Abstract
Storage of potato tubers at low temperatures leads to the degradation of starch into reducing sugars, resulting in cold-induced sweetening (CIS). These sugars react with free amino acids when processed at high temperatures, resulting in brown, bitter-tasting products. Sucrose is one of the intermediate steps of conversion of starch into reducing sugars. Vacuolar invertase breaks down sucrose to glucose and fructose, thereby largely contributing to the accumulation of reducing sugars. Targeted knockout of the potato vacuolar invertase (Pain-1) gene by Cas9/gRNA-mediated site-directed mutagenesis was performed in the tetraploid potato cultivar Symfonia. A vector with expression units for cas9 and three Pain-1-specific gRNAs was constructed, and its mutagenic activity demonstrated in potato protoplasts. Eleven mutant lines were then obtained by Agrobacterium-mediated transformation, with nine of them carrying mutations in all four gene copies present in tetraploid potato. Tubers from four Pain-1 mutant lines were analyzed for sugar accumulation during cold storage. The two lines with knockout of all four gene copies had significantly reduced glucose and fructose levels and lighter chip color compared to the non-mutated control line. This work demonstrates that CIS resistance can be established in a potato elite cultivar by targeted Pain-1 mutagenesis.
