In vitro cell cultures of Cannabis sativa for secondary metabolism manipulation and cannabinoid production

Cannabis sativa is a dicotyledonous dioecious annual herbaceous plant from the family of the Cannabaceae, mainly known for the bioactive properties of the secondary metabolites identified as cannabinoids, abundant in the secretory glandular trichomes (GTs), densely located in the female inflorescence of cannabis. Because of the presence of well-known psychoactive cannabinoid Δ9-tetrahydrocannabinol (THC) and the consequent abuse of cannabis as a drug, the cultivation and commercialization of cannabis products has been and still is severely regulated or prohibited, depending on the country. However, interest in this species has been now growing with the emergence of the pharmaceutical and medical properties of a non-psychoactive cannabinoid known as cannabidiol (CBD). This discovery made way for several recent studies on the cannabinoid biosynthetic pathway, integrating multi-omics methodologies, and on the possibility of increasing productivity via synthetic biology and metabolic engineering, in order to face the many challenges presented by cannabis, such as a lack of knowledge of genetic and molecular mechanisms, low cannabinoid yields and recalcitrant transformation with conventional protocols. Following the recent trend, this research project was developed with the goal of exploring the potential of a novel cannabinoid production method, namely the in vitro cell culture of Cannabis sativa. Direct transformation of cannabis suspension cultures through a protocol based on the biolistic technique, allowed to avoid the ineffective plant in vitro regeneration and achieve stable transformation of undifferentiated cells of cannabis. Based on different studies on the regulation of trichome development and secondary metabolism in other species, such as Solanum lycopersicum (tomato), Prunus persica (peach) and Artemisia annua (sweet wormwood), three transcription factors (TFs) from R3R2-MYB MIXTA and HD-ZIP IV families, involved in the positive regulation of trichome formation and secondary metabolite production were selected for the transformation of in vitro cannabis cell lines, with the aim of inducing cellular differentiation into trichome cells, event that naturally takes place in the plant epidermis, and therefore activating the cannabinoid biosynthetic pathway. The glandular trichomes are hair-like structures that emerge from the epidermis of aerial plant tissues, described as biofactories with the capacity to biosynthesize specialized metabolites like the cannabinoids, critical for the ability of plants to adapt to their environment and to overcome biotic and abiotic stresses. Firstly, the corrected dexamethasone inducible expression system of the transgenes was verified, thanks to a bidirectional promoter and a GUS reporter gene, then the induction was optimized, looking for the best induction treatment in different conditions. Secondly, to understand the capacity of the transgenes in manipulating the secondary metabolism when expressed in undifferentiated cannabis cells, gene expression variation analyses by qRT-PCR were compared between transformed cells and wild-type cells. Studying the cannabinoid biosynthesis pathway, several genes were selected as targets for the TFs, in order to demonstrate the pathway activation. The results highlighted two cell lines that demonstrated some levels of transactivation of endogenous genes related to trichome development and cannabinoid biosynthesis pathways, through the expression respectively of SlCD2, an HD-ZIP IV from tomato, and of PpMYB25, a MIXTA from peach. Complementary, PpMYB25 was also expressed in Nicotiana tabacum plants to study the phenotype and demonstrate the functions of the transgene in a different platform at organism level. Results supported the idea of a regulatory system partly conserved among different species.