Agrobacterium-mediated transfer of the Fusarium graminearum Tri6 gene into barley using mature seed-derived shoot tips as explants

Authors: Gao, Dongying; ABDULLAH, SIDRAT - Oak Ridge Institute For Science And Education (ORISE); BALDWIN, THOMAS - North Dakota State University; Caspersen, Ann; WILLIAMS, EDWARD - Wisconsin Crop Innovation Center; CARLSON, ALVAR - Wisconsin Crop Innovation Center; PETERSEN, MIKE - Wisconsin Crop Innovation Center; Hu, Gongshe; Esvelt Klos, Kathy; Bregitzer, Paul

Submitted to: Plant Cell Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/8/2023
Publication Date: 1/20/2024
Citation: Gao, D., Abdullah, S., Baldwin, T., Caspersen, A.M., Williams, E., Carlson, A., Petersen, M., Hu, G., Esvelt Klos, K.L., Bregitzer, P.P. 2024. Agrobacterium-mediated transfer of the Fusarium graminearum Tri6 gene into barley using mature seed-derived shoot tips as explants. Plant Cell Reports. (2024) 43:40. https://doi.org/10.1007/s00299-023-03129-z.
DOI: https://doi.org/10.1007/s00299-023-03129-z

Interpretive Summary: Barley is the fourth most important cereal crop in the world. In the United States, barley is mostly used for malted beverages. It is grown in more than 23 states, but three states, Idaho, Montana, and North Dakota, produce nearly 80% of the country's barley. Fusarium head blight (FHB) is a devastating disease of barley and wheat that may cause severe yield loss and significantly reduce the market values of grains. Despite extensive studies that have been conducted on this disease, no high-level FHB resistant resources have been found in cultivated barley and its wild progenitor which makes it a big challenge to use host resistance for barley FHB improvement. To overcome the problems, we explored an alternative strategy for improving barley FHB resistance known as RNAi (RNA interference). We targeted the Tri6 gene, a key gene for the accumulation of a toxin known as deoxynivalenol (DON) in the FHB fungus, Fusarium graminearum. The Tri6 gene was successfully transferred into a malting barley cultivar ‘GemCraft’. Importantly, we developed a new meristem transformation method which only needs about 10 weeks to generate transgenic plants and significant shortens the barley transformation cycle compared to the current transformation system with immature embryos (7-12 months or long). Our efforts also provide Tri6 transgenic materials that can be used for improving barley FHB resistance and other studies.

Technical Abstract: Despite its agronomic and economic importance, barley transformation is still very challenging for many elite varieties. In this study, we used direct shoot organogenesis to transform the elite barley cultivar GemCraft with the RNAi constructs containing Tri6 gene of Fusarium graminearum, which causes fusarium head blight (FHB). We isolated 4432 shoot tips and co-cultured these explants with Agrobacterium tumefaciens. A total of 25 independent T0 transgenic plants were generated including 15 events for which transgene-specific PCR amplicons were observed. To further determine the presence of transgenes, the T1 progenies of all 15 T0 plants were analyzed, and the expected PCR products were obtained in 10 T1 lines. Droplet digital (dd) PCR analysis revealed various copy numbers of transgenes in the transgenic plants. We determined the insertion site of transgenes using long-read sequencing data and observed the rearrangements of transgenes. We found phenotypic variations in both T1 and T2 generation plants. FHB disease was evaluated under growth chamber conditions, but no significant differences in disease severity or deoxynivalenol accumulation were observed between two Tri6 transgenic lines and the wildtype. Our results demonstrate the feasibility of the shoot tip transformation and may open the door for applying this system for genetic improvement and gene function research in other barley genotypes.