Interplay between Agrobacterium T-DNA and backbone DNA in transgenic plant cells

Agrobacterium-mediated transformation of plants often results in the integration of multiple copies of T-DNA and backbone DNA from binary vectors into the host genome. However, the interplay between T-DNA and backbone DNA remains elusive. In this study, 70.8% of T₁ Arabidopsis transformants exhibited integration of both T-DNA and backbone DNA, and no cases of only backbone integration were observed. To elucidate the integration patterns, we employed bulk-genome resequencing in Arabidopsis and identified 20 integration sites across 10 T₁ transgenic plants, most of which were flanked by left borders of T-DNA at both ends. On average, each integration site contained 6.3 copies of T-DNA and 2.65 copies of backbone DNA. The junction structures between T-DNA and the backbone were highly variable, revealing a previously underappreciated frequency of readthrough at both the left and right borders. Transient expression studies in Nicotiana benthamiana leaves demonstrated that T-DNA and backbone DNA were simultaneously transferred into transformed cells, although the backbone DNA had lower copy numbers than T-DNA. These findings suggest a close relationship between T-DNA and backbone DNA during their transfer and integration, thus offering new insights into the mechanism underlying Agrobacterium-mediated transformation.