The selection of chlorsulfuron-resistant cell lines of independent origin from an embryogenic cell suspension culture of Brassica napus L.

Abstract

A simple method has been developed to isolate chlorsulfuron-resistant cell lines of independent origin from an embryogenic cell suspension culture of Brassica napus cv Jet Neuf. Clonal cell cultures were used as the source of cells for the isolation of resistant variants. The selection procedure involved plating of cells, mutagenized with ethyl methane sulfonate, on a medium supplemented with 10 −8 or 5 × 10 −8 M chlorsulfuron and rescuing the cell colonies which survived after 7 weeks of incubation. The selected cell lines were 10–1000-fold more resistant than the wild-type to chlorsulfuron and another sulfonylurea herbicide sulfometuron methyl. The activity of the enzyme acetolactate synthase, the site of action of sulfonylureas, isolated from resistant cells was not inhibited by those concentrations of both the herbicides which inhibited the wild-type enzyme. Subculture of variant calli on agarose-solidified medium was necessary for the induction of embryogenesis, the further development of embryos, and the maintenance of resulting shoot cultures. Stem explants from regenerated shoots of the resistant variant formed callus on chlorsulfuron-supplemented medium indicating that the resistance was not lost during organogenesis.