AUTHOR=Aliu Ephraim , Ji Qing , Wlazlo Anna , Grosic Sehiza , Azanu Mercy K. , Wang Kan , Lee Keunsub 

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

JOURNAL=Frontiers in Plant Science

VOLUME=15

YEAR=2024

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2024.1429353

DOI=10.3389/fpls.2024.1429353

ISSN=1664-462X

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