WO2022066946A1 - Methods for production of cannabinoids - Google Patents

Abstract

Provided are in vitro cannabinoid-rich callus of Cannabis sp., developed from a tissue culture system containing one or more plant hormones, and cultured and optionally subcultured from flower explants containing capitate stalked trichomes during a stage prior to an inflorescence maturation threshold of a Cannabis plant. The Cannabis plant can be a species selected from Cannabis sativa, Cannabis indica, or Cannabis ruderalis, or can be a hybrid thereof. Also provided are methods for culturing callus and callus cell suspensions for producing cannabinoid molecules.

Description

METHODS FOR PRODUCTION OF CANNABINOIDS

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No. 63/082,063 filed on September 23, 2020, which is incorporated herein by reference in its entirety as if fully set forth herein.

FIELD OF THE DISCLOSURE

[0002] The disclosure provides new methods of production of cannabinoids (including CBD, CBN and THC) from callus and callus cell suspensions formed from capitate stalked trichomes during a stage prior to an inflorescence maturation threshold of a Cannabis plant.

BACKGROUND OF THE DISCLOSURE

[0003] One of the critical factors for the safe use of medicinal plants concerns the standardization of their cultivation and the consequent content of active components according to the geoclimatic characteristics of the land where they grow. For example, specimens that grow in different latitudes, despite being the same species, are not chemically comparable. As a result, the chemical composition of plants grown in a given condition may need to be determined to establish an effective and safe dose.

[0004] Given the medicinal importance of Cannabis, in recent years efforts have been made to generate in vitro culture systems using biotechnological tools for large-scale production of phytocannabinoids, including through the cultivation of cells of this species. Over one hundred cannabinoids have been identified, some of which have proven or suspected therapeutic potential. However, the extensive use of land for the plantation of this genus of plants is a concern, due to the legality in each country. In addition, the psychotropic effect of the constituent tetrahydrocannabinol (THC) can be a disadvantage, detracting from the commercial therapeutic use of many current cannabinoid-based formulations.

[0005] The methods of the present disclosure provide a solution to these disadvantages, by providing materials and methods for repeated cultivation of callus material and cell suspensions derived from Cannabis, thereby allowing the production of specific plant products at predictable levels.

SUMMARY OF THE DISCLOSURE

[0006] Provided are methods including producing or culturing an in vitro cannabinoid-rich callus or callus cell suspension derived from a Cannabis plant. The in vitro cannabinoid-rich callus is induced in a tissue culture system containing at least one auxin hormone and at least one cytokinin and cultured from flower explants including capitate stalked trichomes during a stage prior to an inflorescence maturation threshold of the Cannabis plant. The Cannabis plant can be a species selected from: Cannabis sativa, Cannabis indica or Cannabis ruderalis.

[0007] The cannabis species can be the result of a cross, such as a hybrid of Cannabis sativa and Cannabis indica.

[0008] The cannabidiol (CBD) yield of the culture is preferably at least 5.8 mg CBD/kg dry weight callus in one month of culture.

[0009] In the methods, the callus and callus cell suspension can be subcultured from the original culture, wherein the subculture culture conditions include at least one auxin hormone and at least one cytokinin.

[0010] In the method of culturing cannabinoid-rich callus in a cell suspension, the amount of CBD produced can be at least 13 mg CBD/kg dry weight callus to at least 100 mg CBD/kg dry weight callus.

[0011] In a method of culturing cannabinoid-rich callus in a tissue culture system containing at least one auxin hormone and at least one cytokinin, where the callus is cultured from flower explants including capitate stalked trichomes during a stage prior to an inflorescence maturation threshold of a Cannabis plant, the cannabinoid includes CBD.

[0012] In a method of culturing cannabinoid-rich callus in a tissue culture system containing at least one auxin hormone and at least one cytokinin, where the callus is cultured from capitate stalked trichomes during a stage prior to an inflorescence maturation threshold of a Cannabis plant, the cannabinoid is at least one of CBD, cannabinol (CBN), and tetrahydrocannabinol (THC). [0013] Further provided is a cannabinoid-yielding callus line obtained from at least one of callus and callus cell suspension, wherein the callus is formed from culturing flower explants including capitate stalked trichomes during a stage prior to an inflorescence maturation threshold of a Cannabis plant, wherein the CBD yield is at least 5.8 mg CBD/kg dry weight callus in one month of culture.

[0014] In the cannabinoid-yielding callus line, the Cannabis plant can be a species selected from the group consisting of Cannabis sativa, Cannabis indica and Cannabis ruderalis.

