Genetic engineering technology has emerged as a powerful tool to elucidate mechanisms of plant disease resistance and develop disease resistant plants. This technology has potential in the battle against Fusarium graminearum (Fg) in barley, especially as no resistant barley cultivars are currently available. Nevertheless, the potential of this technology is constrained by the ability to transform and regenerate gene-edited plants in vitro, with barley proving to be particularly challenging. The established protocols for producing genetically engineered barley are genotype dependent, exhibit low efficiency, and are labor-intensive. To address this issue, we established the Barley Genetic Engineering Facility for FHB Research Community since 2022. This facility, supported by the USWBSI, provides services to develop tissue culture protocols for customer-based barley cultivars and for optimizing transformation efficiency to produce transgenic barley plants with customer-provided constructs. After testing different explants such as mature barley seeds to save the time to grow up plants with immature embryos, or germinated seedlings to provide the meristematic tissues, we demonstrate that immature scutellum explant proves to be the best material for the production of stable transgenic barely plants. We have shown that different barley cultivars require different hormone regimes at the initial stage to produce regenerable embyogenic calli. We have developed the tissue culture protocols for several barley cultivars, including the two-rowed spring barley “Genesis”, the winter cultivar “Thunder”, and the six-rowed barley “Morex”. We have been able to generate barley embryogenic calli in 3 weeks to be transformed either by gene gun bombardment or by Agrobacterium. In 8 weeks, transformed barley calli can be selected and transgenic plantlets can be regenerated. Aided with our improved barley transformation and regeneration protocols, we have been able to transform Morex embryogenic calli with our integrating CRISPR-gene editing vector and produce transgenic Morex plants that are phenotypically normal and producing seeds. Our optimized barley tissue culture and transformation protocols will aid in the production of transgenic and gene-edited barley plants for the FHB Research Community.

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.