1. Imidazolinone Tolerance<br>Imidazolinone herbicides are active against the enzyme acetolactate synthase (ALS), also known as acetohydroxyacid synthase (AHAS) or acetolactate pyruvate-lyase. <br>This enzyme catalyzes the first step in the biosynthesis of the essential branched chain amino acids isoleucine, leucine and valine. Herbicide induced ALS inhibition results in a lethal decrease in protein synthesis. Unmodified canola is not tolerant to imidazolinones, while soybeans, peas, white beans and kidney beans are naturally tolerant to them. <br>The genome of Brassica napus contains an ALS multigene family comprising five genes, two of which are constitutively expressed, share extensive homology with ALS genes from Arabidopsis thaliana and Nicotiana tabacum, and are assumed to encode the primary ALS activities necessary for plant growth and development. <br>Modifications of ALS genes in various plant species can result in herbicide tolerant phenotypes and typically consist of one amino acid substitution, sufficient to alter the binding site for imidazolinones such that the herbicide no longer inactivates the ALS enzyme. <br>Pioneer Hi-Bred International Inc. selected two B. napus lines, P1 and P2, with altered ALS enzymes not inhibited by the herbicide. The modifications in P1 and P2 occur at different and unlinked loci, presumably representing different ALS genes. Lines NS1471 and NS1473 contain both mutations, while line NS738 contains the P2 mutation only. The novel ALS enzyme in line P2 has been isolated and sequenced: it differs from the native gene by one amino acid substitution. <br>The novel imidazolinone resistance is constitutively expressed and synthesis of the branched chain amino acids isoleucine, leucine and valine, is not affected by the ALS modifications in NS1471, NS1473 and NS738.<br>2. Development Method <br>NS738, NS1471 and NS1473 were developed from the in vitro culture of microspores, from the B. napus cultivar Topas, mutagenized with ethyl nitrosourea and selected on imidazolinone containing culture medium. The regenerated haploid plantlets were recovered and treated with colchicine to induce chromosome duplication resulting in doubled-haploid plants. <br>The two imidazolinone tolerant lines P1 and P2 were selected and crossed reciprocally then crossed with B. napus cultivars Topas and Regent, and the progeny subjected to repeated cycles of selfing and further breeding. <br>3. Stable Expression<br>NS738, NS1471 and NS1473 are now several generations removed from the original modified materials, and still consistently show imidazolinone resistance.