The recent discoveries of a variety of medical cannabis with different preparations to treat and or cure a number of serious disorders and its newly discovered industrial applications have given momentum to the quest for exploring, exploiting, and protecting natural resources with commercial potential. The quantity and composition of cannabinoids, in particular, THC and CBD, have been targeted by extensive research by breeders, the scientific community, and legislative authorities for almost half a century. Here, we considered that concentration and the ratio of THC/CBD could be the crucial key points for the initiation of a breeding program towards different end products in the huge cannabis/hemp market. Although cannabis has a long history of cultural use in Iran, there is little detailed information about the Iranian cannabis market, including the chemical characteristics of the locally available cannabis land races, which are stored in the CGRC gene bank. To complete the puzzle, the current study was conducted as the primary report of screening THC and CBD contents of 20 selected native cannabis populations collected from different locations in Iran towards initiating anew breeding program for different industrial and medical purposes. The populations under study differed in morphological features including total height , thin or squat growth, leaf shape, phyllotaxy, number of nodes, number of lateral inflorescences, internode length, compact or non-compact inflorescence, flowering time , seeding time , seed features, etc., and although not reported here,dry rack cannabis this is in line with earlier research reported that chemical phenotypes can be characterized by different morphological features. Danziger and colleagues reported architecture of cannabis plant may considerably affect the cannabinoids profile, which has significant pharmaceutical and economic importance.
Additionally, phenotype markers that can facilitate preliminary identification and selection as a supplement to chemical and genetic analysis developed in 2021. They showed significant morphological differences in terms of leaf color, leaflet shape, large and compact inflorescences, and dense and resinous trichomes, which were identified between 21 cultivars covering three chemical phenotypes . They also reported that modern cannabis cultivars are morphologically distinguished by a morphological feature by users and breeders. Among the studied populations, three dwarf populations including Saq-01, Pir-01, and Naq-01 distributed in the north-west along with 10 other populations were located in the group of Type I . These three unique populations were assumed to be marijuana, as they have similar features to those reported recently with thinner stems, more branches, and a higher density of floral tissues than industrial hemp plants. In addition, the plants of population of Ard-01 were morphologically and chemically distinct.Cannabis is an economically important species and is predicted to become a significant commercial crop with unprecedented market growth potential. Recent publication showed among individual cannabis plant parts including roots, leaves, stem bark and inflorescence, cannabis inflorescence was characterized by the highest concentration of cannabinoids in three chemovars. They stated that the comprehensive profile of bioactive metabolites can rediscover therapeutic potential for each part of cannabis from their traditional use. Chemical screening of natural populations can help identify chemical diversity, which is a primary step for improving breeding programs in this plant. Taken together, the cannabis definitions are different based on scientific and political assignations. The significant difference in cannabinoid content of cannabis is supported by numerous studies showing that the most important classification of cannabis types that vary widely among political jurisdictions is that of the drug type and the fiber type .
THC is the major cannabinoid in marijuana types, while CBD predominates in fiber-type hemps. The cultivation of cannabis varieties containing up to 0.2% ∆-9-THC with no indication of permitted percentage of the other compounds, first of all being non-addictive psychoactive cannabidiol , have been recently allowed by two European regulations and Italian law. These varieties have been used for food, oils, fiber, powder, and bioengineering. Recent studies on chemical composition and quantification of hemp industrial varieties indicated ∆9-THC content was lower compared to other cannabinoids. Additionally, ∆8-THC was detected only in one hemp oil sample at too low a concentration. Additionally, a report in 2019 indicated that, among several cannabinoids, only CBDA was determined to show a different concentration in hemp inflorescences samples. Additionally, according to their study, THCV was not found in the hemp inflorescence samples analyzed, and ∆-9-THC and ∆-8-THC were detected at low concentrations, below the legal limit. Therefore, their results confirmed the classification of the studied samples as fiber . In order to grow consumer interest in hemp oilseed supplements, four main cannabinoids of CBD, CBDA, CBN, and ∆-9-THC in an oil matrix of seven commercial hemp oil supplements have been determined. They reported that the cannabinoid composition is required to be monitored in such supplements, as in some cases, the cannabinoids concentration in analyzed samples differed significantly from those declared by the manufacturers. In addition, cannabis chemotypes have been reported using the biochemical composition, in particular, the THC/CBD ratio in many publications.
