Transfer and expression of npt II and bar genes in cucumber (Cucumis sativus L.)

The generation of transgenic Cucumis sativus cv. Greenlong plants resistant to phosphinothricin (PPT) was obtained using Agrobacterium tumefaciens-mediated gene transfer. The protocol relied on the regeneration of shoots from cotyledon explants. Transformed shoots were obtained on Murashige and Skoog medium supplemented with 4.4 muM 6-benzylaminopurine, 3.8 muM abscisic acid, 108.5 muM adenine sulfate, and 2 mg 1(-1) phosphinothricin. Cotyledons were inoculated with the strain EHA105 harboring the neomycin phosphotransferase, II (npt II), and phosphinothricin resistance (bar) genes conferring resistance to kanamycin and PPT. Transformants were selected by using increasing concentrations of PPT (2-6 mg 1(-1)). Elongation and rooting of putative transformants were performed on PPT-containing (2 mg 1(-1)) medium with 1.4 muM gibberellic acid and 4.9 muM indolebutyric acid, respectively. Putative transformants were confirmed for transgene insertion through PCR and Southern analysis. Expression of the bar gene in transformed plants was demonstrated using a leaf painting test with the herbicide Basta. Pre-culture of explants followed by pricking, addition of 50 muM acetosyringone during infection, and selection using PPT rather than kanamycin were found to enhance transformation frequency as evidenced by transient beta-glucuronidase assay. Out of 431 co-cultivated explants, 7.2% produced shoots that rooted and grew on PPT, and five different plants (1.1%) were demonstrated to be transgenic following Southern hybridization.