An evaluation of new and established methods to determine T-DNA copy number and homozygosity in transgenic plants

Abstract

Stable transformation of plants is a powerful tool for hypothesis testing. A rapid and reliable evaluation method of the transgenic allele for copy number and homozygosity is vital in analysing these transformations. Here the suitability of Southern blot analysis, thermal asymmetric interlaced (TAIL-)PCR, quantitative (q)PCR and digital droplet (dd)PCR to estimate T-DNA copy number, locus complexity and homozygosity were compared in transgenic tobacco. Southern blot analysis and ddPCR on three generations of transgenic offspring with contrasting zygosity and copy number were entirely consistent, whereas TAIL-PCR often underestimated copy number. qPCR deviated considerably from the Southern blot results and had lower precision and higher variability than ddPCR. Comparison of segregation analyses and ddPCR of T1 progeny from 26 T0 plants showed that at least 19% of the lines carried multiple T-DNA insertions per locus, which can lead to unstable transgene expression. Segregation analyses failed to detect these multiple copies, presumably because of their close linkage. This shows the importance of routine T-DNA copy number estimation. Based on our results, ddPCR is the most suitable method, because it is as reliable as Southern blot analysis yet much faster. A protocol for this application of ddPCR to large plant genomes is provided.

Keywords: Southern blot; TAIL-PCR; ddPCR; digital droplet PCR; qPCR; segregation analysis; selectable marker; transformation.

Figure 1

(a) Southern blot (b) TAIL‐PCR analyses for T₀ plant VPZ‐23, five segregating T₁ plants, two homozygous T₂ plants and wild type control (WT). The final three lanes show 25 and 50 pg digested VPZ plasmid DNA with 10 μg of digested WT DNA and 50 pg of VPZ plasmid without WT DNA. Arrows in panel b indicate the bands that were absent in WT and show a size shift between reaction 2 and 3 in the TAIL‐PCR and scored in Table 1. TAIL‐PCR was performed with AD3 and T‐DNA specific primers RB3.

Figure 2

The segregation of nonphotochemical quenching (NPQ) in 10‐day‐old seedlings transformed by NbPsbs plants of N. tabacum. (a) Imaged NPQ for PsbS‐43 T₁ (segregating T₁ progeny of T₀ plant carrying one T‐DNA copy) and wild type control (WT); (b) distribution of NPQ in 11 T₁ segregating populations and WT. Presented values of NPQ were recorded after 10 min of induction at 1000 μmol quanta m^‐2 s^‐1. Bar on panel (a) represents 2.5 cm.