Cannabinoids production in Cannabis sativa L.: An in vitro approach

Abstract

Cannabis sativa L. (Cannabaceae) is the oldest known medicinal plant. For millennia, the plant has also been used for fibre and oil production.The most prominent feature of C. sativa is the psychoactive effect ascribed to its secondary metabolites, cannabinoids (mainly to tetrahydrocannabinol, THC). However, many other pharmacological properties of the aforementioned specialized compounds have been described. Currently, the demand for THC for various medical applications is substantial, while cultivation and breeding of Cannabis in most countries is strictly regulated and limited to serving research purposes and meeting therapeutic needs. Therefore, the hereby proposed and discussed production of THC using in vitro cultures could be a viable alternative. In the work presented here, in vitro organogenesis from callus cultures (undifferentiated plant cell masses grown on solid media) was successfully established, ultimately resulting in regeneration of the complete C. sativa plant. Further, production of THC as well as other important cannabinoids was achieved in cell suspension, hairy root and trichome cultures of Cannabis. The optimal combination of phytohormones, as applied to the B5 growth medium, fostering the development of meristemoids from callus cultures was: 1-naphthaleneacetic acid (NAA), 6-benzylaminopurine (BA) and adenine hemisulfate salt (AS) in respective concentrations of 0.5, 5 and 40 mg/l. Concurrently, the most favourable augmentation protocols of the B5 medium for the induction and differentiation of shootlets (small plants with leaves but without roots) were: 0.5 mg/l of gibberelic acid (GA3) or 0.25 mg/l of thidiazouron (TDZ) and 3 mg/l of GA3 (8.5 ± 1.73 and 7.25 ±1.03 shootlets/callus, respectively). The subsequent root formation of shootlets was most prominent after supplementation with 1.5 mg/l of indole-3-acetic acid (IAA). In vitro acclimatized plants growing in Erlenmeyer flasks formed tetrahydrocannabinolic acid (THCA), cannabigerolic acid (CBGA) and cannabidiolic acid (CBDA), retrieved at respective concentrations of about 0.33, 0.45 and 157.1 mg/g fresh weight. In contrast, ex vitro acclimatized plants (grown hydroponically for 8 weeks) synthesized THCA, THC, CBGA, cannabigerol (CBG) and CBDA at corresponding concentrations of 1.54, 28.30, 6.0, 0.125 and 1121.4 mg/g fresh weight. The obtained results confirmed the generation of pharmacologically important cannabinoids; however, the biosynthetic abilities of the investigated cell and hairy root cultures did not provide sufficient levels of the valuable metabolites to warrant scaling-up of the proposed in vitro production platform.