In Vivo and in Vitro Mutagenesis of a Brassica Napus Acetolactate Synthase Gene for the Production of Herbicide Resistance

Abstract

Herbicide resistance in plants may occur as a result of point mutations in the gene encoding the herbicide target. Such mutations have been well characterized for resistance to herbicides which affect photosynthesis (1) and amino-acid metabolism (2,3). A better understanding of how these mutant enzymes interact with various herbicides will aid the development of new herbicide/plant systems. The enzyme acetolactate synthase (ALS (or AHAS)) has been the focus of several studies recently because it is the target for three classes of herbicides: sulfonylureas (4,5), imidazolinones (6) and triazolopyrimidines (7). Resistance to these herbicides has been produced, by mutagenesis of seeds or cultured tissure, for a number of plant species including: tobacco, Arabidopsis thaliana, oilseed rape, and sugar beet. Determining the structural basis of these mutations becomes more complex as crop species with multiple copies of ALS are used. For example, Brassica napus (oilseed rape) has 4 to 5 genes for ALS (8) and appears to regulate these sequences differentially. Characterization of new mutations requires isolation and sequencing of all ALS genes in that plant and then demonstration that the specific mutations found can cause a resistant phenotype upon re-introduction into plants. Since it is necessary to re-introduce these in vitro mutated genes into a suitable host to determine their phenotype, we sought to develop a bacterial expression system. This would enable us to take advantage of the convenience and simplicity of prokaryotic genetics and transformation.