Engineering a Gibberellin‐Switchable Dual‐Use Line Based on Ent‐Kaurene Oxidase Gene <scp> <i>ZmKO1</i> </scp> Enables Two‐Line Hybrid Seed Production in Maize

Male-sterile systems are pivotal for maize hybrid seed production, yet existing systems are constrained by significant limitations. Here, we identified a novel single-gene-controlled recessive genic male sterility (GMS) mutant, defective in filament elongation 1 (def1), which exhibits non-elongated filaments, unopened glumes, and non-exserted anthers, yet undergoes normal microsporogenesis and produces viable pollen. Map-based cloning revealed that def1 is caused by a loss-of-function mutation in the ZmKO1 gene. Previous studies have identified ZmKO1 and ZmKO2 as the two putative KO-encoding genes in maize. Although heterologous expression evidence suggested that only ZmKO1 possesses KO activity, we demonstrate that both ZmKO1 and ZmKO2 are functional KOs for GA biosynthesis. The def1 mutant or ZmKO1 knockout lines exhibited reduced plant height during early seedling development but recovered rapidly to wild-type levels. In contrast, ZmKO2 knockout lines displayed severe dwarfism and pollen-less male sterility. These contrasting phenotypes demonstrate a clear functional divergence between the two genes. Utilising marker-assisted backcrossing and CRISPR/Cas9 editing, we generated ZmKO1-based GMS lines across diverse genetic backgrounds. These lines maintained stable male sterility and could be efficiently propagated via exogenous GA₃ application under different genetic backgrounds and environments, demonstrating robust "dual-use" properties. Data indicated that this dual-use trait has no significant effect on other agronomic traits of the male sterile line and its derived hybrid combinations. Based on this, we established a novel two-line hybrid seed production system in maize, termed the Gibberellin-switchable Dual-use Line (GDL), and successfully applied it in the hybrid seed production of Chuandan 99.