin .The putative transformed shoots were analyzed through PCR test for the presence of npt11Gene by using npt11 gene specific primer and all the transformants that selected on the media were formed to be positive for npt11.In contrast the control and untransformed plants did not show the presence of npt11 gene approximately 700bp fragment specific to npt11 was amplified with PCR. The transgenic explants that were produced by this agro bacterium mediated transformation of cotyledons in the field level further analysis to asses the degree of insect protection is in progress. Table.1 Shoot initiation in cotyledon explants of S-16 Cultured on MS medium containing Zeatin riboside in various concentrations S.No Growth regulator Zeatin No of No of Regeneration frequency riboside explants shoots/explants 1 0.25 10 Callus 2 0.5 10 Callus 3 0.75 10 2-3 60 4 1.0 10 5-6 80 5 1.25 10 3-4 50 6 1.5 10 Callus 7 1.75 10 Callus 8 2.0 10 Callus Table .2 Rooting response of in vitro regenerated shoots of S-16 cultured in MS medium containing IBA in various concentrations S.No Growth regulator IBA Rooting frequency No of das 1 0.25 2 0.5 3 0.75 50 17 4 1.0 80 17 5 1.25 60 21 6 1.5 40 24 7 1.75 8 2.0 CONCLUSION The normal cultivation methods gives only resistance towards only for increasing the yielding and quality improvement. But in this standard protocol gives better regeneration than the normal conventional breeding. Although by using agrobacterium mediated gene transformation give a better regeneration compared to grown in normal medium without using any type transformation methodology. From this standard protocol, in future we can develop new varities and that can be helpful to study the phylogentic relation and genetic variation in between the different varities. This protocolwidely applicable and improve the brinjal varities , it showed resistance towards the different insects and also gives better yielding.in future this study explored to identify the which gene responsible for the resistance.

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