Cannabis Cultivars Research Articles

Gene copy number is associated with phytochemistry in Cannabis sativa.

Gene copy number (CN) variation is known to be important in nearly every species where it has been examined. Alterations in gene CN may provide a fast way of acquiring diversity, allowing rapid adaptation under strong selective pressures, and may also be a key component of standing genetic variation within species. Cannabis sativa plants produce a distinguishing set of secondary metabolites, the cannabinoids, many of which have medicinal utility. Two major cannabinoids—THCA (delta-9-tetrahydrocannabinolic acid) and CBDA (cannabidiolic acid)—are products of a three-step biochemical pathway. Using whole-genome shotgun sequence data for 69 Cannabis cultivars from diverse lineages within the species, we found that genes encoding the synthases in this pathway vary in CN. Transcriptome sequence data show that the cannabinoid paralogs are differentially expressed among lineages within the species. We also found that CN partially explains variation in cannabinoid content levels among Cannabis plants. Our results demonstrate that biosynthetic genes found at multiple points in the pathway could be useful for breeding purposes, and suggest that natural and artificial selection have shaped CN variation. Truncations in specific paralogs are associated with lack of production of particular cannabinoids, showing how phytochemical diversity can evolve through a complex combination of processes.

Extractions of Medical Cannabis Cultivars and the Role of Decarboxylation in Optimal Receptor Responses.

Introduction: Phytocannabinoids, characteristic compounds produced by medical cannabis, interact with cannabinoid (CB) receptors (CB1 and CB2) as well as other receptor systems to exhibit their corresponding pharmacological effects. In their natural form, CBs such as Δ9-tetrahydrocannabinolic acid and cannabidiolic acid are inactive at these receptors, while their decarboxylated forms (Δ9-tetrahydrocannabinol and cannabidiol, respectively) are potent ligands at CB receptors. Thus, extraction and processing of medical cannabis for active constituents are important.Purpose and Methods: Patients consuming medical cannabis often have limited alternative treatment options and in recent years, medical cannabis extracts have been popular as a substitute for dried cannabis plants, despite limited studies on these derivatives. We investigated three disparate cannabis cultivars and compared four chemical extraction methods head to head, viz. Soxhlet, ultrasound-assisted supercritical fluid, and microwave-assisted extractions, for their efficiency. We further characterized the chemical compositions of these extracts.Results: Microwave extraction consistently produced completely decarboxylated phytocannabinoid extracts. Factors such as temperature and exposure time play important roles in the decarboxylation of phytocannabinoids, thereby generating pharmacologically active CBs, and these conditions may differ for each cannabis cultivar.Conclusion: Chemical consistency and potency due to active compounds are in turn important in producing consistent and reliable medical cannabis extracts and their derivatives. These processes must be subject to higher levels of scientific rigor as the patient population around the world are seeking the help of such extracts for various clinical conditions, and as medical cannabis industry is receiving acceptance in various countries.

Terpenoid Chemoprofiles Distinguish Drug-type Cannabis sativa L. Cultivars in Nevada

An unintended consequence of state-mandated cannabis testing regulations has been the resulting database from the analysis of thousands of individual cannabis flower samples from artificially restricted geographical regions. The resulting detailed chemical database can serve as the basis for the development of a chemotaxonomic classification scheme outside of conjectural cultivar naming by strain. Chemotaxonomic classification schemes for cannabis cultivars have previously been reported by others based largely on cannabis strains grown in California under an unregulated testing environment or in Europe from strains grown by a single cultivator. In this study 2,237 individual cannabis flower samples, representing 204 individual strains across 27 cultivators in a tightly regulated Nevada cannabis testing market, were analyzed across 11 cannabinoids and 19 terpenoids. Even though 98.3% of the samples were from Type I cannabis strains by cannabidiolic acid/tetrahydrocannabinolic acid (THCA) ratio of <0.5 CBDA, principal component analysis (PCA) of the combined dataset resulted in three distinct clusters that were distinguishable by terpene profiles alone. Further dissection of individual strains by cultivators within clusters revealed striking fidelity of terpenoid profiles and also revealed a few outliers. We propose that three terpenoid cluster assignments account for the diversity of drug type cannabis strains currently being grown in Nevada.

Identification of Terpenoid Chemotypes Among High (-)-trans-Δ9- Tetrahydrocannabinol-Producing Cannabis sativa L. Cultivars.

