Mingzheng Ma, Peifeng Liu, Jinjie Zhu, Zhaoxu Gao, Xiantao Qi, Chuanxiao Xie, Changlin Liu

Plant Biotechnology Journal; 2025; IF: 11.2

DOI:10.1111/pbi.70140

Abstract

Maize starch with enhanced freeze-thaw stability is critical for improving the quality of frozen foods. Here, we employed CRISPR/Cas9-mediated targeted mutagenesis to simultaneously disrupt Waxy1 (encoding granule-bound starch synthase) and Sugary2 (encoding soluble starch synthase SSIIa) in maize. Eight transgene-free homozygous mutants—including single-gene (Waxy1 or Sugary2) and double-gene mutants—were generated and characterized. The double mutants exhibited near-complete loss of amylose and altered amylopectin branching, resulting in starch with exceptional water-holding capacity. After six freeze-thaw cycles, double mutants showed <10% water separation (vs. ~60% in wild type), minimal retrogradation, and high viscosity. Notably, these improvements were achieved without significant agronomic penalties in plant height or kernel weight. Our study demonstrates that dual mutation of Waxy1 and Sugary2 significantly enhances maize starch freeze-thaw stability, offering a promising strategy for breeding elite maize lines tailored for frozen food applications.