Efficient production of transgenic tomatoes via Agrobacterium-mediated transformation

Cotyledonary leaves of 9-d-old tomato (Lycopersicon esculentum Mill.) were co-cultivated with Agrobacterium tumefaciens GV 3101 harboring binary vector pBI101 containing kanamycin resistance gene (npt II) as selection marker. Murashige and Skoog (MS) inorganic salts with Gamborg's B5 vitamins supplemented with optimized concentrations of zeatin riboside and indole-acetic acid resulted in enhanced regeneration efficiency. Under optimized conditions of plant regeneration, transformation frequency in cvs. Pusa Ruby, Pusa Uphar and DT-39 was greater than 37 %. Transformed shoots were selected on kanamycin medium and the presence of the transgene in the primary transformants was confirmed by PCR. Integration of the npt II gene in the tomato genome was further confirmed by Southern blot analysis. RT-PCR analysis using neomycin phospho-transferase (npt II) gene-specific primers confirmed the expression of the transgene in transgenic plants. Transformed plants were successfully transferred to phytotron, where these plants grew to maturity and produced flowers and fruits.