Plant Cell Rep (2006) 25: 1362–1368 DOI 10.1007/s00299-006-0192-8 GENETICS AND GENOMICS Nina Papazova · Pieter Windels · Ann Depicker · Isabel Taverniers · Isabel Roldan-Ruiz · Anne Milcamps · Erik Van Bockstaele · Guy Van Den Eede · Marc De Loose Sequence stability of the T-DNA – plant junctions in tissue culture in Arabidopsis transgenic lines Received: 20 February 2006 / Revised: 30 May 2006 / Accepted: 6 June 2006 / Published online: 30 June 2006 C  Springer-Verlag 2006 Abstract The stability of the inserted transgenes and particularly the junction regions of transgenic events is a concern of food labeling, traceability and post release monitoring, as these regions are used for development of event-specific DNA-based detection methods. During the standard agricultural breeding practices, the transgenic lines can be exposed to completely different conditions than those in the laboratory environment. Some of these conditions have the potential to affect the stability of the transgenic locus and the surrounding DNA. As tissue culture is recognized as a stressful and mutagenic factor, we have analyzed the effect of this process on the stability of the junction regions at nucleotide level in five Arabidopsis thaliana transgenic lines in comparison with the respective integration loci in ColO and C24 ecotypes. No indication of any kind of alteration at nucleotide level Communicated by P. Puigdomenech N. Papazova () · P. Windels · I. Taverniers · M. De Loose Unit Technology and Food, Institute for Agricultural and Fisheries Research, Burg. Van Gansberghelaan 115, 9820 Merelbeke, Belgium e-mail: nina.papazova@ilvo.vlaanderen.be A. Depicker VIB, Ghent University, Technologie Park 927, 9052 Zwijnaarde, Belgium I. Roldan-Ruiz · E. Van Bockstaele Unit Plant, Institute for Agricultural and Fisheries Research, Caritasstraat 21, 9090 Melle, Belgium A. Milcamps · G. Van Den Eede European Commission – Joint Research Center, Institute of Health and Consumer Protection, 21020 Ispra, VA, Italy E. Van Bockstaele Department for Plant Production, Faculty of Agricultural and Applied Biological Sciences, Ghent University, Coupure Links 653, 9000 Gent, Belgium of the junctions was found. The relevance of the stability of the plant–T-DNA junction regions for application of the DNA-based methods in commercial transgenic plants is discussed. Keywords Arabidopsis thaliana . GMO . Plant–T-DNA junctions . Sequencing . SNPs . Tissue culture Introduction The integration of the T-DNA into the plant genome is very often accompanied with rearrangements of the target locus. Deletions, insertions and chromosomal alterations have been reported for the model species Arabidopsis thaliana (Forsbach et al. 2003; Gorbunova and Levy 1997; Kohli et al. 2003; Windels et al. 2003), as well for some crop plants (Arencibia et al. 1998; Nakano et al. 2005). The in- tegration locus of each transgenic event is unique and the junction at the integration locus between the plant-DNA and the T-DNA can be used for unequivocal identifica- tion of the transgenic event. On this basis the DNA-based assays for event-specific detection of GM plants are devel- oped (Holst-Jensen et al. 2003). Moreover, the European legislation on traceability and labeling of GM-derived food and feed requires the provision of event-specific detec- tion methods allowing distinguishing between the different transgenic events (641/2004/EC). Therefore, the long-term stability of the transgenic locus (including the T-DNA and the junction regions) is an important issue in the context of the European legislation requirements for labeling of GM-derived foods, as it is a crucial factor regarding the utilization of event-specific DNA-based methods. It is however well known that during the maintenance, breeding and cultivation the transgenic plants are often exposed to different stress conditions which in some cases can have significant mutagenic effects. One example of such stress conditions is tissue culture involving an undifferentiated callus phase (reviewed in Madlung and Comai 2004) and known to induce somaclonal variation in