A molecular analysis in leaves of in vitro-cultivated commercial and non-commercial Cannabis sativa genotypes

Cannabis sativa is a multipurpose crop valued for its bioactive compounds, strong and long cellulosic fibres, as well as nutritional benefits. With applications spanning different sectors, from medicine to biocomposites, it presents significant potential for pharmaceuticals, cosmetics, sustainable materials, and functional foods. This study provides a molecular characterisation of leaves from both commercial genotypes of C. sativa and non-commercial ones maintained in the germplasm collection of the Vavilov Institute of Plant Genetic Resources. The leaves were sampled from in vitro -propagated shoots cultivated on phytohormone-free medium. The targeted gene expression analysis revealed higher expression of late stage phenylpropanoid biosynthetic genes (chalcone synthase- CHS7 , flavone synthase- FNS6 , flavonoid 3′-hydroxylase- F3’H3 ) in non-commercial genotypes. In contrast, commercial genotypes showed increased expression of genes involved in the early steps of the pathway (phenylalanine ammonia lyases- PAL4 , PAL7 and 4-coumarate:coenzyme A ligase- 4CL1 ). Untargeted metabolite profiling identified > 100 differentially abundant metabolites, 92 of which belonging to flavonoids, phenolic acids, cannabinoids, lignanamides and lignans. Flavonoids, their derivatives, and cannabinoids were more abundant in non-commercial genotypes, indicating prioritisation of defence compound production. These findings highlight the potential of non-commercial genotypes as natural factories of bioactive molecules, paving the way for future applications. • Micropropagation was optimised for commercial and non-commercial Cannabis genotypes. • Leaf gene expression and metabolomics revealed distinct profiles across genotypes. • Late-step phenylpropanoid pathway genes were upregulated in non-commercial genotypes. • High flavonoid and cannabinoid levels distinguished non-commercial genotypes. • Non-commercial genotypes offer potential as factories for valuable phytochemicals.