Genetics and Plant Physiology – 2019, Volume 9(1–2), pp. 75–98 ©2019 Published by the Institute of Plant Physiology and Genetics – Bulgarian Academy of Sciences Available online at http://www.ifrg-bg.com Review Article BIOLOGY AND ECOLOGY OF GENUS CANNABIS: GENETIC ORIGIN AND BIODIVERSITY. IN VITRO PRODUCTION OF CANNABINOIDS Vassilevska-Ivanova R. Institute of Plant Physiolgy and Genetics, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 21, 1113 Sofia, Bulgaria Received: 25 August 2019 Accepted: 27 October 2019 Contents: Taxonomy and genetic origins of Cannabis landraces Genetic variability of genus Cannabis Breeding of Cannabis Indoor production In vitro cell culture of Cannabis to produce cannabinoids Cannabinoids biosynthesis Control of gender (male flowering control) Flowering and harvesting Life cycle of Cannabis Use of colchicine to produce polyploid plants of Cannabis Cannabis-based medicine and medicaments Keywords: Breeding; Cannabis sativa; cannabinoids; cannabidiol; cell culture; genetic variability; tetrahydrocannabidol. Abbreviations: BLD – broad leaf hemp; CBD – cannabidol; 2,4-D – 2,4-dinitrophenoxy acetic acid; IAA – indole-3-acetic acid; K – kinetin; NAA – 1-naphthalene acetic acid; NLH – narrow leaf hemp; THC – delta-9-te trahydrocannabidol. Citation: Vassilevska-Ivanova R., 2019. Biology and ecology of genus Cannabis: genetic origin and biodiversity. In vitro production of cannabinoids. Genetics and Plant Physiology, 9(1–2): 75–98. TAXONOMY Cannabis sativa L. is the botanical glands, containing 5 to 20% Δ9-THC. name and Latin binomial of hemp. Nowdays debates about cannabis are not Marijuana (marihuana) is colloquial confined to its value as a medicine or name for dried leaves and flowers of to its possible hazards as a recreational cannabis varieties rich in THC (1 to 20% drug. Something much more fundamental Δ9-THC). Hashish is an Arabic name has been engaging the experts for years: for cannabis resin or compressed resin its taxonomy (Schulters et al., 1974; *Corresponding author: [email protected] 76 Vassilevska-Ivanova R. Small and Conquist, 1976; Hilling, 2005; 2013). The first indication of dissent came McPartland, 2018). Are all the plants in 1785 when another eminent biologist, belonging to the genus Cannabis mere Jean-Baptiste Lamarck, was given some varieties of a single species-or is it correct plant specimens collected in India. On the to recognise at least three separate species? basis of several characteristics including In his original 1753 classification, Carl their firm stems, thin bark, and the shape of Linnaeus identified just one, Cannabis their leaves and flowers, Lamarck felt that sativa. The term sativa means “cultivated” they should be distinguished from C. sativa. and describes the common hemp plant Accordingly he invoked a new species, grown widely across Europe in during its C indica. Cannabis indica, meaning the life cycle. C. sativa is native to Europe and Cannabis from India where the first samples western Eurasia where it has been grown of the plant reaching Europe originated. for millennia as a fiber and seed crop, and C. indica is native to eastern Eurasia and was introduced to the New World during was spread by humans around the world European colonization (Clarke and Merlin, primarily as a source of psychoactive Genetics & Plant Physiology 2019 vol. 9(1–2) Biology and ecology of genus Cannabis 77 THC (Small, 2015). C. indica is used for confusion and disagreement reigned, but marijuana and hashish production, but better science would prevail (Gould, 2015) in many regions of eastern Asia it has a Hillig grouped C. indica varieties long history of cultivation for its strong into four subspecies—three based on their fibers and nutritious seeds. Compared to diverse morphological and biochemical the essential oil of C. indica varieties, C. traits (C. indica ssp. indica, C. indica sativa produces less quantity and variety ssp. afganica, C. indica ssp. chinensis), of terpenes, which are of importance in the and another characterized largely by its efficacy of Cannabis medicines (Small, spontaneous growth habit (C. indica ssp. 2007). C. sativa represents a very small Kafiristanica) (Hilling and Mahlberg, portion of the genetic diversity seen in 2004; Hilling 2005). Presently, almost Cannabis worldwide, and it is not divided all modern drug Cannabis varieties are into subspecies based on differing origins hybrids between members of two C. indica and uses like C. indica (Gould, 2015; Clark subspecies: subspecies indica, representing and Merlin, 2016; McPartland, 2018) the traditional and geographically The third and least well founded widespread NLD landrace marijuana species is C ruderalis. This was the name varieties, and subspecies afghanica, that a