Male sterility can reduce cost and enable high seed purity during hybrid seed production. However, the commercial application of male sterility in hybrid seed production has not been widely used in tomatoes. CRISPR/Cas9-mediated gene editing can facilitate acceleration for the practical application of male sterility in hybrid seed production. Here, by using the CRISPR-Cas9 system, two genes DYSFUNCTIONAL TAPETUM1 (SlDYT1) and Glutathione S-transferase (SlGSTAA), which underly the two closely linked loci Male sterile 10 (Ms10) and Anthocyanin absent (AA), were knocked out simultaneously in two tomato parental lines. The generated dyt1gstaa double mutants developed green hypocotyl owing to anthocyanin deficiency and exhibited stable male sterility. Up to 92% effectiveness in selecting male sterility was achieved using green hypocotyl as a morphological marker, and thereafter an efficient and stable propagation strategy of male sterility with the aid of the morphological marker selection was developed. Furthermore, dyt1gstaa-derived hybrid seeds were produced and found to have comparable yield, weight, and germination rate with the corresponding WT-derived F1 seeds. The dyt1gstaa system not only increased hybrid seed purity to 100% but also facilitated its rapid and cost-effective determination. Moreover, this system was discovered to have no evident side effects on important agronomic traits. This study suggested that our CRISPR/Cas9-created dyt1gstaa system can be deployed in tomato hybrid seed production.

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