Enhancing -mediated plant transformation efficiency through improved ternary vector systems and auxotrophic strains.

-mediated transformation is an essential tool for functional genomics studies and crop improvements. Recently developed ternary vector systems, which consist of a T-DNA vector and a compatible virulence () gene helper plasmid (ternary helper), demonstrated that including an additional gene helper plasmid into disarmed strains significantly improves T-DNA delivery efficiency, enhancing plant transformation. Here, we report the development of a new ternary helper and thymidine auxotrophic strains to boost -mediated plant transformation efficiency. Auxotrophic strains are useful in reducing overgrowth after the co-cultivation period because they can be easily removed from the explants due to their dependence on essential nutrient supplementation. We generated thymidine auxotrophic strains from public strains EHA101, EHA105, EHA105D, and LBA4404. These strains exhibited thymidine-dependent growth in the bacterial medium, and transient expression assay using Arabidopsis seedlings showed that they retain similar T-DNA transfer capability as their original strains. Auxotrophic strains EHA105Thy- and LBA4404T1 were tested for maize B104 immature embryo transformation using our rapid transformation method, and both strains demonstrated comparable transformation frequencies to the control strain LBA4404Thy-. In addition, our new ternary helper pKL2299A, which carries the gene from pTiBo542 in addition to other gene operons (, , , , , and ), demonstrated consistently improved maize B104 immature embryo transformation frequencies compared to the original version of pKL2299 (33.3% vs 25.6%, respectively). Therefore, our improved system, including auxotrophic disarmed strains and a new ternary helper plasmid, can be useful for enhancing plant transformation and genome editing applications.