Somaclonal variation is a proven source of genetic diversity and is of particular interest in cases where breeding is not a suitable route for crop improvement. Novel sequencing techniques can enhance this approach by enabling the rapid genotypic characterisation of large populations of regenerated plants before phenotypes are expressed. We have been producing novel grapevine somaclones using somatic embryogenesis. However, the stress of tissue culture can alter the epigenetic state of plant cells in a way that may persist in regenerated plants. To study the epigenetic changes induced by this process, we performed bisulphite sequencing on embryogenic callus (EC) and plants regenerated from EC. Compared with leaf material, we found that EC had increased cytosine methylation, particularly in the asymmetrical CHH context, which is indicative of de novo methylation. However, genes and transposable elements (TEs) within genes showed reduced TEspecific CHG methylation. Plants regenerated from EC showed hypermethylation across all cytosine contexts three years after tissue culture. Interestingly, when EC were treated with the demethylating compound 5-Azacytidine, regenerated plants showed higher methylation than those from untreated callus, particularly in CG and CHG contexts. These data suggest that demethylation of TEs during somatic embryogenesis triggers de novo hypermethylation that is stabilised across symmetric cytosine contexts, and which persists years into the future. Based on these results, nanopore-based genotyping efforts are being adapted to include methylation calling, so that the functional impact of epigenetic changes can be determined in somaclone