[0015] In the cannabinoid-yielding callus line, the Cannabis plant can be a hybrid of Cannabis sativa and Cannabis indica.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Some of the drawings submitted herein may be better understood in color. Applicant considers the color versions of the drawings as part of the original submission and reserves the right to present color images of the drawings in later proceedings.

[0017] FIGs. 1A-1G. The planting and germination process of the different varieties of Cannabis. (FIG. 1A) Germination of cannabis seeds; (FIG. 1 B) Cannabis seedling of one month transplanted to definitive pot; (FIG. 1C) Vegetative stage of Cannabis plants with high cannabidiol (CBD) content at 2 months of cultivation; (FIG. 1 D) Start and (FIG. 1 E) end of the flowering stage under controlled conditions in an indoor marquee exclusively for Cannabis; (FIG. 1 F) Harvest of Cannabis inflorescences (Swiss Dream variety); and (FIG. 1G) capitate stalked trichomes during a stage prior to inflorescence maturation threshold of Cannabis.

[0018] FIGs. 2A-2D. The callus induction process derived from capitate trichomes of inflorescence of Cannabis plants on MS medium with the addition of 1mg/L of 1-naphthalacetic acid (ANA) and 0.5mg/L of 6-benzylaminopurine (BAP) after (FIG. 2A) second week; (FIG. 2B) third week; (FIG. 2C) fourth week; and (FIG. 2D) fifth week.

[0019] FIG. 3. A bar graph showing a comparison of percentage of callogenesis according to the variety of Cannabis under in vitro conditions (temperature 22°C/dark). CBDT: CB Dutch Treat variety; MB: Mataro Blue variety; SD: Swiss Dream variety. Data are the mean of ten independent replicates (plates with 10 explants) ± SD.

[0020] FIG. 4. The appearance of induced buds from Cannabis callus CBDT variety after three weeks of subculture in MS medium with a combination of plant hormones (auxin: Cytokinin) incubated at 22°C under photoperiod 16 hours light/8 hours dark.

[0021] FIGs. 5A-5C. The appearance of callus induced from flowers of different Cannabis varieties after three weeks of cultivation in MS medium with addition of NAA to a final concentration of 1 mg/L and BAP to a final concentration of 0.5mg/L. (FIG. 5A) Mataro Blue friable (falling apart easily) and compact callus; (FIG. 5B) CB Dutch Treat friable callus; and (FIG. 5C) Swiss Dream friable callus.

[0022] FIGs. 6A-6C. The morphology of Cannabis cell suspension cultures. (FIG. 6A) Comparison of appearance of the growth of cells in 250 mL flask cultures containing 50 mL of liquid medium MS treated with combination hormones; incubated with (flask on left) and without light (flask on right) at 22°C at a constant speed of 90 rpm in a shaking incubator. (FIG. 6B) The subculture of cell suspension after 1 cycle of 28 days. (FIG. 6C) The cells were observed dyed with lactophenol cotton blue under a light microscope DM500 (Leica Microsystem). Scale bar indicates 20 pm.

DETAILED DESCRIPTION

[0023] There is a need in the art for natural compounds useful for therapeutic purposes, including clinical use against certain pathologies (neurodegenerative, anorexia, epilepsy, psychiatric disorders), and chronic pain control for cancer-related and neuropathic processes, obtained directly from plant cells of Cannabis.

[0024] Given this context, the present disclosure provides a strategy to enhance the production of cannabinoids from callus and callus cell suspensions formed from capitate stalked trichomes during a stage prior to an inflorescence maturation threshold of different cannabis varieties. Methods are provided in the Examples for obtaining cannabinoids from callus, and from cell cultures derived from callus. “Derived from” in the present disclosure refers to obtaining a composition (e.g., a callus or callus cell suspension) from another composition (e.g., a Cannabis plant) by methods described herein, including germinating Cannabis seed and growing to Cannabis plants, obtaining explants of flowers including capitate stalked trichomes during a stage prior to an inflorescence maturation threshold of a Cannabis plant, culturing the flower explants to develop into callus that produces cannabinoids, and culturing the cannabinoid-producing callus as a cell suspension.

[0025] For cannabinoid production, a high CBD content variety can be used for in vitro cultures of plant cells in callus form to produce CBD of high purity. In this way, the goal is to dispense with the planting and exploitation of cannabis crops and favor the production of these phytocannabinoids in a controlled and inducible environment, obtaining a standardized product to meet the growing demand for these compounds in the area of health.

