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 forpomegranate (Punica granatum L.), a difficult-to-transform plant. In vitro shoot segments wereinoculated with Agrobacterium tumefaciens strain LBA4404 harboring the binary vectorpBI121 carrying the neomycin phosphotransferase (nptII) gene as a selectable marker and β-glucuronidase (gus) gene as a reporter. After 28 d in WPM selection medium containing 50 mgL-1 kanamycin, 59 new shoots proliferated. gus analysis was performed on these putativetransgenic shoots, of which 32 stained positive. Positive staining shoots were cut and culturedin selection medium for 2 subsequent subcultures until final gus analysis. After three months ofthe selection period, 6 putative transgenic shoots were obtained. Presence of the gus and nptIIgenes was confirmed by polymerase chain reaction. Southern blot analysis confirmed that TDNAwas stably integrated into the genome of three out of six PCR-positive plants. Thetransgenic plants were rooted and successfully acclimatized.