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

Abstract

<i>Agrobacterium</i>-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 (<i>vir</i>) gene helper plasmid (ternary helper), demonstrated that including an additional <i>vir</i> gene helper plasmid into disarmed <i>Agrobacterium</i> strains significantly improves T-DNA delivery efficiency, enhancing plant transformation. Here, we report the development of a new ternary helper and thymidine auxotrophic <i>Agrobacterium</i> strains to boost <i>Agrobacterium</i>-mediated plant transformation efficiency. Auxotrophic <i>Agrobacterium</i> strains are useful in reducing <i>Agrobacterium</i> 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 <i>Agrobacterium</i> strains EHA101, EHA105, EHA105D, and LBA4404. These strains exhibited thymidine-dependent growth in the bacterial medium, and transient <i>GUS</i> 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 <i>virA</i> gene from pTiBo542 in addition to other <i>vir</i> gene operons (<i>virG</i>, <i>virB</i>, <i>virC</i>, <i>virD</i>, <i>virE</i>, and <i>virJ</i>), 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 <i>Agrobacterium</i> system, including auxotrophic disarmed <i>Agrobacterium</i> strains and a new ternary helper plasmid, can be useful for enhancing plant transformation and genome editing applications.