Cannabinoid biosynthesis in plants occurs in specialized biosynthetic organs called glandular trichomes on female flowers and leaves. Several studies use metabolic profiling of trichomes to demonstrate variation in trichome size, density, and relative concentration of cannabinoids . However, the genetic mechanisms underlying the developmental changes in trichomes and consecutive cannabinoid content are still unknown. Apart from natural and chemical biosynthesis methods , heterologous biosynthesis of cannabinoids has also been reported . However, the considerable amount of side products is still one of the major bottlenecks in cannabinoid production. This review highlights the latest research developments and challenges in cannabis plant sciences, the role of trichomes as biosynthetic sites, with a special focus on plant biology. In addition, we discuss the existing legal practices with patent information for the C. sativa L. We also discuss the new potential use of cannabinoids for COVID-19 treatment. Finally, we address the available genomic and transcriptomic resources and discuss their potential toward the genetic improvement of cannabis. Overall, we provide the first indepth review of diverse aspects of C. sativa L. from traditional medicinal use to genomics insights and research perspective to broad industrial applications.Based on the research during the past two centuries, we divide the scientific era into four periods . The period zero marked the first-ever mention of Cannabis in the category of medicinal plants in the years 1783 and 1787 . There were only 52 articles and 38 reviews in the next five decades . Most reports mentioned the botanical aspects of hemp,indoor grow tent the quality of fiber, and few observations about its use in traditional medicines.
The first period started with the detailed evidence-based report of chemical properties and medicinal potential of Cannabis indica by William O’Shaughnessy followed by an array of medicinal reports in 1923 articles and 183 reviews in the next 96 years. scientific endeavors to experiment, observe, and understand the diverse medicinal applications of cannabis were still in the early stages. However, 1900s witnessed a series of legal regulation in the direction of the criminalization of cannabis. Cannabis was starting to be categorized into the list of narcotic drugs and Poisons Rules including the Pure Food and Drug Act pushed for stricter measures for cannabis distribution. Later International opium Convention called for measures to regulate Indian hemp. Exports unless exclusively for medical or scientific purposes or European hemp were banned. Uniform State Narcotic Drug Act , Geneva Trafficking Conventions resulted in criminalizing the cultivation, possession, manufacture, and distribution of cannabis derivatives. Marijuana Tax Act levied heavy taxes on the possession and selling of cannabis, excluding medical, and industrial use. As a consequence, the cultivation and procurement of cannabis for research purpose became increasingly difficult and severely limited the research of medicinal cannabis during this era . During the second period , cannabis research suffered major restrictions owing to legal regulations in the first two decades until the identification of the first cannabinoid—cannabidiolic acid in 1954 , isolation of the most psychoactive component of cannabis, the THC in 1964 . Isolation of the THC, discovery of CB1 , and CB2 receptors, followed by the Compassionate Investigational New Drug program paved the way for decriminalization laws. The discovery of endocannabinoid and the role of cannabis in the medicinal field have been reviewed in As a consequence a steep surge was observed in the number of cannabis-related articles from 445 articles and 25 reviews during 1937–1964 to nearly 8,888 articles and 773 reviews during 1964–1996 , although with a short period of decline between 1973 and 1982. Finally, the third period began with the historical Compassionate Use Act of 1996 in California approving medical cannabis.
Postlegalization , cannabis has been extensively explored for its diverse potential in the pharmaceutical and medicinal industries. During the third period, cannabis research witnessed an unprecedented growth with nearly 67,777 articles, 13,202 reviews, and 493 preprints , of which 97.01% articles were published in the last two decades since 2000 and the first draft of the cannabis genome in 2011 in this era were the two major accomplishments that exponentially accelerated the research development. The trends of cannabis study in the diverse array of research articles and journals indicate the core interests of the scientific community. To further investigate the most researched field, the journals of cannabis articles were categorized into scientific and social areas. The journals related to social, law, and policy-based studies were merged into the subject category of social research. Although the majority of broad scientific subjects were grouped into the following seven major categories: medicinal , pharmaceutical-comprised of pharmacology, pharmaceuticals, drug, toxicology, and chemical studies, neurosciences-comprised of neurological, brain-related, psychiatry, psychology, and cognitive studies, biochemistry-included biotechnology, microbiology, immunology, virology, and biochemistry, genomics-grouped genetical and genomic studies, plant biology-included plant sciences, agricultural, botanical aspects, plant-pathogen and environment studies, and lastly, bioinformatics . Journals that could not be classified into either of the aforementioned categories or social research categories were excluded from downstream evaluation. The scientific subject areas were further compared for the corresponding number of articles and journals . A distinct pattern was observed for the Clinical aspects of cannabis which remained a major focus since the very beginning with nearly 94.76% published articles including 64.51% articles in medicinal subject areas, 19.55% in pharmaceutical sciences, and 10.70% in neurosciences.
In contrast, plant biology and agricultural sciences comprised only 2.62% of articles, followed by 0.71% genomics,and 0.07% bioinformatics-based cannabis research. Genomics and bioinformatics are relatively new subjects growing at a fast pace since the release of the first Cannabis draft genome in 2011 together. Recent advances in sequencing technologies have further propelled genomic and transcriptomic studies with the purpose of dissecting the regulatory networks. The growth of genomic data in public space has met with the fast-paced development of bioinformatics tools for data analysis. In addition, ongoing developments of machine-learning and artificial intelligence -based genomic tools will facilitate genetic-level understanding of cannabis metabolism for the selective breeding of genetically modified cannabis with improved metabolic traits.Physiological, morphological, and developmental aspects of Cannabis are key in understanding the plant growth patterns and chemical profiles. However, plant growth and function are substantially influenced by abiotic factors and nutrient availability. Although botanical aspects , plant architecture, and florogenesis of female C. sativa plants with detailed trichome morphogenesis provided crucial insight into plant biology. However, it also became increasingly important to determine the effect of abiotic factors on Cannabis growth and chemical yield, especially for large-scale commercial breeding programs. Hence, in-depth analysis of the effect of soil fertilization, salinity, temperature, and light conditions, as well as nutrient and water use efficiency is key in establishing industrial-scale systems for the cultivation of hemp and marijuana varieties. The first available records about the mineral nutrition of hemp plants were published by Tibeau et al., in 1936 . Later in 1944, Clarence H Nelson published the effect of varying soil temperature on hemp growth .
