CRISPR/Cas9-mediated genome editing technology is a powerful tool for targeted gene mutagenesis in various crops. In wheat, most of the genome editing studies so far have directly or indirectly relied on callus induction and plant regeneration from the explants receiving gene construct or preassembled Cas9/guide RNA (gRNA) ribonucleoprotein complex delivered by particle bombardment or Agrobacterium-mediated methods. Unfortunately, the efficiency of callus induction and plant regeneration is generally very low for most wheat genotypes, limiting the application of genome editing in commercially grown wheat varieties. Recently, a new approach named “Haploid Inducer-Mediated Genome Editing” or “HI-Edit” has been developed, which combines haploid induction with genome editing to generate transgene-free doubled haploid plants with the target genes edited. However, few studies have been reported in wheat using this approach. In this study, we aimed to use HI-Edit to mutate two wheat genes, TaHRC and Tsn1, which confer susceptibility to Fusarium head blight (FHB) and three foliar diseases (tan spot, Septoria nodorum blotch and spot blotch), respectively. For each gene, two target sites were designed to make a guide RNA, which was cloned into a CRISPR/Cas9-mediated genome editing plasmid. The gene constructs were then transformed into the hybrid maize variety Hi-II by Agrobacterium-mediated transformation. The resulting T₀ and T₁ maize plants with high expression of Cas9 and gRNA were used to pollinate emasculated florets of wheat variety Dayn. Haploid wheat plants were generated from the tissue-culture rescued embryos derived from the wide crosses. PCR amplification and sequencing indicated that 15-33% of the haploid plants contained mutations at the target sites of the targeted gene. Doubled haploid plants with mutations at the target genes were generated and phenotype data on FHB inoculation or ToxA infiltration in the greenhouse will be presented. This wheat × maize hybridization combined with genome editing approach provides a very useful tool for targeting wheat genes of interest for functional characterization and trait improvement without regulatory issues.

ACKNOWLEDGEMENT AND DISCLAIMER

This material is based upon work partially supported by North Dakota Wheat Commission and the U.S. Department of Agriculture under Agreement No. 59-0206-0-162 (USWBSI). This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture.