Mutagenesis Analysis of Acetolactate Synthase (ALS) Genes from Vitis vinifera for Use as a Selectable Marker to Facilitate Precision Breeding of Grapevine

Acetolactate synthase (ALS) is a critical enzyme in the biosynthesis pathway of amino acids leucine, isoleucine and valine. Many mutations in this gene have been found to confer resistance to various inhibiting herbicides. However, the reliability/usefulness of herbicide resistance provided by single site-mutant ALS genes in plants is often limited by the lack of stringent selectivity. In this study, multi-site mutagenesis was studied in order to exploit the potential of ALS gene(s) from grapevine as a selectable marker for in vitro selection of improved elite cultivars via precision breeding. Seven grapevine genes homologous to AtALS of Arabidopsis were identified from the genome of 'Pinot Noir'. Among these genes, VvALSc and VvALSg were utilized as candidates for mutagenesis analysis. A total of eight mutations standardized to AtALS were incrementally introduced into the candidate genes to evaluate their efficacy in inducing herbicide resistance in grapevine and tobacco. These mutations were reported previously to be associated with novel resistance to five herbicide families including imidazolinones (IMI), pyrimidinylthiobenzoates (PTB), sulfonyl-aminocarbonyl-triazolinone (SCT), sulfony lureas (SU), and triazolopyrimidines (TP). Mutant genes were placed in a binary vector under the direction of a double CaMV 35S-derived bi-directional dual promoter complex along with an EGFP-NPTII fusion marker gene. Following Agrobacterium-mediated gene delivery to somatic embryos of grapevine and leaf disks of tobacco, selective culture using either kanamycin or related herbicides yielded stable GFP-expressing transgenics. Progress is underway to evaluate comparatively the selection efficiencies of mutant VvALS genes harboring various mutations. The implication of using a multi-site mutagenesis approach for the improvement of an herbicide-based selectable marker will be discussed.