Improvement of Flowering Stage in Japonica Rice Variety Jiahe212 by Using CRISPR/Cas9 System

The flowering period of rice significantly impacts variety adaptability and yield formation. Properly shortening the reproductive period of rice varieties can expand their ecological range without significant yield reduction. Targeted genome editing, like CRISPR/Cas9, is an ideal tool to fine-tune rice growth stages and boost yield synergistically. In this study, we developed a CRISPR/Cas9-mediated multiplex genome-editing vector containing five genes related to three traits, <i>Hd2</i>, <i>Ghd7</i>, and <i>DTH8</i> (flowering-stage genes), along with the recessive rice blast resistance gene <i>Pi21</i> and the aromatic gene <i>BADH2</i>. This vector was introduced into the high-quality <i>japonica</i> rice variety in Zhejiang province, Jiahe212 (JH212), resulting in 34 T₀ plants with various effective mutations. Among the 17 mutant T₁ lines, several displayed diverse flowering dates, but most exhibited undesirable agronomic traits. Notably, three homozygous mutant lines (JH-C15, JH-C18, and JH-C31) showed slightly earlier flowering dates without significant differences in yield-related traits compared to JH212. Through special Hyg and Cas marker selection of T₂ plants, we identified seven, six, and two fragrant glutinous plants devoid of transgenic components. These single plants will serve as sib lines of JH212 and potential resources for breeding applications, including maintenance lines for <i>indica</i>-<i>japonica</i> interspecific three-line hybrid rice. In summary, our research lays the foundation for the creation of short-growth-period CMS (cytoplasmic male sterility, CMS) lines, and also provides materials and a theoretical basis for <i>indica</i>-<i>japonica</i> interspecific hybrid rice breeding with wider adaptability.