Shoot tips of Solanum tuberosum 'Désirée' were successfully cryopreserved by the DMSO droplet method and stored for almost 7 years, while control material was maintained in vitro for the same period of time. To analyse potential epigenetic changes, the DNA methylation status was assayed by methylation-sensitive amplified polymorphism (MSAP) analysis using restriction endonucleases MspI and HpaII. An amount of 93.6% of the analysed MSAP signals were stable among all cryopreserved and in vitro maintained samples tested, indicating extensive stability of DNA methylation. Only 0.9% of MSAP signals showed results that differed between the two treatments and at the same time matched for all three biological replications within each treatment. These can be seen as indicating directed effects of the two treatments on the DNA methylation. Cryopreserved samples displayed in comparison to in vitro stored samples consistent hypomethylation for 0.6% (3 of 469) of MSAP signals (Table 4, pattern 4) and consistent hypermethylation for 0.2% (1 of 469), respectively. For 5.6% of all MSAP signals, inconsistent results were observed among the three biological replications at least for one of the two treatments. These were interpreted as resulting from stochastic DNA methylation changes in individual samples. As results for two biological replications were identical and different from the result for the third biological replication, the direction of methylation change could be determined in those cases. Cases of stochastic loss of CG methylation in cryopreserved samples were most frequent among them, adding up to 3.4% of MSAP signals. Stochastic loss of CG methylation was also found in material maintained in vitro, only for 0.6% of all MSAP signals. In conclusion, methylation changes occurred in long-term cryopreservation of potato, in a random rather than directed fashion. Hence, cryopreservation and long-term in vitro maintenance both induce limited changes of DNA methylation status. The order of magnitude of methylation changes observed was consistent with other studies, where similar rates of DNA methylation changes have been found.
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DMSO DROPLET METHOD;
Document Type: Research Article
Publication date: September 1, 2010
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CryoLetters is a bimonthly international journal for low temperature sciences, including cryobiology, cryopreservation or vitrification of cells and tissues, chemical and physical aspects of freezing and drying, and studies involving ecology of cold environments, and cold adaptation
The journal publishes original research reports, authoritative reviews, technical developments and commissioned book reviews of studies of the effects produced by low temperatures on a wide variety of scientific and technical processes, or those involving low temperature techniques in the investigation of physical, chemical, biological and ecological problems.