Amino acid substitutions in grapevine ( Vitis vinifera ) acetolactate synthase conferring herbicide resistance

Acetolactate synthase genes (ALS) have successfully been modified for providing resistance to ALS-inhibiting herbicides in many plant species. Based on sequence and expression analyses, we confirmed VvALS1 as the best functional ALS candidate in grapevine. To develop an ALS-based herbicide selection system for facilitating grape transformation, we firstly evaluated the responses of Vitis vinifera cv Chardonnay callus and young in vitro shoots of Vitis vinifera cv Thompson Seedless to several representative ALS-inhibiting herbicides and found a typical linear response curves to some of the herbicides, including chlorsulfuron and imazapyr belonging to the sulfonylurea or imidazolinone families, respectively. Secondly, we created constructs containing amino acid substitutions in the domains which are known to be critical to herbicide resistance and generated transgenic plants for 3 amino acid substitutions using Agrobacterium-mediated transformation of meristematic bulk tissues of Thompson Seedless. Finally, we showed that ectopic expression of two amino acid substitutions (P191S and P191T) at the N-terminal region and another (W568L) at C-terminal region in VvALS resulted in high resistance to chlorsulfuron or imazapyr herbicides in transgenic in vitro shoots. Our work highlighted the potential use of VvALS mutations imparting herbicide resistance as a selectable marker in grapevine transformation research and as a means in fostering grapevine improvement via cisgenesis, paving the way for developing a selectable co-editing system to facilitate transgene-free gene-editing.