Generation of Transgene-Free Maize Male Sterile Lines Using the CRISPR/Cas9 System.

Male sterility (MS) provides a useful breeding tool to harness hybrid vigor for hybrid seed production. It is necessary to generate new male sterile mutant lines for the development of hybrid seed production technology. The CRISPR/Cas9 technology is well suited for targeting genomes to generate male sterile mutants. In this study, we artificially synthesized gene with biased codons of maize. A CRISPR/Cas9 vector targeting the gene of maize was constructed and transformed into maize using an -mediated method, and eight T independent transgenic lines were generated. Sequencing results showed that genes in these T transgenic lines were not mutated. However, we detected mutations in the gene in F and F progenies of the transgenic line H17. A potential off-target site sequence which had a single nucleotide that was different from the target was also mutated in the F progeny of the transgenic line H17. Mutation in the gene and the male sterile phenotype could be stably inherited by the next generation in a Mendelian fashion. Transgene-free male sterile plants were obtained by screening the F generation of male sterile plants, and the MS phenotype could be introduced into other elite inbred lines for hybrid production.