Evaluation of parameters for high efficiency gene transfer via Agrobacterium tumefaciens and production of transformants in Indian mulberry, Morus indica cv. K2

Agrobacterium-mediated transformation of Indian mulberry, Morus indica cv. K2, employing the highly regenerative hypocotyl, cotyledon, leaf and leaf callus explants is reported. Preculture of explants on regeneration medium for 5 days and co-cultivation for 3 days was found optimal. With the GUS-histochemical assay up to 40% hypocotyl, 50% cotyledon, 50% leaf and 54% leaf callus explants tested positive. Agrobacterium strain LBA4404 was more infective than GV2260 and A281 and amongst the plasmids tested, pBI121 successfully transformed 100% of the explants followed by p35SGUSINT (90-100%). Approximately, 50% of the explants survived selection. Up to 35.7% hypocotyl, 38.5% cotyledon, 23% leaf and 40% leaf callus derived shoots tested positive for GUS gene activity by the spectrofluorometric analysis. Transgene integration in the regenerated transformants was confirmed by polymerase chain reaction (PCR) and Southern hybridization making this the first report of stable transformation of mulberry