An Agrobacterium tumefaciens EHA105-Based GAANTRY Recipient Strain Generates High-Quality Transgenic Arabidopsis and...

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, whereas rates were higher in Arabidopsis (37.5% vs. 48.9%). 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.