Rapid generation of a tomato male sterility system and its feasible application in hybrid seed production

Male sterility enables reduced cost and high seed purity during hybrid seed production. However, progress toward its commercial applicationhas been slow in tomato due to the disadvantages of most natural male-sterile mutants. Here, we developed a practical method for efficient tomato hybrid seed production using a male sterile system with visible marker, which was rapidly generated by CRISPR/Cas9-mediated gene editing. Two closely linked genes, TM6 and DFR, which were reported to be candidates of ms15 (male sterile-15) and aw (anthocyanin without) locus respectively, were knocked out simultaneously in two elite tomato inbred lines. Mutagenesis of both genes generated green hypocotyl male-sterile (GHMS) lines, exhibiting male-sterility across different genetic backgrounds and environmental conditions and showing green hypocotyl due to defective anthocyanin accumulation, which serves as a reliable visible marker for selecting male-sterile plants at the seedling stage. We further proposed a strategy for multiplying the GHMS system and verified its high efficiency in stable male sterility propagation. Moreover, elite hybrid seeds were produced using GHMS system for potential side effects evaluation, and no adverse influences were found on seed yield, seed quality as well as important agronomic traits. This study provides a practical approach for the rapid generation and feasible application of male sterility in tomato hybrid breeding.