Harnessing haploid-inducer mediated genome editing for accelerated maize variety development

The integration of haploid induction and genome editing, termed HI-Edit/IMGE, is a promising tool for generating targeted mutations for crop breeding. However, the technical components and stacking suitable for the maize seed industry have yet to be fully characterised and tested. Here, we developed and assessed three HI-Edit/IMGE maize lines: Edit^Wx, Edit^Sh, and Edit^Wx&Sh, using the haploid inducer CHOI3 and lines engineered using the CRISPR-Cas9 system targeting the Waxy1 (Wx1) and Shrunken2 (Sh2) genes. We meticulously characterised the HI-Edit/IMGE systems, focusing on copy numbers and the mutant alleles mtl and dmp, which facilitate haploid induction. Using B73 and six other parental lines of major commercial varieties as recipients, HI-Edit/IMGE demonstrated maternal haploid induction efficiencies ranging from 8.55% to 20.89% and targeted mutation rates between 0.38% and 1.46%. Comprehensive assessment verified the haploid identification, target gene editing accuracy, genome background integrity, and related agronomic traits. Notably, Edit^Wx&Sh successfully combined distinct CRISPR-Cas9 systems to induce multiple desired mutations, highlighting the potential of HI-Edit/IMGE in accelerating the integration of edited traits into commercial maize varieties. Our findings underscore the importance of meticulous Cas9 copy number characterisation and highlight potential challenges related to somatic chimerism. We also validated the performance of single-cross haploids derived using the HI-Edit/IMGE process. Our results confirm the industrial applicability of generating targeted mutations through pollination and provide critical insights for further optimising this technology.