T‐<scp>DNA</scp> orientation, distance between two T‐<scp>DNAs</scp>, and the transformation target cells significantly impact vector backbone integration and efficiency of generating marker‐free transgenic plants in a co‐transformation system

The development of marker-free transgenic plants is essential to address biosafety concerns and facilitate regulatory approval. Co-transformation strategies involving separate T-DNAs for the gene of interest and selectable marker gene offer a clean approach but are often hampered by linked integration and vector backbone incorporation. In this study, we designed and evaluated a series of double T-DNA vectors with varying intervening sequence lengths and orientations to determine their impact on co-transformation efficiency and integration patterns in different plant species. Our results showed that shorter spacer regions increased the likelihood of linked T-DNA integration, while an ~3 kb intervening region minimized this risk. Contrary to previous findings, inverse orientation of T-DNAs with respect to each other in the vector significantly increased the frequency of linked and closely spaced integrations compared to tandem arrangements. Co-transformation efficiency and integration outcomes varied across species and transformation methods, with Arabidopsis exhibiting higher rates of linked integration possibly due to germline transformation via floral dip, in contrast to somatic cell transformation in tobacco, lettuce, and tomato. Incorporation of a GFP reporter gene within the intervening region enabled easy identification of unlinked integration events in the T0 generation, reducing downstream screening efforts. Marker-free plants were successfully recovered in the T1 generation, confirming the effectiveness of this approach. These findings emphasize the importance of T-DNA design, orientation, and target cell type in optimizing co-transformation strategies for generating marker-free transgenic plants.