Transformation of pomegranate (Punica granatum l.) a difficult-to-transform tree

In this study, an Agrobacterium-mediated transformation method was developed for pomegranate, a difficult-to-transform plant. We first optimized callus induction and shoot regeneration efficiency. Induction of calluses was best achieved from the intermodal stem sections on WPM medium supplemented with 12 μM BA and 8 μM NAA. The highest frequency of shoot regeneration (69.33%) and number of shoots (7.16) per piece of callus were obtained when calluses were incubated on WPM medium supplemented with 12 μM BA and 2 μM NAA. Callus pieces were inoculated with Agrobacterium tumefaciens strain EHA105 harboring the binary vector pBin19 carrying the neomycin phosphotransferase (nptII) gene as a selectable marker and green fluorescent protein (GFP) gene as a reporter. After 4 weeks in WPM selection medium (the best callus induction medium supplemented with 50 mg L−1 kanamycin), putative transgenic calluses were obtained. From such calluses transformed shoots began to appear approximately 3 week after the kanamycin resistance transgenic callus pieces had been placed on the best shoot regeneration medium containing 50 mg L−1 kanamycin. GFP fluorescence was observed in putatively transformed calluses and shoots. Molecular analysis confirmed the integration of the nptII transgene in transformed shoots. The transgenic shoots were rooted and successfully acclimatized.