Abstract
Key messageTransformation resulted in deletions and translocations at T-DNA inserts, but not in genome-wide small mutations. A tiny T-DNA splinter was detected that probably would remain undetected by conventional techniques.We investigated to which extent Agrobacterium tumefaciens-mediated transformation is mutagenic, on top of inserting T-DNA. To prevent mutations due to in vitro propagation, we applied floral dip transformation of Arabidopsis thaliana. We re-sequenced the genomes of five primary transformants, and compared these to genomic sequences derived from a pool of four wild-type plants. By genome-wide comparisons, we identified ten small mutations in the genomes of the five transgenic plants, not correlated to the positions or number of T-DNA inserts. This mutation frequency is within the range of spontaneous mutations occurring during seed propagation in A. thaliana, as determined earlier. In addition, we detected small as well as large deletions specifically at the T-DNA insert sites. Furthermore, we detected partial T-DNA inserts, one of these a tiny 50-bp fragment originating from a central part of the T-DNA construct used, inserted into the plant genome without flanking other T-DNA. Because of its small size, we named this fragment a T-DNA splinter. As far as we know this is the first report of such a small T-DNA fragment insert in absence of any T-DNA border sequence. Finally, we found evidence for translocations from other chromosomes, flanking T-DNA inserts. In this study, we showed that next-generation sequencing (NGS) is a highly sensitive approach to detect T-DNA inserts in transgenic plants.
Highlights
Authorisation for import or cultivation of genetically modified (GM) plants requires detailed risk evaluations for food, feed and environmental safety
Deviations in the GM plant genomes can be caused by the transformation process itself, or can be a consequence of somaclonal variation, i.e. spontaneous mutations occurred during tissue culture, regeneration and propagation of the GM plant
In our floral dip-mediated transgenic Arabidopsis plants, we detected an average of two small mutations compared to their common parent, disregarding the insert sites
Summary
Authorisation for import or cultivation of genetically modified (GM) plants requires detailed risk evaluations for food, feed and environmental safety At genomic level, this comprises characterization of T-DNA and vector sequence, copy number of inserts, assessment of flanking genomic regions, endogenous host gene interruptions by the T-DNA insert, and evaluation of homology between inserted and junction sequence to genes known to encode toxins or allergens (EFSA 2011). Whole genome re-sequencing of GM plants does provide information about T-DNA inserts and their flanking DNA, but delivers additional genome-wide sequence information This enables comparative genomics between genomes of GM versus the non-GM plants. We used the floral dip method (Clough and Bent 2008) for Arabidopsis thaliana transformation, which circumvents in vitro propagation and regeneration, thereby excluding mutations due to somaclonal variation
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