Accelerated breeding for early-maturing and aromatic glutinous restorer lines with CRISPR/Cas9-mediated targeted editing for hybrid rice.

Hybrid rice breeding depends on the development of elite parental lines with favorable traits such as grain quality, heading time, and plant architecture. However, improving restorer lines through conventional breeding is time-consuming and labor-intensive. Here, we employed a multiplex CRISPR/Cas9 editing strategy to simultaneously target Wx, Badh2, and Se14 in the elite restorer line FH676, aiming to generate glutinous, aromatic, and early-maturing lines. Through Agrobacterium-mediated transformation, we obtained Wx/Badh2 double mutants (Dm) and Wx/Badh2/Se14 triple mutants (Tm). Grain quality analysis revealed significantly reduced amylose content and enhanced aroma content in the edited lines, consistent with Wx and Badh2 knockouts. The triple mutants also exhibited significantly earlier heading compared to the wild type. The early-maturing Tm lines and the Dm lines achieved grain yields of 39.2 ~ 39.4 g and 42.9 ~ 43.0 g per plant, respectively, both exceeding yields of conventional glutinous cultivars used at present. To evaluate hybrid performance, we crossed Tm and Dm lines with two sterile lines: LX (aromatic) and NX (glutinous). The LX/Tm hybrid headed 4.8 days earlier than its wild-type counterpart, with no reduction in plant height or yield. The NX/Tm hybrid showed an advance of ~ 6.3 days in heading but a 6.2 ~ 7.0% reduction in yield due to decreased grain number per panicle. The Se14 knockout likely relieves repression of RFT1 expression during the floral transition under long-day conditions. Variations in phenotypic response across different maternal backgrounds suggest epistatic interactions affecting the dosage response of Se14. In summary, Se14 is a promising target for engineering early-maturing hybrid rice. The edited FH676 lines provide valuable germplasm resource for developing early-maturing, aromatic, and glutinous hybrids through CRISPR-based genome editing.