Fusarium head blight (FHB) caused by Fusarium graminearum (Fg) is major disease in barley, causing significant yield losses in the United States and other parts of the world. Most cultivars in the US are susceptible to Fg infection. Transgenic and gene editing technologies allow us to study underlying molecular mechanisms of host FHB resistance, uncover host FHB susceptibility genes, and produce barley cultivars that are resistant to FHB.  All these approaches rely on developing efficient protocols for tissue culture, transformation and regeneration. Cereals are notoriously recalcitrant to in vitro culture, and the protocols established for barley are genotype-dependent. To address this problem and assist the USWBSI community of barley researchers, we have established a barley genetic engineering facility to develop tissue culture and transformation protocols for a wide range of agronomically important barley cultivars. Our improved plant tissue culture protocols permit the regeneration of multiple barley plantlets from a single scutellum explant from the two-rowed cultivar Genesis and the six-rowed cultivar Morex. Transgenic barley plants have been produced using these cultivars using both biolistic and Agrobacterium-mediated transformation methods. Additionally, we have improved our CRISPR-gene editing platform to generate knock-outs (KO) for several host genes involved in conditioning Fg susceptibility. We have adopted the polycistronic tRNA-gRNA (PTG) platform which enhances gRNA production and mutation efficiency in transformed plants by creating double cuts at targets in the barley genome. Our studies with the model plant Arabidopsis have shown that CRISPR-mediated knock-outs of the AtEIN2 (ethylene insensitive 2) gene result in an augmented Fg resistance. Additionally, the HvUGT (uridine diphosphate glycosyltranferase) promoter has been shown to play a vital role in FHB susceptibility. We have constructed transient and integrating gene editing vectors based on the PTG platform to disrupt HvEIN2 and modulate regulation of the promoter of barley HvUGT. Results using barley protoplasts and calli confirm that this modified CRISPR approach reliably produces site-specific mutations within the HvEIN2 coding region and the HvUGT promoter.

ACKNOWLEDGEMENT AND DISCLAIMER

This material is based upon work partially supported by the U.S. Department of Agriculture, under Project ID #FY22-GD-003. 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.