Micropropagation of Cannabis sativa: Genetic and epigenetic stability assessment over multiple generations

Background: Micropropagation is an increasingly sought-after propagation method in the cannabis industry as growers seek to maximize production efficiency, improve multiplication rates and cultivate plants that are free from biotic stress. However, knowledge about the impact of micropropagation on cannabis is still limited in the scientific literature, as most studies have primarily focused on optimizing environmental parameters and growth conditions. Methods: In vitro cultures of three cannabis cultivars (Critical Purple Kush, Green Crack, and Gelato) were initiated and maintained for 60 weeks with subcultures every three weeks. Leaf samples were collected at the start and after every five subcultures for DNA extraction, and the genomes were sequenced using 3D-GBS and EM-seq to identify genetic (SNPs) and epigenetic (DMPs) variations, followed by GO and KEGG pathway analyses. Results: The results revealed that genomic variants from our CPK (16,169), GC (15,472) and GEL (16,605) samples were mostly located within the intergenic regions. Mutations seem to mostly occur during culture initiation and the first five subcultures, plateauing afterwards up to the 20 ^th subculture. Methylation sequencing revealed that DMPs were less frequent than SNPs, with differential methylation levels showing cultivar-specific patterns varying between CPK, GC and GEL with respectively 22%, 9% and 13% for promoter regions, 13%, 5% and 6% for exon regions, 4% and 3% for intron regions and 61%, 82% and 78% for intergenic regions. These results indicate a strong cultivar dependency of epimutation and suggest their potential phenotypic impact. Gene ontology (GO) and KEGG pathway enrichment analyses of genes harboring SNPs and DMPs revealed functional associations relevant to cannabis micropropagation. Conclusion: Our results highlight the importance of monitoring both genetic and epigenetic stability for long-term cannabis cultivation.