Generating homozygous mutant populations of barley microspores by ethyl methanesulfonate treatment

Induced mutations are important for genetic research and breeding. Mutations induced by physical or chemical mutagenesis are usually heterozygous during the early generations. However, mutations must be fixed prior to phenotyping or field trials, which requires additional rounds of self-pollination. Microspore culture is an effective method to produce double-haploid (DH) plants that are fixed homozygotes. In this study, we conducted ethyl methanesulfonate (EMS)-induced mutagenesis of microspore cultures of barley (Hordeum vulgare) cultivar 'Hua30' and landrace 'HTX'. The EMS concentrations were negatively correlated with the efficiency of callus induction and the frequency of mutant plant regeneration. The two genotypes showed different regeneration efficiencies. The phenotypic variation of the regenerated M-1 plants and the presence of genome-wide nucleotide mutations, revealed by whole-genome sequencing, highlight the utility of EMS-induced mutagenesis of isolated microspore cultures for developing DH mutants. Genome-wide analysis of the mutation frequency in the regenerated plants revealed that a considerable proportion of mutations resulted from microspore culture (somaclonal variation) rather than EMS-induced mutagenesis. In addition to producing a population of 1972 homozygous mutant lines that are available for future field trials, this study lays the foundation for optimizing the regeneration efficiency of DH plants and the richness of mutations (mainly by fine-tuning the mutagen dosage).