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Published on January 7, 2009; 10.1104/pp.108.133165

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Received November 25, 2008
Accepted December 22, 2008

Cell culture-induced gradual and frequent epigenetic reprogramming of invertedly repeated tobacco transgene epialleles

Katerina Krizova , Miloslava Fojtova *, Ann Depicker , and Ales Kovarik

Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., 612 65 Brno, Czech Republic; Department of Plant Systems Biology, Flanders Institute for Biotechnology, and Department of Molecular Genetics, Ghent University, B-9052 Ghent, Belgium

* Corresponding author; email: fojtova{at}ibp.cz.

Using a two component transgene system involving two epiallelic variants of the invertedly repeated transgenes in locus 1 (Lo1) and a homologous single copy transgene locus 2 (Lo2) we have studied stability of the methylation patterns and trans-silencing interactions in cell culture and regenerated plants. The posttranscriptionally silenced (PTGS) epiallele of the Lo1 trans-silences and trans-methylates the target Lo2 in a hybrid (Lo1/Lo2 line) while its transcriptionally silenced (TGS) variant (Lo1E) does not. This pattern was stable over several generations in plants. However, in early Lo1E/Lo2 callus decreased transgene expression and partial loss of Lo1E promoter methylation compared to leaf tissue in the parental plant were observed. Analysis of small RNA species and coding region methylation suggested that the transgenes were silenced by a PTGS mechanism. The Lo1/Lo2 line remained silenced, but the non-methylated Lo1 promoter acquired partial methylation in later callus stages. These data indicate that a cell culture process has brought both epialleles to a similar epigenetic ground. Bisulfite sequencing of the 35S promoter residing the Lo1 silencer revealed molecules with no, intermediate and high level of methylation demonstrating, for the first time, cell to cell methylation diversity of callus. Regenerated plants showed high inter- but low intra-individual epigenetic variability indicating that the callus-induced epiallelic variants were transmitted to plants and became fixed. We propose that epigenetic changes associated with dedifferentiation might influence regulatory pathways mediated by trans-PTGS processes.






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