Standardization of Agrobacterium tumefaciens-mediated genetic transformation protocol in Punica granatum L. cv. Kandhari Kabuli

A protocol for Agrobacterium tumefaciens-mediated genetic transformation in Punica granatum cv. Kandhari Kabuli has been developed using cotyledon and hypocotyl explants via both direct and indirect organogenesis pathways. Agrobacterium tumefaciens strain LBA 4404 harboring gus and npt-II genes in a binary vector pBI 121 had been used for genetic transformation studies. Pre-culturing of explants for 48 h (2 days) on regeneration medium before agrobacterial infection significantly increased the transformation frequency. Co-cultivation period of 48 h (2 days) was found to be optimum for genetic transformation. Cefotaxime in a concentration of 500 mg/l was found optimal for controlling the excessive bacterial growth and transformed shoots were selected using 50 mg/l kanamycin. Only the transformed cells were able to grow on the selective regeneration medium. Transgenic shoots were rooted on half-strength MS medium supplemented with 0.10 mg/l NAA, 0.02% activated charcoal, 50 mg/l kanamycin and 500 mg/l cefotaxime and subsequently acclimatized to the culture room conditions. Stable transformation and expression were confirmed on the basis of GUS histochemical and spectrophotometric assays. This genetic transformation protocol will provide a platform for genetic manipulation of pomegranate for incorporation of genes governing various horticulturally important traits in future.