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1 January 2005 TRANSFER AND EXPRESSION OF NPTII AND BAR GENES IN CUCUMBER (CUCUMIS SATIVUS L.)
G. VENGADESAN, R. PREM ANAND, N. SELVARAJ, R. PERL-TREVES, A. GANAPATHI
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Abstract

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 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 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.

G. VENGADESAN, R. PREM ANAND, N. SELVARAJ, R. PERL-TREVES, and A. GANAPATHI "TRANSFER AND EXPRESSION OF NPTII AND BAR GENES IN CUCUMBER (CUCUMIS SATIVUS L.)," In Vitro Cellular and Developmental Biology - Plant 41(1), 17-21, (1 January 2005). https://doi.org/10.1079/IVP2004602
Received: 13 July 2004; Accepted: 1 September 2004; Published: 1 January 2005
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KEYWORDS
Acetosyringone
agro-transformation
cucumber
Phosphinothricin
pre-culture
β-glucuronidase
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