Sandwich Ct real-time PCR identifies single-copy T-DNA integration accumulating in backbone-free transgenic T1 Arabidopsis

A sandwich Ct real-time PCR (SC-PCR) was used to detect single-copy T-DNA plants by visualizing Ct patterns of T-DNA and two reference amplicons. Detecting the T-DNA copy number directly by visualizing the Ct pattern eliminates the errors introduced by multistep calculations of relative Ct values. Using SC-PCR, we found that single-copy T-DNA integrations were more frequent in transgenic T-1 Arabidopsis without a vector backbone. On the basis of this phenomenon, we combined the negative screen of the vector backbone and SC-PCR to efficiently identify single-copy T-DNA plants. We found that T-DNA copy number detection was underestimated in trans genic plants containing inverted T-DNA repeats due to hairpin structures formed during PCR, indicating that PCR-based methods for detecting T-DNA copy number should be reevaluated. We solved this problem by releasing T-DNA from the complex structures using restriction enzymes before performing SC-PCR. We also demonstrated that latent Agrobacterium contamination in the T-1 transgenic Arabidopsis generated by the floral dip method was exceedingly low and may not affect the detection of T-DNA copy number. Overall, our method provides a whole-set procedure for detecting single-copy T-DNA plants more efficiently than other screening methods including Southern blotting.

Automated summary

The study utilized SC-PCR to detect single-copy T-DNA plants and found that single-copy T-DNA integrations were more frequent in transgenic T-1 Arabidopsis without a vector backbone. By combining negative screening of the vector backbone and SC-PCR, they efficiently identified single-copy T-DNA plants. Additionally, the study highlighted the need to reevaluate PCR-based methods for detecting T-DNA copy number in plants containing inverted T-DNA repeats.