Abstract
Background: Agrobacterium mediated genetic transformation experiments were carried out in Dolichos bean Cv. (Konkan Bhushan) showing better regenerability. Methods: Three cry genes viz. cry1Aabc, cry1Fa1 and cry2Aa were used for the transformation each of which were linked to CaMV35S promoter and nptII gene under control of nos promoter and terminator. A vector system consisting of the disarmed hyper virulent Agrobacterium tumefaciens strain EHA-105 harboring pBinAR or BinBt3 was used. Mature embryo axis with single cotyledon was used as explant. Kanamycin as well as PCR screening was carried out to assess the transformation frequency. Progeny analysis using PCR was also carried out to assess the transgene segregation and stable transformation.Result: Kanamycin concentration of 500 mg/l was found as optimum for selection of a transgenic turning leaf blades albino. Among five methods of colonization used, the method employing mild injury to explant with dipping in Agrobacterium culture for 20 minutes followed by co-cultivation for 48 hours, cefotaxime washing and sowing in soil resulted in maximum survival (74.80%) associated with maximum transformation frequency through PCR analysis (2.13%). Among three cry genes, the gene cry2Aa was found the most effective in transforming Dolichos bean. The progeny analysis of transformants has shown the 3:1 mendelian segregation ratio confirming stable transformation of transgene.
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