Chemical phenotypes can be used to define cannabis varieties with different chemical variants and different morphological features to classify C. sativa into three principal classes differ in their THC/CBD ratios: chemotype I ; chemotype II ; and chemotype III. Additionally, cannabis varieties were classified into three groups: chemotype I , intermediate type , which has an intermediate ratio close to 1.0, and chemotype III, which exhibit a low total THC/total CBD ratio ≤ 1.0. On the other hand, three main classes of cannabis have been suggested based on THC/CBD ratio: THC-type plants with THC/CBD ≥ 10, intermediate-type plants with THC/CBD ≈ 1, and CBD-type plants with THC/CBD ≤ 0.1. Marchei and colleagues stated that, while the THC content in light cannabis has to be within 0.2%, CBD content is highly variable, ranging from 2 to 40%. Additionally, serum THC/CBD concentration ratio was used as a useful biomarker to identify use of light cannabis , illegal THC cannabis , and medical cannabis. Another study on chemotaxonomic discrimination indicated significant chemical differences in three chemotypes, so that CBD dominant varieties had higher amounts of total CBD, while THC dominant varieties had higher total THC, and intermediate varieties were generally equal to or in between those in CBD-dominant and THC-dominant varieties. They finally showed that chemotype markers could be used as chemical fingerprints for quality standardization or variety identification for clinical studies and cannabis product manufacturing. THC and CBD variations among populations of this Iranian collection enabled us to define studied populations as three different groups: Type I , Type II , and Type III with a prevalence of THC, both THC and CBD in an approximately equal proportions, and CBD, respectively . Genetic diversity in Iranian cannabis germplasm has been assessed by merging the data with the marijuana and hemp data prepared by to elucidate the relationship of Iranian cannabis with marijuana and fiber type accessions. Finally, they categorized Iranian cannabis populations into marijuana and hemp clusters and reported that natural populations of cannabis in Iran in general more closely fit the profile of marijuana than hemp. Additionally, in this study, we have used the same sources to fingerprint THCand CBD, and these populations were defined as Type I, Type II, and Type III, thus revealing, on the basis of chemistry, these three distinct types. In this research, most plants of population Ard-01, which were also morphologically distinct with a THC/CBD ratio around one due to equivalent THC and CBD concentrations, were assigned to class Type II,roll bench defined as an intermediate THC/CBD ratio. This supports the findings of a genetic diversity study reported earlier, indicating Ard-01 was failed to group with either the marijuana or hemp clusters. This is an interesting finding and may have immediate significance for commercialization, promising for therapeutic purposes in the production of medications with formula requirement of THC/CBD ratio around the unity such as Sativex®. This is an important medication for the suppression of spasticity and pain associated with multiple sclerosis. It is worth noting that sequencing the whole genome of this morphologically, chemically, and genetically distinct population would be of value, as this should provide further insight into the genetic basis of the three chemotypes described here. Furthermore, a concentration of THC ≤ 0.3% and higher amount of CBD in the plants from the location Sam-01 that was located in class Type III is another significant finding in this study, worthy of further investigation, and may reflect a tight control of cannabinoid type and content for cultivation and could provide pre-breeding germplasm resources for future development of hemp crops, particularly in nations where there is a strict regulatory environment around the production of high THC crops.
Although most of the focus has been on identifying plants with higher concentrations of THC for the recreational drug industry, and those with higher concentrations of CBD for medicinal, fiber, and grain purposes, there is also evidence reporting therapeutic benefits for CBD with anti-convulsive, anti-epileptic, antimicrobial, and anti-parkinsonian properties, which are more important recently due to the lack of psychotropic effects associated with CBD consumption, as well as FDA-approved CBD drugs such as Epidiolex® . Berman and colleagues found that, despite the similarity in CBD contents, not all equally high-CBD cannabis extracts produced the same effects. They stated that, as cannabinoids profiling of diverse medical cannabis plants are different, analyzing the effects of specific cannabis compositions for pharmacological-based research is critical. Therefore, it seems likely in the future that both THC and CBD content may be of wide relevance for further development within the pharmaceutical industry. This is supported by a previous study reported that the combination of the psychoactive cannabis ∆9-THC with other non-psychotropic cannabinoids such as CBD demonstrated a higher activity than THC alone. Additionally, previous findings showed that all three major products—food, fiber, and medicine—were extracted from the same crop of the accessions from Darchula district in the northwest of Nepal. In addition, some Iranian cannabis populations were evaluated using wood and fiber anatomy and stem biometry characteristics. They suggested that both populations of Bsh-01 and Zah-01 are significant candidates in terms of fiber anatomy, fiber length, and stem biometry and can be considered for textile and paper industries, while in this study, the aforesaid populations were defined as Type I and, despite the prevalence of THC, also have eligible fiber anatomy characteristics. Our research showed that populations of Nhv-01, Ban-01, Sir-01, Rmhz-01, Ark-01, and Sam-01 are defined as Type III. However, in the earlier study, some populations are expected to be putative high-potential fiber populations, indicating that both populations Ban-01 and Nhv-01 have strong fiber characteristics such as a higher average of bast and woody cores and, alongside populations of Rmhz-01 and Ark-01, can be considered an option for breeding programs towards producing fiber. The results of this study contribute important pre-breeding information for cannabis breeders to improve breeding programs utilizing this collection. Although the uses of these populations cannot be predicted with certainty, as accessions high in THC or high in CBD are Type I and III, respectively, they are not necessarily “hemp” or “medicinal marijuana” in the classic sense; however, according to previous study using genomic data, Iranian populations were located in two distinct marijuana and hemp clusters. Therefore,populations of this collection assumed to offer a range of measurable health benefits in the pharmaceutical, dietary supplement industries, dual-proposal , and even renewable and sustainable feed stock for the production of bio-fuels. Additionally, fiber anatomy is required to assess for fiber production purposes.In general, correlation analysis and PCA results revealed a positive correlation between temperature variables and the two target chemical metabolite contents, and a negative correlation between latitude as well as elevation and metabolites content . It is clear that higher temperatures promote cannabinoid biosynthesis, as found in this research. We also found a negative correlation between THC and CBD concentrations, but it was not significant. The THC and CBD biosynthesis pathways have been elucidated and show that cannabigerolic acid is a prerequisite for both CBD and THC biosynthesis and then follows two pathways to synthesize carboxylic acids , and these acidic forms of cannabinoids, upon heating or smoking, decarboxylate to their neutral forms. THCA synthase and CBDA synthase enzymes that catalyze the reaction of THCA to THC and CBDA to CBD, respectively, compete with each other for CBGA and expedite neutral cannabinoids creation and their levels.