Fusarium head blight (FHB) is one of the most destructive diseases of wheat worldwide. Developing and growing FHB resistant wheat cultivars is one of the most practical and effective approaches to minimize FHB damage. Recently, manipulating susceptibility genes (S-genes) in crops has become one of the promising breeding strategies to create new sources of resistance. CRISPR/Cas genome editing technology provides a powerful tool to generate resistant mutants by knocking out S-genes precisely for functional validation of candidate genes and the creation of new resistant sources for breeding. However, this editing technology relies on gene transformation to deliver the CRISPR/Cas and gRNA into wheat plant cells and only a few wheat genotypes can be used for transformation because most wheat cultivars have very low rates of callus induction and tissue regeneration in the transformation process. Therefore, a novel efficient gene delivery system that bypasses tissue culture and regeneration is urgently needed for its application in wheat genomic research and breeding. Nanoparticle (NP)-mediated gene delivery system has been evaluated for potential application in crop improvement owing to its ability to reduce immune responses and cytotoxicity without host range restriction. In this study, we evaluated the feasibility of NP to deliver Cas9 and gRNA into wheat tissues using the floral dip method. We optimized the NP binding capacity, identified the best ratio of NPs with Cas9/gRNA editing reagents, and then directly dripped NPs-binding editing reagents into the immature spikes at preanthesis (Feekes 10.0). Sanger sequencing of the target gene from the progenies of the treated plants identified sequence mutations from the Fhb1 susceptibility allele of Bobwhite and the resistance allele of Wangshuibai and Sumai3. These mutants will be phenotyped for FHB resistance to validate their functions on FHB resistance. This work demonstrated that NP directly delivered the editing reagents to target cells and generated inheritable mutations in the target gene regions, therefore the new NP-mediated genome-editing technology has high potential to be used to develop new FHB resistant wheat varieties.

ACKNOWLEDGEMENT AND DISCLAIMER 

This material is based upon work supported by the U.S. Department of Agriculture. 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 author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture.