Knockout of SSIIa in waxy barley blocks starch biosynthesis and increases soluble sugar and Fructan accumulation

Starch is the main storage carbohydrate in barley, and disrupting starch biosynthesis can alter the type of storage carbohydrate accumulated. Here, the starch synthase gene, SSIIa, which is associated with amylopectin biosynthesis, was knocked out in waxy (GBSSI-knockout) barley through genome editing to generate a double mutant (gbssI ssIIa) of both SSIIa and GBSSI genes. Wild-type Golden Promise (GP), gbssI ssIIa, and waxy plants exhibited similar phenotypes in terms of plant height, tiller number, spike length, and grains per spike. The starch content in gbssI ssIIa grains was only 3.87%, significantly less than in waxy (33.13%) and GP (62.83%). Consequently, the soluble sugar content of gbssI ssIIa was increased to 23.03%, reaching the level typically classified as super sweet. The fructan content in gbssI ssIIa was 5.09%, significantly higher than in waxy (0.95%) and GP (0.47%). The elevated expression of 6-FFT may partially explain the increased fructan content. β-glucan levels were only marginally affected, showing comparable content in gbssI ssIIa and waxy. Additionally, monosaccharide and oligosaccharide levels were increased, as revealed by GC-MS analysis. This study provides a new strategy for generating sweet cereal crops with higher soluble sugar, β-glucan, and fructan content by simultaneously knocking out GBSSI and SSIIa genes.