Agrobacterium-mediated transformation is the dominant method for plant transgenesis, yet it frequently produces multi-copy, structurally complex T-DNA insertions associated with transgene silencing, unpredictable expression, and genome instability. Here, leveraging a high-throughput phenotypic reporter, we systematically dissect how T-DNA vector architecture, plasmid biology, and regulatory element choice shape transformation outcomes in Arabidopsis thaliana. We discover a pronounced trade-off between transformation efficiency and T-DNA copy number, uncovering the virulence enhancing overdrive sequence as a major determinant of this relationship. Guided by these insights, we engineered a new T-DNA vector that balances efficient transformation with predominantly single-copy integration. Additionally, we replaced viral elements, such as the widely used CaMV 35S promoter, with Arabidopsis-derived regulatory elements to minimise undesired enhancer effects, and developed a streamlined workflow for efficient T-DNA insertion mapping in the genome. Together, these advances form the T1 vector series, an Arabidopsis-optimised T-DNA vector system that enables clean, single-copy, and readily mappable transgene integration with predictable expression in the first generation after transformation.
Shaw, W. M., Gajendiran, A., Tchantouridze, E. I., Bechen, L. L., Clarke, S. G., Guiziou, S., Gehring, M., Khalil, A. S.
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