Epidemics of Fusarium head blight (FHB) cause a serious reduction in wheat grain yield and quality, which results in significant economic losses to wheat producers. Growing resistant varieties is the most sustainable and effective approach to minimize FHB damage. 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 system can precisely knock out S-genes, therefore, is a powerful tool to generate resistant mutants for functional validation of candidate genes and the creation of new resistant sources for breeding. However, conventional genome editing requires transgenic process to deliver gRNA and Cas gene, which raises GMO regulatory concerns, and a new method is urgently needed to produce transgene-free edited cultivars for commercial production. Transient expression of CRISPR/Cas RNP (ribonucleoprotein) complex is the most effective way to avoid transgene integration and off-target editing and nanoparticles (NPs) has proven to be a very promising material for gene delivery in numerous biotechnological applications. Previously, we used NPs to deliver CRISPR/Cas9 DNA constructs into wheat cells through the floral dip method, which generated several inheritable mutations in the targeted regions of a histidine rich calcium binding protein (TaHRC), the causal gene for Fhb1. In this study, we evaluated the possible application of NPs to deliver RNP complexes to the meristematic cells that grown directly from imbibed wheat seeds, therefore, the edited mutants derived from the regrown plants are transgene-free. Our preliminary results showed that the GFP and GUS report genes were successfully delivered into wheat tissues by NPs and successfully expressed in both wheat leaves and shoot. We evaluated the binding capacity and ratio of NPs to RNP complexes and validated the RNP activity to edit the targeted genes by in vitro DNA cleavage assay. We delivered specific RNP complexes targeting Fhb1 susceptible allele into embryos of imbibed dry ‘Bobwhite’ seeds and will screen the regrown seedlings for Fbh1mutants. This new genome editing system will provide a new tool for routine gene editing to create transgene-free, new sources of FHB resistance for wheat breeding programs after further optimization.

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.