The time course of cannabinoid accumulation in the leaves of individual plants of three Cannabis accessions was determined by gaschromatographic analysis in greenhouse-grown plants. The total amounts and the concentration ratios of CBD, THC and CBG were determined; two accessions (an experimental hybrid, (21R £ 15R) £ NL, and plants from a seized seed lot) were found chemotypically uniform, with all plants belonging to chemotpe II (mixed) and I (high THC) respectively. The Carmagnola accession showed chemotypic heterogeneity, with a majority of plants belonging to chemotype III. The CBD/THC and CBG/CBD ratios were shown to be largely constant in the leaves, since 28 and until 103 days after sowing, and consistent with the ratios determined on mature inXorescences. CBD and THC maximum amounts in the leaves showed a peak in the leaves around 80 days from sowing, and were shown to be simultaneous during the growth period, irrespective of the chemotypes. Callus cultures were obtained from all the Wve diVerent chemotypes (I, II, III, IV, V), and GC analyses were performed. Independently of the type and amount of cannabinoids in the mother plants, it was conWrmed that callus cultures of Cannabis were not able to produce detectable amounts of any cannabinoids.
The Yanghai Tombs near Turpan, Xinjiang-Uighur Autonomous Region, China have recently been excavated to reveal the 2700-year-old grave of a Caucasoid shaman whose accoutrements included a large cache of cannabis, superbly preserved by climatic and burial conditions. A multidisciplinary international team demonstrated through botanical examination, phytochemical investigation, and genetic deoxyribonucleic acid analysis by polymerase chain reaction that this material contained tetrahydrocannabinol, the psychoactive component of cannabis, its oxidative degradation product, cannabinol, other metabolites, and its synthetic enzyme, tetrahydrocannabinolic acid synthase, as well as a novel genetic variant with two single nucleotide polymorphisms. The cannabis was presumably employed by this culture as a medicinal or psychoactive agent, or an aid to divination. To our knowledge, these investigations provide the oldest documentation of cannabis as a pharmacologically active agent, and contribute to the medical and archaeological record of this pre-Silk Road culture.
Four crosses were made between inbred Cannabis sativa plants with pure cannabidiol (CBD) and pure Δ-9-tetrahydrocannabinol (THC) chemotypes. All the plants belonging to the F1’s were analyzed by gas chromatography for cannabinoid composition and constantly found to have a mixed CBD-THC chemotype. Ten individual F1 plants were self-fertilized, and 10 inbred F2 offspring were collected and analyzed. In all cases, a segregation of the three chemotypes (pure CBD, mixed CBD-THC, and pure THC) fitting a 1:2:1 proportion was observed. The CBD/THC ratio was found to be significantly progeny specific and transmitted from each F1 to the F2’s derived from it. A model involving one locus, B, with two alleles, BD and BT, is proposed, with the two alleles being codominant. The mixed chemotypes are interpreted as due to the genotype BD/BT at the B locus, while the pure-chemotype plants are due to homozygosity at the B locus (either BD/BD or BT/BT). It is suggested that such codominance is due to the codification by the two alleles for different isoforms of the same synthase, having different specificity for the conversion of the common precursor cannabigerol into CBD or THC, respectively. The F2 segregating groups were used in a bulk segregant analysis of the pooled DNAs for screening RAPD primers; three chemotype-associated markers are described, one of which has been transformed in a sequence-characterized amplified region (SCAR) marker and shows tight linkage to the chemotype and codominance.
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