Russian, Janischevsky, gave to the representing the geographically limited cannabis plants he found growing in the BLD hashish landraces of Afghanistan. It south eastern central region of his country. is through combining landraces from such The differences he noted were mostly in geographically isolated and genetically the size, shape, and casing of the seeds. diverse populations that the great variety And even Janischevsky himself seems of modern-day hybrid recreational and not to have been totally convinced that medical Cannabis varieties blossomed. these justified a new species (McPartland, Cannabis is open-pollinated, 2018). with male and female flowers borne Since the 1960s taxonomists have on separate plants, and therefore to championed several different naming produce a seed usually two plants must systems. Many preferred a three species be involved. Random combinations of concept by recognizingC. ruderalis as alleles and accompanying variation are to a wild species possibly ancestral to both be expected. Cannabis landrace varieties C. sativa and C. indica. Others chose are a work in progress (Hilling, 2005; to reduce C. indica and C. ruderalis to Small, 2015). They are maintained by subspecies or varieties of a single species repeated natural and human selection in C. sativa. In the late 1970s markedly situ—nature selecting for survival and different appearing hashish varieties were humans selecting for beneficial traits— introduced to the West from Afghanistan and without persistent human selection and considered by some to be the true C. and maintenance they drift back to their indica and by others as a fourth species atavistic, naturally selected survival level. C. afghanica, while all the other drug The landraces strains of Cannabis are varieties were held to be members of C. the native, indigenous or heirloom strains sativa following the single species model. found on every continent of the planet. A By the dawn of the new millennium landrace strain is pure, never crossed and Genetics & Plant Physiology 2019 vol. 9(1–2) 78 Vassilevska-Ivanova R. always grown in its natural environment: over millennia from ancient lineages that this isolation and the resulting inbreeding survived in isolation, but may also develop means these varieties are highly stable from semi-feral populations of cultivars that and exstremely vigorous. In most cases, have escaped from cultivation sites. Via the the real landraces are found within poor, latter route, it may take just a hundred years isolated rural communities in remote areas or so for a landrace to develop. Landraces of third world countries (McPartland, are of crucial importance to crop-breeding 2018). Landraces are always dominant programs the world over, and have been plant in their own environment and always for centuries. However, modern agriculture overpower any “intrusion”. A landrace depends far too heavily on uniform crop results from the plant’s natural adaptation varieties, and due to their encroachment to the environment and from the thorough onto vast areas of arable land, biodiversity selection made by the grower. Their gene the world over is threatened. Cultivars are pool is still large because they reproduce very often bred directly from landraces. through pollination out in the open and In fact, this process is the backbone of many males and female take part in commercial cannabis breeding. Cannabis the process, resulting in a high genetic seed breeders have repeatedly drawn from variability. In spite of their heterogeneity, pools of landrace cannabis varieties the Landraces share many morphological world over, and hybridised them to create traits that distinguish them as a group, unique cultivars. The more effort is put into for growers make selections according multiple generations of careful crossing to their objectives. They are regarded as and backcrossing, the more stable and true- the first step in cannabis domestication. breeding the final variety will prove to be These locale-specific varieties of cannabis (Salentijn et al., 2015). “landraces” are the backbone of modern However, when a disease emerges, commercially bred cannabis seeds. But having a diverse gene pool means there is these landraces are under threat from habitat more chance that individuals with genes loss, government eradication programs, and for resistance exist among the population. invasion of foreign varieties. Landrace crop If all individuals are identical, and are varieties are those that have adapted over susceptible to a particular pathogen, all time to local environmental conditions, individuals in the population are equally in isolation from other populations of the at risk. As cannabis is often propagated same species, to a point where they have by clones, this is