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).

A survey was made to evaluate sanitary status of grapevines in the Czech Republic with regard to occurrence of economically important viruses. Propagation material of 109 grapevine clones was tested for presence of Grapevine fanleaf virus, Arabis mosaic virus, Grapevine leafroll-associated virus 1, Grapevine leafroll-associated virus 3, Grapevine virus A, Grapevine virus B and Grapevine fleck virus. Dormant canes were collected and cortical scrappings were analyzed by DAS-ELISA. All seven viruses tested were found to be widely spread in Czech propagation material of grapevine. From 330 individual vines tested, 148 vines were found to be infected with at least one virus. From 109 clones tested, in 98 clones at least one vine negative for tested pathogens was found. Such vines were promoted as candidate plants into screenhouse in Faculty of Horticulture Lednice and will be further tested by other methods. Sanitation of infected grapevine clones is needed in near future.

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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A specific dot-blot hybridization assay was developed using an in vitro-transcribed non-radioactive RNA probe for the routine detection of Grapevine virus A (GVA) for use as an alternative diagnostic tool for large-scale surveys. GVA was detected in RNA isolated from infected vine plants, plant extracts and tissue prints using this probe however no reaction was observed with healthy plants of genus Vitis.