CRISPR/Cas9 Knock-Out of Slphd_ms1 (Solyc04g008420) Gene Results in Complete Male Sterility in Tomato

Abstract

Tomato is one of the world's most commercially important fruit crops and a critical source of vitamins, minerals, and phytochemicals in the human diet. In addition to being an important plant for agriculture, the tomato is a model plant for understanding plant gene functions and regulation and for developing plants with beneficial agricultural properties. Due to their superior agronomic performance, the hybrids of vegetable crops are commercially important. The use of male sterility in hybrid seed production reduces the cost, and the purity of tomato varieties is high, as it does not produce pollen. In this study, we generated CRISPR/Cas9 edited lines into male sterility phenotype by knock-out of SlPHD_MS1 (Solyc04g008420), encoding a PHD-type transcription factor in pollen formation and tapetum development. Nine mutant lines were generated, and knock-outs of SlPHD_MS1 created male-sterile tomato plants. There were two homozygous, one heterozygous, and six biallelic mutant plants. Two types of mutational changes were observed in mutant plants: insertion and deletion. Indel frequency created by gRNA19 varied between 30 and 100, and those done by gRNA25 ranged between 45 and 96. Although one, three, and four base pair deletions were common, 13-bp deletions were also detected in line 7. The mutated lines generated by CRISPR/Cas9 did not produce pollen and induced a parthenocarpic fruit set. The SlPHD_MS1 and the three most co-expressed genes were found to have decreased expression in mutant lines, according to qRT-PCR analyses. Taken together, our results suggest a role for SlPHD_MS1 (Solyc04g008420) in male sterility and may aid research into the regulatory processes of tomato pollen and tapetum growth.