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Online ISSN : 2349-8080 Issues : 12 per year Publisher : Excellent Publishers Email : editorinchiefijcrbp@gmail.com |
2Center for Research and PG studies, Department of Life Sciences, Kristu Jayanti College (Autonomous), K. Narayanapura, Kothanur (P.O), Bangalore, Karnataka-560 077, India
An improved protocol for Agrobacterium-mediated transformation was developed for tomato plant. In this study, transformation was carried out using disarmed A. tumefaciens strain LBA4404 harboring a binary vector pGPTV-kan (13400bp) containing the CP-gene, was used in this study. Factors that affect transformation/regeneration protocols were optimized in a series of experiments. Results indicated that exposure of cotyledonary/leaf explants to Agrobacterium inoculums of 0.7 OD at 600nm for 10 mins, co-cultivation in MS-liquid media, selection on kanamycin (kanamycin 50mg/ml), Augmentin, and cefotaxime (250mg/ml)-containing medium and subsequent regeneration on MS medium supplemented with 2.5mg/L BAP and 0.1 mg/L IAA resulted in transformation efficiency of 24.50%. Coat protein (CP) expression was observed in transformed tomato shoots but never in the control plants. PCR amplification of DNA extracted from the transformed tissues demonstrated the generation of the expected amplicon, corresponding to CP gene. This result strongly verifies the successful transformation of tomato plant. Moreover, this protocol may pave the way for problem solving-applications encompassing other Indian crops of economic importance.
