Selection of Transformed Callus in a Liquid Medium and Regeneration of Transgenic Plants in Cucumber (Cucumis sativus L.)

Abstract

The effects of three antibiotics and culture methods were investigated to find out reliable selection procedure for Agrobacterium -mediated transformation in cucumber (Cucumis sativus L.). When cotyledonary explants without inoculation of Agrobacterium were cultured on agar medium containing kanamycin, geneticin (G 418) or hygromycin, callus formation and greening of explants were suppressed more effectively by G 418 or hygromycin than by kanamycin. However, G 418 and hygromycin could suppress effectively callus formation only at the part of explants contacting with an agar medium. In order to expose the whole surface of explants to antibiotics, tissues were cultured in a liquid medium. Combination of liquid culture and addition of G 418 or hygromycin were highly effective in suppressing callus formation from uninfected explants. Explants infected with Agrobacterium were cultured in a liquid medium with each antibiotic. Hygromycin or G 418 resistant calli were selected under the presence of 20-80 mg/l hygromycin or 20 mg/l G 418, respectively. Calli formed at 20 mg/l hygromycin were transferred to a regeneration medium, and regenerated plants were obtained via embryogenesis. PCR analysis demonstrated integration of hygromycin phosphotransferase (HPT) gene to all regenerated plantlets and transmission of HPT gene to their progeny.