Introduction: With laws changing around the world regarding the legal status of Cannabis sativa (cannabis) it is important to develop objective classification systems that help explain the chemical variation found among various cultivars. Currently cannabis cultivars are named using obscure and inconsistent nomenclature. Terpenoids, responsible for the aroma of cannabis, are a useful group of compounds for distinguishing cannabis cultivars with similar cannabinoid content.Methods: In this study we analyzed terpenoid content of cannabis samples obtained from a single medical cannabis dispensary in California over the course of a year. Terpenoids were quantified by gas chromatography with flame ionization detection and peak identification was confirmed with gas chromatography mass spectrometry. Quantitative data from 16 major terpenoids were analyzed using hierarchical clustering analysis (HCA), principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and orthogonal partial least squares discriminant analysis (OPLS-DA).Results: A total of 233 samples representing 30 cultivars were used to develop a classification scheme based on quantitative data, HCA, PCA, and OPLS-DA. Initially cultivars were divided into five major groups, which were subdivided into 13 classes based on differences in terpenoid profile. Different classification models were compared with PLS-DA and found to perform best when many representative samples of a particular class were included.Conclusion: A hierarchy of terpenoid chemotypes was observed in the data set. Some cultivars fit into distinct chemotypes, whereas others seemed to represent a continuum of chemotypes. This study has demonstrated an approach to classifying cannabis cultivars based on terpenoid profile.

Classification of Cannabis Cultivars Marketed in Canada for Medical Purposes by Quantification of Cannabinoids and Terpenes Using HPLC-DAD and GC-MS

For over a century, research on cannabis has been hampered by its legal status as a narcotic. The recent legalization of cannabis for medical purposes in North America requires rigorous standardization of its phytochemical composition in the interest of consumer safety and medicinal efficacy. To utilize medicinal cannabis as a predictable medicine, it is crucial to classify hundreds of cultivars with respect to dozens of therapeutic cannabinoids and terpenes, as opposed to the current industrial or forensic classifications that only consider the primary cannabinoids tetrahydrocannabinol (THC) and cannabidiol (CBD). We have recently developed and validated analytical methods using high-pressure liquid chromatography (HPLC-DAD) to quantify cannabinoids and gas chromatography with mass spectroscopy (GC-MS) to quantify terpenes in cannabis raw material currently marketed in Canada. We classified 32 cannabis samples from two licensed producers into four clusters based on the content of 10 cannabinoids and 14 terpenes. The classification results were confirmed by cluster analysis and principal component analysis in tandem, which were distinct from those using only THC and CBD. Cannabis classification using a full spectrum of compounds will more closely meet the practical needs of cannabis applications in clinical research, insdustrial production, and patients’ self-production in Canada. As such, this holistic classification methodology will contribute to the standardization of commercially-available cannabis cultivars in support of a continuously growing market.

Cannabis Domestication, Breeding History, Present-day Genetic Diversity, and Future Prospects

ABSTRACTHumans and the Cannabis plant share an intimate history spanning millennia. Humans spread Cannabis from its Eurasian homelands throughout much of the world, and, in concert with local climatic and human cultural parameters, created traditional landrace varieties (cultivars resulting from a combination of natural and farmer selection) with few apparent signs of domestication. Cannabis breeders combined populations from widely divergent geographical regions and gene pools to develop economically valuable fiber, seed, and drug cultivars, and several approaches were used with varying results. The widespread use of single plant selections in cultivar breeding, inbreeding, and the adoption of asexual reproduction for commercial drug production, reduced genetic diversity and made many present-day cultivars susceptible to pathogens and pests. The great majority of drug Cannabis cultivars are now completely domesticated, and thus are entirely dependent on humans for their survival. Future ramifications remain to be realized.

A segment of rbcL gene as a potential tool for forensic discrimination of Cannabis sativa seized at Rio de Janeiro, Brazil.

Cannabis sativa, known by the common name marijuana, is the psychoactive drug most widely distributed in the world. Identification of Cannabis cultivars may be useful for association to illegal crops, which may reveal trafficking routes and related criminal groups. This study provides evidence for the performance of a segment of the rbcL gene, through genetic signature, as a tool for identification for C. sativa samples apprehended by the Rio de Janeiro Police, Brazil. The PCR amplified and further sequenced the fragment of approximately 561 bp of 24 samples of C. sativa rbcL gene and showed the same nucleotide sequences, suggesting a possible genetic similarity or identical varieties. Comparing with other Cannabaceae family sequences, we have found 99% of similarity between the Rio de Janeiro sequence and three other C. sativa rbcL genes. These findings suggest that the fragment utilized at this study is efficient in identifying C. sativa samples, therefore, useful in genetic discrimination of samples seized in forensic cases.

