The methylation-sensitive amplified polymorphism (MSAP) technique using HpaII and MspI isoschizomers was used to analyse DNA-methylation alterations in stressed grapevine plants. The stress used was in vitro propagation via nodal segments and in vitro thermotherapy for virus elimination. A set of pertinent grapevine plants derived from two cultivars (18 plants each for Müller Thurgau and Riesling) was used as stressed variants for analyses. A total of 695 and 700 MSAP bands were recognised and evaluated as present/absent for all analysed variants derived from both cvs. Müller Thurgau and Riesling. Average computed similarity of MSAP banding between analysed variants (Dice/Nei and Li coefficient) was 0.935 for both cultivars. Clustering of variants within resulting dendrograms showed significant differences between woody cuttings despite originating from the one plant. Further, there was a strong 'donor' effect of maternal plants on future arrays of DNA methylation in their regenerants. The 'donor' effect even seemed to prevail in the effect of stress on final DNA-methylation state in stressed regenerants. Additional MSAP evaluation suggests that thermotherapy induced an additional array of methylation changes when compared with stress caused by in vitro cultivation. From the viewpoint of whether methylation of CCGG loci increased/decreased due to stress, the results showed moderate prevalence for decreasing CCGG loci methylation.

There is relatively little information concerning long-term alterations in DNA methylation following exposure of plants to environmental stress. As little is known about the ratio of non-heritable changes in DNA methylation and mitotically-inherited methylation changes, dynamics and reversibility of the DNA methylation states were investigated in grapevine plants (Vitis vinifera) stressed by in vitro cultivation. It was observed that significant part of induced epigenetic changes could be repeatedly established by exposure to particular planting and stress conditions. However, once stress conditions were discontinued, many methylation changes gradually reverted and plants returned to epigenetic states similar to those of maternal plants. In fact, in the period of one to three years after in vitro cultivation it was difficult to distinguish the epigenetic states of somaclones and maternal plants. Forty percent of the observed epigenetic changes disappeared within a year subsequent to termination of stress conditions ending and these probably reflect changes caused by transient and reversible stress-responsive acclimation mechanisms. However, sixty percent of DNA methylation diversity remained after 1 year and probably represents mitotically-inherited epimutations. Sequencing of regions remaining variable between maternal and regenerant plants revealed that 29.3% of sequences corresponded to non-coding regions of grapevine genome. Eight sequences (19.5%) corresponded to previously identified genes and the remaining ones (51.2%) were annotated as “hypothetical proteins” based on their similarity to genes described in other species, including genes likely to undergo methylation changes following exposure to stress (V. vinifera gypsy-type retrotransposon Gret1, auxin-responsive transcription factor 6-like, SAM-dependent carboxyl methyltransferase).

ABSTRACT:In this paper, results of the in vitro treatment are compared with those of in vivo therapy (i.e. treatment of plants in a peat substrate) when eliminating GFLV (Grapevine fanleaf virus) from three grapevine rootstocks. Therapy took 45 days under the temperature of 37°C in both cases. As far as the health condition of treated plants was concerned, no differences were found between the two methods. The differences were manifested in numbers of plants dying during the therapy and in the course of cultivation of apical segments treated with thermotherapy. Based on these results, it can be recommended to apply the thermotherapy in vivo, which -as compared with the in vitro method -enables us to obtain a greater number of apical segments from treated plants and to establish a smaller number of in vitro cultures in the course of the treatment. A shorter period of in vitro cultivation of plants is another advantage of this method because it helps to reduce the risk of somaclonal variability.

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