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 β- 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 TDNA was stably integrated into the genome of three out of six PCR-positive plants. The transgenic plants were rooted and successfully acclimatized

کلیدواژه ها:

genetic transformation ، polymerase chain reaction ، selectable marker ، transgenic plants