Sweet potato (Ipomoea batatas) is a major crop and an important industrial starch source; however, its hexaploid genome has hindered the generation of complete gene knockouts. Because the amylose-to-amylopectin ratio determines starch functionality, the production of amylose-free (waxy) starch is of considerable interest for food, pharmaceutical, and industrial applications. It was hypothesized that the complete knockout of all six alleles of IbGBSS1, which encodes granule-bound starch synthase I, would abolish amylose biosynthesis without compromising plant growth or yield. CRISPR-Cas9 mutagenesis combined with the Hi-Tom high-throughput mutation detection platform was used to generate homozygous Ibgbss1 mutants with confirmed edits across all alleles. These mutants contained < 1% amylose and exhibited normal growth and unchanged yield under both greenhouse and field conditions. Physicochemical analyses showed that amylose-free starch displayed larger granules, an altered amylopectin chain-length distribution (reduced DP 6-12 and enriched > DP 36), and numerous surface pores. Differential scanning calorimetry indicated increased gelatinization onset and peak temperatures, along with higher gelatinization enthalpy. Transcriptome analysis revealed broad reprogramming of starch and sucrose metabolism, accompanied by increased accumulation of glucose, fructose, and sucrose in storage roots. These results demonstrate that IbGBSS1 is essential for amylose biosynthesis and establish a strategy for generating complete multi-allelic knockouts in hexaploid sweet potato. Amylose-free germplasm was obtained without a yield penalty, providing potential for food and industrial applications.
