One-step creation of CMS lines using a BoCENH3-based haploid induction system in Brassica crop

Heterosis utilization in a large proportion of crops depends on the use of cytoplasmic male sterility (CMS) tools, requiring the development of homozygous fertile lines and CMS lines¹. Although doubled haploid (DH) technology has been developed for several crops to rapidly generate fertile lines^2,3, CMS lines are generally created by multiple rounds of backcrossing, which is time consuming and expensive⁴. Here we describe a method for generating both homozygous fertile and CMS lines through in vivo paternal haploid induction (HI). We generated in-frame deletion and restored frameshift mutants of BoCENH3 in Brassica oleracea using the CRISPR/Cas9 system. The mutants induced paternal haploids by outcrossing. We subsequently generated HI lines with CMS cytoplasm, which enabled the generation of homozygous CMS lines in one step. The BoCENH3-based HI system provides a new DH technology to accelerate breeding in Brassica and other crops.