Targeted mutation of / combined with the RUBY reporter enables an efficient two-line system for hybrid seed production in .

The recessive genic male sterility (RGMS) method has several benefits in hybrid seed production; however, it is seldom employed in industrial hybrid seed production owing to the difficulty of producing an ample number of pure male-sterile seeds. In this study, we present an efficient methodology for developing a two-line strategy to produce hybrid seed through targeted mutation of and in conjunction with the reporter in . In this method, male-sterile lines were successfully created directly from different elite rapeseed breeding lines through CRISPR/Cas9-mediated mutagenesis and enhanced -mediated transformation. To establish an efficient transgenic maintainer, three seed production technology (SPT) cassettes carrying a functional gene linked to different reporters (, and ) were tested and compared in rapeseed. The results indicated that the PMR-based reporter possesses advantages such as phenotypic stability and ease of identification at early stages, making it an ideal tool for rapid and efficient screening. Subsequently, ideal transgenic maintainer lines with a single hemizygous copy of the SPT cassette were successfully developed in the context of double mutants. The progeny from crossing the maintainer line with its male-sterile counterpart exhibited a 1:1 segregation pattern of nontransgenic male-sterile and male-fertile maintainer plants, distinguishable by seedling color. This biotechnological approach to male sterility offers promising prospects for improving the propagation of recessive genic male-sterile plants and the development of hybrid seeds in rapeseed. Furthermore, it is simple to adapt this technique to more crops.