Understanding cultivar-specificity and soil determinants of the cannabis microbiome.

Understanding microbial partnerships with the medicinally and economically important crop Cannabis has the potential to affect agricultural practice by improving plant fitness and production yield. Furthermore, Cannabis presents an interesting model to explore plant-microbiome interactions as it produces numerous secondary metabolic compounds. Here we present the first description of the endorhiza-, rhizosphere-, and bulk soil-associated microbiome of five distinct Cannabis cultivars. Bacterial communities of the endorhiza showed significant cultivar-specificity. When controlling cultivar and soil type the microbial community structure was significantly different between plant cultivars, soil types, and between the endorhiza, rhizosphere and soil. The influence of soil type, plant cultivar and sample type differentiation on the microbial community structure provides support for a previously published two-tier selection model, whereby community composition across sample types is determined mainly by soil type, while community structure within endorhiza samples is determined mainly by host cultivar.

A qualitative and quantitative HPTLC densitometry method for the analysis of cannabinoids in Cannabis sativa L.

Cannabis and cannabinoid based medicines are currently under serious investigation for legitimate development as medicinal agents, necessitating new low-cost, high-throughput analytical methods for quality control. The goal of this study was to develop and validate, according to ICH guidelines, a simple rapid HPTLC method for the quantification of Delta(9)-tetrahydrocannabinol (Delta(9)-THC) and qualitative analysis of other main neutral cannabinoids found in cannabis. The method was developed and validated with the use of pure cannabinoid reference standards and two medicinal cannabis cultivars. Accuracy was determined by comparing results obtained from the HTPLC method with those obtained from a validated HPLC method. Delta(9)-THC gives linear calibration curves in the range of 50-500 ng at 206 nm with a linear regression of y = 11.858x + 125.99 and r(2) = 0.9968. Results have shown that the HPTLC method is reproducible and accurate for the quantification of Delta(9)-THC in cannabis. The method is also useful for the qualitative screening of the main neutral cannabinoids found in cannabis cultivars.

Direct NMR analysis of cannabis water extracts and tinctures and semi-quantitative data on Δ 9-THC and Δ 9-THC-acid

Cannabis sativa L. is the source for a whole series of chemically diverse bioactive compounds that are currently under intensive pharmaceutical investigation. In this work, hot and cold water extracts as well as ethanol/water mixtures (tinctures) of cannabis were compared in order to better understand how these extracts differ in their overall composition. NMR analysis and in vitro cell assays of crude extracts and fractions were performed. Manufacturing procedures to produce natural remedies can strongly affect the final composition of the herbal medicines. Temperature and polarity of the solvents used for the extraction resulted to be two factors that affect the total amount of Δ 9-THC in the extracts and its relative quantity with respect to Δ 9-THC-acid and other metabolites. Diffusion-edited 1H NMR (1D DOSY) and 1H NMR with suppression of the ethanol and water signals were used. With this method it was possible, without any evaporation or separation step, to distinguish between tinctures from different cannabis cultivars. This approach is proposed as a direct analysis of plant tinctures.

Metabolomic Differentiation of Cannabis sativa Cultivars Using 1H NMR Spectroscopy and Principal Component Analysis

The metabolomic analysis of 12 Cannabis sativa cultivars was carried out by 1H NMR spectroscopy and multivariate analysis techniques. Principal component analysis (PCA) of the 1H NMR spectra showed a clear discrimination between those samples by principal component 1 (PC1) and principal component 3 (PC3) in cannabinoid fraction. The loading plot of PC value obtained from all 1)H NMR signals shows that Delta9-tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA) are important metabolites to differentiate the cultivars from each other. The discrimination of the cultivars could also be obtained from a water extract containing carbohydrates and amino acids. The level of sucrose, glucose, asparagine, and glutamic acid are found to be major discriminating metabolites of these cultivars. This method allows an efficient differentiation between cannabis cultivars without any prepurification steps.

Chemotaxonomic Features Associated with Flavonoids of Cannabinoid-Free Cannabis (Cannabis sativa subsp. sativa L.) in Relation to hops (Humulus Lupulus L.)

The major flavonoids present in the leaves and flowers of the cannabinoid-free cannabis (Cannabis sativa subsp. sativa L.) cultivars Felina and Futura are orientin (1), vitexin (2), luteolin-7-O-g-D-glucuronide (3), and apigenin-7-O-g-D-glucuronide (4), while prenylated flavonoids, to which the potent estrogenicity of hops (Humilus lupulus L.) is associated, are absent. The different composition of flavonoids has chemotaxonomic value.