Optimization of micropropagation protocols and assessment of epigenetic changes in tissue cultures of diverse medical cannabis (Cannabis sativa L.) genotypes

Micropropagation is a crucial technique for the large-scale production of genetically uniform and pathogen-free Cannabis sativa . However, genotype-dependent responses and low multiplication rates often limit its efficiency. This study investigated the effects of basal media (Driver and Kuniyuki Walnut (DKW) and Murashige and Skoog (MS), plant growth regulators (thidiazuron (TDZ), indole butyric acid IBA, and gibberellic acid (GA₃)), and the mitogenic factor phytosulfokine-alpha (PSK-α) on the propagation and rooting efficiency of five medical cannabis genotypes. Simultaneous ex vitro rooting and acclimatization were also investigated as a streamlined approach for adapting in vitro plantlets grown under greenhouse conditions. Shoot multiplication was significantly influenced by the interaction between genotype and culture medium. While the number of shoots did not differ significantly between DKW and MS media, DKW promoted a greater shoot length, more nodes per culture, and a higher biomass. TDZ addition increased the shoot multiplication rate but reduced shoot length and increased callus formation. A decline in shoot length and number of nodes was observed across subcultures, particularly on DKW-based media and less on MS-based media. GA₃ effectively promoted shoot elongation and facilitated subsequent rooting and acclimatization. Suboptimal in vitro rooting led to the development of a novel ex vitro rooting protocol that combined rooting and acclimatization and achieved high success rates within 3 weeks, significantly reducing the overall micropropagation time. PSK-α had no significant effect on shoot multiplication or elongation. Furthermore, epigenetic analysis indicated progressive DNA hypomethylation in micropropagated shoots compared to greenhouse-grown plants, which increased with prolonged in vitro culture, possibly affecting gene expression and development. These results provide valuable insights into the processes of cannabis micropropagation and emphasize the need for further research on in vitro -induced epigenetic changes, including high-performance liquid chromatography based global DNA methylation analysis, bisulfite sequencing of differentially methylated genes and their regulatory regions, and the functional consequences of methylation changes on gene expression and plant morphological and physiological traits. • Micropropagation efficiency in cannabis varies by genotype and culture medium. • TDZ enhances shoot proliferation but reduces shoot length and increases basal callus formation. • Ex vitro rooting significantly improves cannabis rooting and acclimatization success. • In vitro culture indicates progressive DNA hypomethylation in cannabis shoots. • MSAP-Seq is a reliable method for analyzing DNA methylation in cannabis.