Effect of explants, bacterial cell density and overgrowth-control antibiotics on transformation efficiency in tomato (Solanum lycopersicum L.)
2013
Pawar, B. D. | Jadhav, A. S. | Kale, A. A. | Chimote, V. P. | Pawar, S. V.
Improved protocol for Agrobacterium mediated transformation of tomato cultivar, "Dhanshree" was developed by optimizing various parameters that affect transformation efficiency. In the present investigation, Agrobacterium strain EHA 105 harboring a pBI121: gus gene construct was used for transformation. The kanamycin concentration was standardized and 50 mg/L was found to be optimum based on lethal effect to the explants. Effect of varying concentration of Agrobacterium on the transformation efficiency of cotyledon explants revealed that the concentration of 0.2 at OD600 was optimum. Cotyledons proved to be better for transformation as compared to hypocotyls and leaf explants. Highest transformation efficiency was obtained in 7-14 days old cotyledon which was precultured for one day on the MS medium containing 2 mg/L zeatin and 0.2 mg/L IAA. It was then co-cultivated with Agrobacterium for 3 days on the same medium composition used for preculture. Subsequently the explants were transferred to selective shooting medium supplemented with 50 mg/L kanamycin, 250 mg/L cefotaxime and 250 mg/L carbenicillin. These explants were maintained for 6-8 weeks which resulted in more than 12 % transformation efficiency as judged by GUS assay technique.
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