The first publication with a detailed response of greenhouse cultivated cannabis to nitrogen , phosphorus , and potassium was published in 1977 . Furthermore, two parallel reports by HMG et al., in 1995 discussed the impact of nitrogen fertilization on sex expression in hemp , and the effect of temperature on leaf and canopy formation . Importantly, most physiological studies in the second and third period were published for hemp with a focus on photosynthetic response and biomass yield with varying conditions such as temperature, water availability, nitrogen, and mineral nutrition . However, the first study to assess the chemical response of hemp plants was published in 1997 . Since the physiological response of drug-type medical cannabis plants may differ from hemp plants owing to the distinct genetic and chemical differences. Hence, a clear understanding of optimum factors for medical cannabis is inevitable for the efficient cultivation of plants with desired chemical composition. Among the first few studies that addressed medical cannabis, photosynthetic response to photon flux densities, temperature, and CO2 conditions were published by Chandra et al., in 2008 and 2011 . Bernstein and the group further addressed the growth and chemical response of medical cannabis to mineral nutrition especially N, P, and K . Saloner and Bernstein reported optimum N concentration at 160 mg L 1 , N with lower levels showed several symptoms inducing necrosis and growth retardation while the higher levels impacted in reducing concentrations of THCA and CBDA. Shiponi and Bernstein showed a negative association of cannabinoid concentrations and yield with increasing P supply. Saloner et al. further determined genotype-dependent effect of K nutrition on medical cannabis reporting 240 ppm K detrimental for the genotype Royal Medic and stimulant for Desert Queen genotype while 15 ppm K was insufficient for both genotypes. Further in 2019 Bernstein et al. discussed the combined effect of NPK nutrition upon cannabinoid concentration. In addition to soil nutrients, heavy metal uptake potential of hemp varieties has also been thoroughly investigated by multiple reports in past years . Industrial hemp varieties of C. sativa have also been shown to grow in soils contaminated with heavy metals and reported for heavy metal accumulation. Several field projects have assessed the phytoremediation potential of hemp plants for the reclamation of contaminated and radioactive soils.
Drug-type cultivars with THC/CBD ratio R10 are classified as chemotype I, while those with THC/CBD ratio ranging from 0.2 to 10 are grouped as chemotype II. In contrast, fiber-type cultivars with THC/CBD ratio <0.2 are categorized as chemotype III. Chemotype IV also has low THC contents but with the potent percentage of CBG. Furthermore, hydroponic grow tent the chemotypes producing very little to almost zero cannabinoid compounds are grouped as chemotype V -was first described by Mandolino et al. . Apart from cannabinoid content, drug and fiber-type plants have significant genetic variation. Sawler et al., 2015 described that marijuana is genetically inclined toward ‘‘sativa’’ and hemp have a similarity with the ‘‘indica’’ type . Moreover, each plant type has unique applications differentiating them from each other. For example, the fiber-type “hemp’’ plant has mostly food and industrial applications, including production dietary products, hemp oil, seeds, and fiber, while the ‘‘marijuana’’ drugtype plant is used exclusively for medicinal and recreational purposes. Despite such a huge genetic and application diversity, both types of cannabis plants were categorized as ‘‘Scheduled 1 drug’’ according to the ‘‘Controlled Substances Act’’ in 1970 . These restrictions had a serious impact on the research preventing the scientific community to study the potential of diverse yielding traits for hemp. However, after 44 years in 2014 the ‘‘agricultural act section 7606’’ was implemented which distinguish hemp from marijuana . Approval of law opened the window for scientific community to conduct research and cultivate hemp. Since then, 33 US states and more than 47 countries around the world have been growing hemp for research and industrial use . On the other hand, Marijuana research and legalization have been expanding at a comparatively slower rate and till now only 16 countries have legalized medicinal cannabis . Furthermore, a detailed study would be desirable to understand the gene function, the genetic composition, and the underlying mechanisms regulating the diversity of cannabinoids in both major varieties.
Availability of the regeneration protocol and transformation studies could be utilized for the expression studies to unravel the mystery of these mechanisms, especially in trichomes.Glandular trichomes are the primary site for cannabinoid biosynthesis and accumulation in C. sativa. The biosynthesis of cannabinoids starts from the plastidial localized methylerythritol 4-phosphate pathway resulting in the formation of geranyl pyrophosphate and the fatty acid pathway leading to the production of olivetolic acid. GPP and OA in the presence of olivetolic acid cyclase and an aromatic prenyltransferase catalyze the reaction to form the cannabigerolic acid, which is the central precursor for cannabinoids biosynthesis. van Bakel et al., 2011 analyzed the transcriptomic and genomic data and described the exclusive presence of the THCAS and CBDAS in the drug and hemp type plant, respectively . It is suggested that the activation of respective enzymes from the central precursor CBGA is responsible for regulating the THC and CBD concentration for each chemotype. However, the precise regulatory mechanism is still unknown. Besides biosynthesis, understanding the trichome physiology is also vital to elucidate the trafficking and localization of metabolites.