Evaluation of green fluorescent protein as a reporter gene and phosphinothricin as the selective agent for achieving a higher recovery of transformants in cucumber (Cucumis sativus L. cv. Poinsett76) via Agrobacterium tumefaciens

Abstract

Efficient Agrobacterium tumefaciens-mediated transformation and a higher recovery of transformed plants of cucumber cv. Poinsett76 were achieved via direct organogenesis from cotyledon explants. Stable transformants were obtained by inoculating explants with A. tumefaciens strains EHA105 or LBA4404, both harboring the binary vector pME508, which contains the neomycin phosphotransferase II (nptII) and phosphinothricin resistance genes (bar) conferring resistance to kanamycin and PPT, respectively, as selectable markers and the sgfp-tyg gene for the green fluorescent protein (GFP) as a visual marker driven by the constitutive CaMV35S promoter in the presence of acetosyringone (50 μM). Transformed shoots were obtained on MS Murashige and Skoog (Plant Physiol. 15: 473–497, 1962) medium supplemented with 1 mg L−1 benzyladenine (BA), 20 mg L−1 l-glutamine and 2 mg L−1 phosphinothricin (PPT) or 100 mg L−1 kanamycin. The regenerated shoots were examined in vivo using a hand-held long wave UV lamp for GFP expression. The GFP screening helped identify escapes and chimeric shoots at regular intervals to increase the growth of transformed shoots on cotyledon explants. Elongation and rooting of putative transformants were achieved on PPT (2 mg L−1) containing MS media with 0.5 mg L−1 gibberellic acid (GA3) and 0.6 mg L−1 indole butyric acid (IBA), respectively. PCR and Southern analyses confirmed the integration of the sgfp gene into the genome of T0 and the progenies. T1 segregation of transgenic progeny exhibited Mendelian inheritance of the transgene. The use of EHA105 resulted in 21% transformation efficiency compared to 8.5% when LBA4404 was used. This higher rate was greatly facilitated by PPT selection coupled with effective screening of transformants for GFP expression, thus making the protocol highly useful for the recovery of a higher number of transgenic cucumber plants.