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.

Soil salinization is a major abiotic constraint on tomato production worldwide. To evaluate adult-stage tomato performance under contrasting saline and non-saline conditions, 23 tomato accessions (13 large-fruited and 10 cherry types) were evaluated at two solar-greenhouse locations in Hebei Province, China: a non-saline site in Shijiazhuang and a saline–alkali site in Huanghua, Cangzhou. Eleven growth, biomass, yield, and quality traits were assessed, and salt tolerance coefficients (STCs) were calculated to characterize relative trait performance between the two sites. Accessions were also genotyped using a PARMS marker linked to SlHAK20. Biomass accumulation was generally lower at the saline site, with above-ground and root dry weights showing the largest relative differences between the two sites, whereas plant height differed comparatively little. Total soluble solids (TSS) were higher at the saline site in most accessions, with the largest relative difference (91.7%) observed in large-fruited accession 305. Correlation analysis revealed strong positive associations between fresh and dry weights of both shoot and root tissues (p < 0.0001). Large-fruited accession 305 and cherry accession 436 showed comparatively favorable biomass retention and relatively small morphological differences between the two sites and therefore represent candidate accessions for further evaluation. The observed SlHAK20-linked marker genotypes were not consistently aligned with phenotypic performance among accessions, indicating that the predictive value of this marker requires further validation. These findings should be interpreted cautiously because salinity was confounded with location, the experiment was conducted during a single growing season, and the marker analysis was not validated in an independent population. This study provides a preliminary comparative evaluation approach and candidate accessions for further investigation and breeding of tomatoes adapted to saline environments.