Hygromycin as selective marker in Agrobacterium-mediated genetic transformation of indica rice MR 219.

The success of Agrobacterium-mediated genetic transformation depends on the efficiency of gene delivery, plant regeneration systems and selection agents. The effects of hygromycin B (hyg B), a selection agent that is commonly used in plant genetic transformation, on embryogenic calli and somatic embryos of indica rice var MR 219 was evaluated. Embryogenic calli and embryos were cultured on pre-regeneration medium containing hygromycin concentrations from 0 to 200 mg/litre for 12 weeks to determine the lethal dose. The potential of the hygromycin phosphotransferase (hpt) gene as the selective marker was also evaluated. The embryogenic calli was co-cultivated with the Agrobacterium strain LBA 4404 that harbours the binary vector pCAMBIA1305.2 with the hpt gene and subsequently cultured on regeneration medium with added hygromycin concentrations at 0 to 50 mg/litre. Results showed that hygromycin concentrations above 30 mg/litre strongly inhibited growth and development of non-transformed embryogenic calli and somatic embryos. Ten to 20 mg/ litre hygromycin concentrations were the most suitable concentrations used for selection of transgenic embryos or plantlets of rice MR 219 with 2.4% efficiency. Hygromycin B is a suitable selection agent and selective marker for genetic transformation of rice MR 219. Keywords: Oryza sativa L.; somatic embryo; phosphotransferase; plant regeneration; tissue culture