Optimizing tissue culture media for efficient transformation of different indica rice genotypes

An efficient system was established for high frequency embryogenesis and regeneration of indica rice, Oryza sativa L. cv. MDU 5. Basic media, carbohydrate sources and concentrations, agar concentrations, amino acids, cytokinins and auxins were evaluated in callus induction and regeneration media to establish the most efficient regeneration protocol for MDU 5 indica rice. The optimized callus induction and regeneration media consisted of the basic MS salt mixtures and vitamin solutions supplemented with 4% maltose, 1g/L casein hydrolysate and 50 mg/L tryptophan, solidified with 1% agar. The callus induction medium was supplemented with 2,4−D 2 mg/L, kinetin 0.5 mg/L, indole acetic acid 1 mg/L, and 6−benzyl aminopurine 0.5 mg/L whereas the media for regeneration phase comprised kinetin 2 mg/L, indole acetic acid 1 mg/L and 6−benzyl aminopurine 2 mg/L. The media optimized for MDU 5 was analyzed for response of 73 other indica genotypes. Of these indica varieties 64 genotypes yielded 98.5% callus induction within eight days, 59% embryogenic calli formation, initiation of multiple green buds within eight days and a regeneration rate of 90%. The embryogenic calli derived were used for transformation with Agrobacterium tumifaciens (LBA 4404 or EHA 101 strains). Two binary vectors (pKHG4 and pIG121Hm) containing hph and GUS genes were used in these transformation studies. Fifty-one genotypes responded to the optimized media by producing hygromycin−resistant calli. The -histochemical test for ß−glucuronidase activity was positive from 32 genotypes with the transformation efficiencies ranging between 7.0% and 8.3%.