Related Experiment Video
Updated: Aug 23, 2026
Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector
Published on: March 28, 2018
Two-fold differences are the detection limit for determining transgene copy numbers in plants by real-time PCR
Ben Bubner1, Klaus Gase, Ian T Baldwin
1Department of Molecular Ecology, Max-Planck-Institute for Chemical Ecology, Hans-Knöll-Str, 8, 07745 Jena, Germany. bubner@ice.mpg.de
Background:
After transformation, plants that are homozygous and contain one copy of the transgene are typically selected for further study. If real-time PCR is to be used to determine copy number and zygosity, it must be able to distinguish hemizygous from homozygous and one-copy from two-copy plants. That is, it must be able to detect two-fold differences.
Results:
When transgenic Nicotiana attenuata plants which had been previously determined by Southern analysis to contain one or two copies of the transgene, were analyzed by real-time PCR (2-delta delta Ct method), the method failed to confirm the results from the Southern analysis. In a second data set we analyzed offspring of a hemizygous one-copy plant, which were expected to segregate into three groups of offspring in a 1:2:1 ratio: no transgene, hemizygous, homozygous. Because it was not possible to distinguish homozygous from hemizygous plants with real-time PCR, we could not verify this segregation ratio.
Conclusions:
Detection of two-fold differences by real-time PCR is essential if this procedure is to be used for the characterization of transgenic plants. However, given the high variability between replicates, a detection of two-fold differences is in many cases not possible; in such cases Southern analysis is the more reliable procedure.
