IMPLANT: a new technique for transgene copy number estimation in plants using a single end-point PCR reaction

Abstract

Background Copy number determination is one of the first steps in the characterization of transgenic plant lines. The classical approach to this, Southern blotting, is time-consuming, expensive and requires massive amounts of high-quality genomic DNA. Other PCR-based techniques are either inaccurate, laborious, or expensive. Results Here, we propose a new technique, IMPLANT (Insertion of competitive PCR calibrator for copy number estimation), a competitive PCR-based technique in which the competitor (based on an endogenous gene) is also incorporated in the T-DNA, which then gets integrated in the genome together with the gene of interest. As the number of integrated competitor molecules directly corresponds to the number of transgene copies, the transgene copy number can be determined by a single PCR reaction. We demonstrate that the results of this technique closely correspond with those obtained by segregation analysis in Arabidopsis and digital PCR In rice, indicating that it is a powerful alternative for other techniques for copy number determination. Conclusions We show that this technique is not only reliable, but is also faster, easier, and cheaper as compared with other techniques. Accurate results are obtained in both Arabidopsis and rice, but this technique can be easily extended to other organisms and as such can be widely adopted in the field of biotechnology.

Genomic consequences associated with Agrobacterium‐mediated transformation of plants

“…While this does occur, it is not as common as might be assumed. Studies across different species have found only between 20-50% of transgenic plants having such simple insertions (Cluster et al, 1996;De Saeger et al, 2022;Flachowsky et al, 2008;Forsbach et al, 2003;Jones et al, 1987;Olhoft et al, 2004). Instead, it has been known for decades that complex insertions frequently arise involving the inclusion of multiple T-DNA copies at a single locus (i.e., T-DNA concatenation), the insertion of T-DNA sequence fragments, or both (Afolabi et al, 2004;De Buck et al, 1999;De Neve et al, 1997;Flachowsky et al, 2008;Gheysen et al, 1990;Holsters et al, 1983;Jones et al, 1987;Jorgensen et al, 1987;Olhoft et al, 2004;Simpson et al, 1986).…”

“…There are also often multiple T‐DNA integration events per transformation event. Notably, the segregation ratios of Arabidopsis plants transformed by floral dip seem to suggest that around 20% of T1 plants have multiple independent T‐DNA insertions (De Saeger et al., 2022; Ondrej et al., 1999). In other species, these estimates are considerably higher but are often limited by small sample sizes.…”