Creation of TGMS Lines of Waxy Rice with Elite Physicochemical Properties of Starch via Waxy Gene Editing

Waxy rice starch (WRS), characterized by low amylose content, high viscosity, and strong gel-forming ability, is highly valued in food and industrial applications. Temperature-sensitive genic male-sterile (TGMS) lines exhibit complete male sterility under low-temperature conditions, a trait widely exploited in hybrid rice breeding. Here, we generated an elite waxy TGMS line, 520S, via CRISPR/Cas9-mediated editing of the Waxy (<i>Wx</i>) gene. The <i>wx</i> mutants displayed robust male sterility, desirable glutinous traits, and favorable physicochemical properties, including gelatinization temperature, gel consistency, paste viscosity, and amylopectin fine structure. Fertility assays confirmed temperature-sensitive pollen sterility consistent with wild-type responses, and T2 generation mutants were transgene-free with stable inheritance of the waxy phenotype. Notably, <i>wx</i> starch maintained gel stability over 48 h, demonstrating superior hydrocolloidal properties and translucency compared with wild-type and commercial WRS. 520S<i>wx1</i> retained gelatinization temperature and amylopectin structure comparable to wild type, highlighting the potential of CRISPR/Cas9-mediated mutagenesis to enhance waxy rice yield while preserving starch quality. These findings establish an efficient strategy to improve both production and functional performance of WRS for industrial and food applications.