ArticlePlant, cell & environment2016
An evaluation of new and established methods to determine T-DNA copy number and homozygosity in transgenic plants.
Article in Plant, cell & environment, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 58 papers.
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Who cites it
58 citing papers in PubMed, 106 citations in OpenAlex.
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- An accurate, reliable, and universal qPCR method to identify homozygous single insert T-DNA with the example of transgenic rice.Frontiers in plant science · 2023Article
- Identification of T-DNA structure and insertion site in transgenic crops using targeted capture sequencing.Frontiers in plant science · 2023Article
- IMPLANT: a new technique for transgene copy number estimation in plants using a single end-point PCR reaction.Plant methods · 2022Article
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- Gene activation via Cre/lox-mediated excision in cowpea (Vigna unguiculata).Plant cell reports · 2022Article
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Corrections and comments
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Authors and funding
6 authors at 3 institutions in 2 countries.
Funding
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.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.