Mutant acetolactate synthase gene is an efficient in vitro selectable marker for the genetic transformation of Brassica juncea (oilseed mustard)

Abstract

We report in this study, the successful deployment of a double mutant acetolactate synthase gene (ALS^dm, containing Pro 197 to Ser and Ser 653 to Asn substitutions) as an efficient in vitro selection marker for the development of transgenic plants in Brassica juncea (oilseed mustard). The ALS enzyme is inhibited by two categories of herbicides, sulfonylureas (e.g. chlorsulfuron) and imidazolinones (e.g. imazethapyr), while the mutant forms are resistant to the same. Three different selection agents (kanamycin, chlorsulfuron and imazethapyr) were tested for in vitro selection efficiency in two B. juncea cultivars, RLM198 and Varuna. For both the cultivars, higher transformation frequencies were obtained using chlorsulfuron (3.8±0.6% and 4.6±0.9% for RLM198 and Varuna, respectively) and imazethapyr (10.2±0.7% for RLM198 and 7.8±1.2% for Varuna) as compared to that obtained on kanamycin (3.1±0.2% and 2.8±0.5% for RLM198 and Varuna, respectively). Additionally, transformation frequencies were higher on imazethapyr than on chlorsulfuron for both the cultivars indicating that imidazolinones are better selective agents than sulfonylureas for the selection of mustard transgenics.