Agrobacterium-Mediated Transformation of Pomegranate (Punica granatum L.) ‘Yousef Khani’ Using the gus Reporter Gene

In this study, an efficient Agrobacterium-mediated transformation method was developed for pomegranate (Punica granatum L.), a difficult-to-transform plant. In vitro shoot segments were inoculated with Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pBI121 carrying the neomycin phosphotransferase (nptII) gene as a selectable marker and B-glucuronidase (gus) gene as a reporter. After 28 d in WPM selection medium containing 50 mg L-1 kanamycin, 59 new shoots proliferated. gus analysis was performed on these putative transgenic shoots, of which 32 stained positive. Positive staining shoots were cut and cultured in selection medium for 2 subsequent subcultures until final gus analysis. After three months of the selection period, 6 putative transgenic shoots were obtained. Presence of the gus and nptII genes was confirmed by polymerase chain reaction. Southern blot analysis confirmed that T-DNA was stably integrated into the genome of three out of six PCR-positive plants. The transgenic plants were rooted and successfully acclimatized.