An Agrobacterium tumefaciens EHA105-based GAANTRY recipient strain generates high-quality transgenic Arabidopsis and potato

Authors: Hathwaik, Upul; Hathwaik, Leyla; Tibebu, Redeat; NOTTHINGHAM, NIC - Jr Simplot Company; TERUKO, OOSUMI - Jr Simplot Company; WEEKS, TROY - Jr Simplot Company; Thilmony, Roger; Thomson, James

Submitted to: Microorganisms
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/5/2026
Publication Date: 2/11/2026
Citation: Hathwaik, U.I., Hathwaik, L.T., Tibebu, R.A., Notthingham, N., Teruko, O., Weeks, T., Thilmony, R.L., Thomson, J.G. 2026. An Agrobacterium tumefaciens EHA105-based GAANTRY recipient strain generates high-quality transgenic Arabidopsis and potato. Microorganisms. 14(2). Article 421. https://doi.org/10.3390/microorganisms14020421.
DOI: https://doi.org/10.3390/microorganisms14020421

Interpretive Summary: In this study we generated and tested a new agrobacterium strain for efficient assembly of large, multi-gene constructs for stable plant transformation which is crucial for advancing plant biotechnology. We previously developed a powerful genome modification technology that use precision site-specific recombinase enzymes in wheat, potato and citrus. This technology has been applied to agrobactrium to produce large, multi-gene constructs for plant genome modification. In this work, we show the applicability of this new technology in Arabidopsis and, more importantly, potato. Our work has the potential to create new crop/potato varieties against diseases such as potato blight, which costs $201 million a year to American farmers.

Technical Abstract: The GAANTRY (Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) system enables efficient gene stacking within an Agrobacterium T-DNA. Using unidirectional site-specific recombinases and alternating selection markers, it allows precise, sequential assembly of multiple genes directly within an Agrobacterium virulence plasmid. Here, we modified Agrobacterium tumefaciens strain EHA105, to create JGT105 as a GAANTRY recipient, and constructed a 15.8 kb T-DNA containing five cargo sequences. We compared the performance of the JGT105 5-stack strain against a conventional binary vector carrying the same cargo sequences in Arabidopsis and potato transformation. The transformation efficiencies were comparable for the GAANTRY strain and the binary vector (potato: 83% vs 82%; Arabidopsis: 1.73% vs 1.95%). Single T-DNA insertion frequencies were also similar between the two systems (17.6% for GAANTRY vs 24.5% for the binary vector construct in potato; 10.3% vs 18.2% in Arabidopsis, respectively). Notably, the GAANTRY construct had significantly reduced vector backbone transfer in potato (10.0% vs 26.5%) for the binary vector, while rates of (37.5% vs 48.9%) were seen in Arabidopsis. These results show that the JGT105 GAANTRY strain is an effective T-DNA delivery system, matching binary vector transformation efficiency while offering lower backbone integration frequency, facilitating the generation of high-quality, multi-gene transgenic plants. when tested. These results demonstrate that the enhanced spectinomycin construct provides strong resistance, comparable to kanamycin in multiple species, offering a superior tool for transgenic plant selection.