Agrobacterium-mediated genetic transformation and production of semilooper resistant transgenic castor (Ricinus communis L.)

Semilooper resistant transgenic castor plants were produced through Agrobacterium-mediated genetic transformation method. Two castor cultivars, Jyothi and VP1 were transformed using the super-binary vector pTOK233 carrying gus A and hpt genes. Putative transformants were regenerated following selection on the hygromycin containing medium. GUS positive primary transformants, when subjected to Southern analysis, revealed stable integration of gus A into their genomes. In the T1 generation, a monogenic segregation ratio of 3 GUS positive: 1 GUS negative plants was observed. Furthermore, transformation experiments were carried out with the Agrobacterium pSB111 super-binary vector carrying a synthetic delta endotoxin gene cryIAb and the herbicide resistance gene bar both driven by cauliflower mosaic virus 35S promoter. Putative transformants were regenerated through selection on the phosphinothricin containing medium and Basta tolerant transformants were subjected to molecular analysis. PCR analysis revealed the presence of both bar and cryIAb genes in the Basta tolerant primary transformants. Southern analysis of PCR positive plants with cryIAb probe showed a 3 Kb band upon HindIII digestion and a > 6 Kb band with BamHI digestion, thus suggesting stable integration of cryIAb intact expression cassette and independent nature of the transformants. The primary transformants subjected to ELISA disclosed varied levels of Cry protein. These transgenics expressing cryIAb – when bioassayed against freshly hatched semilooper larvae – induced substantial (> 88%) insect mortality. Southern analysis of 2T1 plants revealed the presence of cryIAb gene, indicating stable inheritance of the transgene into the next generation. In T1, all the Southern-positive plants for cryIAb invariably exhibited tolerance to Basta, denoting co-segregation of both bar and cryIAb genes. Transgenics, expressing cryIAb exhibited ample resistance against the castor semilooper.