LCMS Spectral Evidence of the Occurrence of Cannabinoid in Cannabis sativa Cell Cultures
Cannabis sativa L. (marijuana; Cannabaceae) is a plant with worldwide distribution, yielding fiber and food, as well as a psychoactive drug. Cannabinoid and in particular the main psychoactive Δ9-THC are promising substances for the development of new drugs and are of high therapeutic potential. In vitro production of cannabinoid using callus and cell cultures (CC) has not been reported yet . We investigated the occurrence and accumulation of cannabinoid in CC of C. sativa. CC was established from leaf derived callus of C. sativa “bedrobinol” cultivar growing in Bedrocan BV Medicinal Cannabis, The Netherlands. The culture medium was modified Gamborg B5  supplemented with different concentration of plant growth regulators; thidiazuron (TDZ), 6-benzylaminopurine (BA), and gibberellic acid (GA3) at 0.5, 1.0 or 1.5 mg/l. CC was kept on rotary shaker 120 rpm under permanent light at 27 ± 1 °C (Fig. 1). Growth parameters were measured weekly during 35 days of growth cycle. In this study, using LCMS we reported for the first time the accumulation of Δ9-tetrahydrocannabinol (Δ9-THC), tetrahydrocannabinolic acid (THCA), cannabinol (CBN), cannabigerol (CBG), cannabigerolic acid (CBGA), and cannabidiol (CBD) in the CC extracts. On the other hand, accumulation of cannabinoid was reduced after 21-days. The results indicate that our cell lines can be used as a model to investigate metabolic pathways of cannabinoid in C. sativa.
Cannabinoid, Cannabis sativa, Cell cultures, Gamborg medium, plant growth regulators, LCMS.
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