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 μM 6-benzylaminopurine, 3.8 μM abscisic acid, 108.5 μM adenine sulfate, and 2 mg l-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 l-1). Elongation and rooting of putative transformants were performed on PPT-containing (2 mg l-1) medium with 1.4 μM gibberellic acid and 4.9 μM indolehutyric 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 μM acetosyringone during infection, and selection using PPT rather than kanamycin were found to enhance transformation frequency as evidenced by transient β-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.
|Number of pages||5|
|Journal||In Vitro Cellular and Developmental Biology - Plant|
|State||Published - Jan 2005|
Bibliographical noteFunding Information:
The senior author gratefully acknowledges the Department of Science and Technology (DST), Government of India for the financial support as Senior Research Fellowship (SRF) and Dr. V. Balaji, Postdoctoral Research Associate, Department of Plant Science, Tel Aviv University, Israel for valuable suggestions. The author also thanks Mr. Danny Shavit, Shavit Professional Scientific Photography, PSP Ltd., Bet Dagan, Israel for his valuable assistance with the photography. The corresponding author is grateful for financial support under the Indo-Israel collaborative programme 8979-1-97 from the Department of Science and Technologies (DST), Government of India.