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

We transferred the Tri6 gene into the elite barley GemCraft via new transformation method through shoot organogenesis and identified the rearrangements of transgenes and phenotypic variations in the transgenic plants. 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.