Studies on growth and cannabinoid composition of callus derived from different strains of Cannabis sativa.

-Callus derived from different plant parts of various strains of Cannabi8 8ativa L. was established and analyzed for growth on different media and for can­ nabinoid content by thin-layer (tic) and gas-liquid chromatography (glc). Plant organs which included bracts and anther-calyx complexes of drug (152) and fiber (150) types and maturing leaves of Mexican (drug) and Turkish (fiber) strains were shown to accumulate characteristic cannabinoids and, therefore, were selected as callus source material. Callus initiated from these plant organs demonstrated different growth re­ sponses to casein hydrolysate (CH) which were different from its growth response to various amino acid mixtures as media supplements. Numerous roots were formed on newly initiated callus and subcultured tissues of several transfers. Addition of 2,4dichlorophenoxyacetic acid at 0.2 mg/liter inhibited root formation. In long-term cultures, different concentrations of naphthaleneacetic acid and kinetin did not initiate root and/or shoot differentiation. However, growth responses of these callus strains differed from each other when maintained on various combinations of these growth regulators. Under these experimental conditions, no major cannabinoids were de­ tected in callus extracts, although numerous unknown metabolites were present. One minor cannabinoid was detected as indicated from a positive Fast Blue Salt B test. Numerous cannabinoids have been reported to occur naturally in Cannabis sativa L. (1). These products, known as terpenophenols or C21 compounds, represent the most distinctive class of compounds in Cannabis (2). Both neutral and carboxylic acid cannabinoids have been detected; however, the cannabinoid acids are believed to be the natural forms in the plant (3, 4, 5). Several studies have shown that there are different types of chemical strains in Cannabis recognizable by their cannabinoid content (6, 7). Terpenophenol­ containing organs of each specific chemical strain possess a cannabinoid pro­ file characteristic of the strain (8). However, bracts and the leaves which subtend these floral organs contain higher levels of cannabinoids than vegetative leaves (8). Roots have been reported to contain no cannabinoids (2). Even though these differences in cannabinoid content within a single plant are well documented, the factors which influence the accumulation and control of the biogenesis of cannabinoids in the different organs remain to be fully investigated (3, 4). Tissue culture procedures have been employed by several investigators to study secondary product formation in tissues other than the intact plant. How­ ever, the profiles of secondary products isolated from cultured cells usually dif­ fer from the natural products. The latter, if present, usually occur in smaller quantities than those present in the parent plant (10). In some instances, the formation of secondary products in cultured tissues has been correlated with organ differentiation on callus (10, 11, 12). In a recent study on cultured cells of Cannabis, Veliky and Genest (13) reported that no major cannabinoids were detected in cell suspension cultures derived from root explants. The purpose of this study was to determine whether callus derived from dif­ ferent cannabinoid-containing shoot organs, as contrasted with the root (13), were capable of synthesizing cannabinoids. Analyses were performed on long­ term callus derived from four Cannabis strains of different geographical orgins and representing several known phenotypes (14). •This research was supported by a grant from the National Institute on Drug Abuse (DA 00981) to P. G. Mahlberg. The authors thank Drs. Ernest Small and Carlton Turner for seeds employed in this investigation. D. E. A. Registration No. PI0043113 (PGM).