Genome Editing Enables Next-Generation Hybrid Seed Production Technology

The next-generation hybrid seed technology enables the successful production of sortable hybrid seeds from genic male sterile (GMS) lines and maintainers; however, it requires multiple laborious and complicated steps. Here, we designed a simple next-generation hybrid seed production strategy that takes advantage of the CRISPR/Cas9 technology to create a Manipulated GMS Maintainer (MGM) system via a single transformation. Under this schema, the maize male fertility gene ZmMS26 was nullified by removal of its fifth exon using the CRISPR/Cas9 system on a vector, and a second vector carrying a functional ZmMS26 cDNA was co-transformed to restore fertility. The second vector also contains a male gametophyte inactivation gene (ZmAA1) encoding maize alpha-amylase driven by the pollen-specific promoter PG47 and an endosperm fluorescent marker (DsRED) driven by the barley endosperm aleurone-specific promoter Ltp2. The derived single-copy hemizygous MGM lines bore a mutated MS26 gene, leading to complete male sterility but normal vegetative growth and grain yield. The MGM system could prevent genetic transmission of the MGM elements via male gametophytes, providing an efficient method for sorting maintainer seeds labeled by DsRED. This strategy can be extended to any GMS gene and to hybrid crops other than maize.