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bioRxiv · 10.64898/2026.06.08.730999

Rational design of T-DNA vectors enables predictable, single-copy integration in Arabidopsis thaliana

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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Abstract

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.

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Shaw, W. M., Gajendiran, A., Tchantouridze, E. I., Bechen, L. L., Clarke, S. G., Guiziou, S., Gehring, M., Khalil, A. S.. 2026-06-10. Rational design of T-DNA vectors enables predictable, single-copy integration in Arabidopsis thaliana. https://doi.org/10.64898/2026.06.08.730999

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