Direct regeneration of transformed shoots in Brassica napus from hypocotyl infections with Agrobacterium rhizogenes.

Abstract

Genetically transformed root clones of rapeseed (Brassica napus) were obtained after in vitro infection of excised hypocotyl segments with a wild type strain of Agrobacterium rhizogenes and two strains of A. rhizogenes harbouring kanamycin resistance. The ability of hairy root formation was affected by light and was highly dependent on the location of the infection site at the hypocotyl. Inoculation of decapitated hypocotyls with an intact root system gave rise to direct shoot formation from the site of inoculation. Histological sections showed that several meristems were initiated at the inoculation site. Root and shoot clones were isolated and subcultured axenically in hormone-free liquid MS medium. Identification of transformed root and shoot clones was based on opine assays. Further selection was carried out in kanamycin-enriched medium. All opine-positive root clones showed NPT II (neomycin phosphotransferase) activity. Nearly half of the shoot clones expressed a strong NPT II activity while the rest gave a weak or no NPT II response.