In Vivo Rice Haploid-Induction System by an Egg Cell-Specific Peptidase Knockout

Doubled haploid (DH) technology is a fast and convenient approach for crop breeding and genetic research. Currently, in vitro anther culture is the main method for rice haploid production. However, genotype dependence remains a major problem in the anther culture of most rice subspecies or cultivars for haploid induction. In this chapter, we describe a protocol for in vivo haploid induction in rice using egg cell-specific peptidase (ECS) knockout lines, including mutation of ECS using CRISPR-Cas9 system, selection of homozygous Osecs mutants, and identification of haploids in the offspring of Osecs by flow cytometry. The ECS mediated maternal in vivo haploid-induction system is a convenient, time-saving and labor-saving technique to produce rice DH lines.