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

Document Type: Research paper


1 Department of Horticultural Science, Faculty of Agriculture, Bu-Ali Sina University, Azadegan Boulevard, Hamedan, Iran

2 Agricultural Biotechnology Research Institute of Iran (ABRII), Mahdasht Road, Karaj, Iran


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.


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