Hemp (Cannabis sativa L.) is a plant of growing economic importance, in which micropropagation enables rapid production of uniform plant material. However, prolonged in vitro cultivation can trigger epigenetic changes, among which global DNA methylation is of central importance. The aim of the thesis was to use the HPLC-UV method to determine the proportion of 5-mC in the genomic DNA of four hemp genotypes, and to compare mother plants, shoots after the first and fourth subculture on DKW medium with and without added TDZ, shoots on elongation medium, acclimatized plants, and vegetative clones. Analysis of the 5-mC proportion in the plant genome showed that differences were already present at the level of the mother plants, while a trend of hypomethylation appeared across subcultures in half of all genotypes. The effect of TDZ was not consistent, neither in the direction nor in the magnitude of methylation and showed strong dependence on both plant genotype and developmental stage. The effect of growth in tissue culture remained detectable after acclimatization and manifested as a higher level of 5-mC in most genotypes after transition to adult plants, compared with vegetatively obtained clones. The results confirm that genotype is a key factor governing both the baseline level and the dynamics of global DNA methylation during hemp micropropagation, and that the HPLC method proved sensitive enough to detect these differences.
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