Cannabinoid production in various Cannabis sativa L. in vitro cultures

Abstract

The main goal of this study was to develop an efficient micropropagation protocol for cannabis (Cannabis sativa L.) and to summarize the influence of plant growth regulators on cannabinoid production in various in vitro cultures. Three in vitro culture lines were obtained during cannabis micropropagation and referred to calli, shoots, and plantlets. Shoot multiplication was examined on apical segments isolated from in vitro seedlings and cultivated on solid MS/B5 medium supplemented with various concentrations (0.05-1.0 mg·L− 1) of thidiazuron (TDZ). Callogenesis was induced from leaf explants of seedlings in the presence of 1.0 mg·L− 1 TDZ and 0.5 mg·L− 1 1-naphthaleneacetic acid (NAA), while further biomass production was evaluated upon TDZ (0.5-2.0 mg·L− 1). The shoot regenerative potential was assessed on various concentrations (0.1-1.0 mg·L− 1) of indole-3-butyric acid (IBA). The HPLC-DAD analysis of cannabinoids revealed that cannabidiolic acid (CBDA), cannabidiol (CBD), cannabinol (CBN), ∆9-tetrahydrocannabinol (∆9-THC), and ∆9-tetrahydrocannabinolic acid (∆9-THCA) were the main identified cannabinoids in calli, shoots and regenerated plantlets. The superior production of Δ9-THC was observed in multiple shoots cultured on 0.05 mg·L− 1 TDZ (49.23 mg·g− 1), callus cultures treated with 1.0 mg·L− 1 TDZ (3.92 µg·g− 1), as well in plantlets regenerated on hormone-free medium (80.1 mg·g− 1). The Δ9-THC content in the leaves of 6-month-old regenerated plantlets was significantly higher (1.6-fold) than that of the mother plant. Cannabinoid contents in the inflorescences of the mother plant were higher or even comparable to those observed for acclimatized plantlets. In conclusion, cannabis in vitro cultures could be proposed as promising systems for cannabinoid production.