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

Authors

Katarzyna Glowacka, University of Nebraska - LincolnFollow
Johannes Kromdijk, University of Illinois at Urbana-Champaign
Lauriebeth Leonelli, University of California - Berkeley
Krishna K. Niyogi, University of California - Berkeley
Tom E. Clemente, Center for Plant Science InnovationFollow
Stephen P. Long, University of Illinois at Urbana ChampaignFollow

Document Type

Article

Date of this Version

2016

Citation

Plant, Cell and Environment (2016) 39, 908–917

Comments

© 2015 The Authors.

Open access

doi: 10.1111/pce.12693

Abstract

Stable transformation of plants is a powerful tool for hypothesis testing.Arapid 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.