[0026] Compared to the traditional process of establishment and management of cannabis cultivation, the use of in vitro callus production and large-scale cell cultures in suspension has the potential to significantly reduce costs and dispense with plant material and the seasonality of flowering.

[0027] Considering the sale price in some countries, the limited number of effective sources for obtaining CBD, and the economic interest in this product, the present technology using in vitro plant cell cultures offers an attractive alternative to the conventional extraction procedure to facilitate the commercial development of cannabinoids.

[0028] Currently, the medicinal uses of Cannabis are already being investigated and developed, due to the growing success that has been shown for the application of its extracts and metabolites for medicinal use. Some countries are working towards national legislation to solve the problem of access to and possession of Cannabis for medicinal purposes, with clear standards to determine its uses, prescription forms, and obtaining Cannabis derivatives. This natural resource for therapeutic purposes is used from the whole plant through home self-cultivation, in its natural state, or through a product obtained directly from its botanical parts such as raw extracts or resins. [0029] Aspects of the current disclosure are now described with additional details and options as follows: (i) Trichomes; (ii) Cannabis and chemical composition of cannabis; (iii) In vitro propagation and cultivation of Cannabis; (iv) Therapeutic potential in the market; (v) Methods to obtain callus and callus cell suspension producing cannabinoids; (vi) Exemplary Embodiments; (vii) Experimental Examples; and (viii) Closing Paragraphs. These headings do not limit the interpretation of the disclosure and are provided for organizational purposes only.

[0030] (i) Trichomes. To carry out the methods of this disclosure, the presence and state of maturity of the trichomes of the inflorescences is essential. Inflorescences are branches that bear flowers on a Cannabis plant. Flowers are the reproductive organs of a plant and can include trichomes. Trichomes are epidermal protuberances covering the leaves, bracts and stems of plants. There are two major classes of trichomes: glandular trichomes and non-glandular trichomes (Happyana et al., 2013). Non-glandular trichomes exhibit low metabolic activity and provide protection to the plant mainly through physical means. By contrast, glandular trichomes are highly metabolically active secretory structures and accumulate metabolites. Glandular trichomes are capable of secreting (or storing) secondary metabolites as a defense mechanism (Andre et al., 2016).

[0031] Researchers can also mine data relative to metabolites, genes, and expression profiles from trichomes (Dai et al., 2010).

[0032] Three types of glandular trichomes appear on a Cannabis plant: bulbous, capitate sessile, and capitate stalked. Capitate stalked trichomes are visible to the human eye. These trichomes include a stalk of 200-300 pm in length and 50-100 pm in width and composed of epidermal and hypodermic cells. A spherical head (or “capitate”) is located at the end of the stalk. The ballshaped head includes a waxy outer cuticle layer. In particular embodiments, the head contains metabolites such as cannabinoids and terpenes. In Cannabis, THCA is accumulated in the heads (glands) of both capitate-stalked and capitate sessile trichomes, but in the former the content is higher (Mahlberg and Kim, 2004). Notably, in the textile variety, the cannabinoids CBDA and CBCA occur at high concentrations instead of THCA, while the reverse is true for drug strains (Mahlberg and Kim, 2004).

[0033] Studies on Cannabis have demonstrated that THCA is synthesized in the storage cavity located in the head of the trichome and that the enzyme responsible for THCA production, i.e., THCA synthase (THCAS), follows a sorting pathway from the secretory cells to the storage cavity (Sirikantaramas et al., 2005).

[0034] Depending on their color, cannabis glandular trichomes show different secretory phases. The mature secreting gland appears translucent (at this stage the cannabinoid content is the highest), while aging glands are yellow and senescing brown (Mahlberg and Kim, 2004).

[0035] According to the present disclosure, the phase in which the capitate stalked trichomes are in a translucent state is optimal for harvesting and subsequent sowing of explants from the inflorescence for the production of calluses. In particular embodiments, after four weeks of callus formation, the formed calluses can be used to produce cannabinoids by means of cell suspensions. In particular embodiments, capitate stalked trichomes during a stage prior to an inflorescence maturation threshold of a Cannabis plant includes capitate stalked trichomes 5 to 6 months after sowing a Cannabis seed. In particular embodiments, capitate stalked trichomes during a stage prior to an inflorescence maturation threshold of a Cannabis plant includes capitate stalked trichomes 6 months after sowing a Cannabis seed.

[0036] W02020084384 describes that: Cannabis flowers including capitate stalked trichomes that are translucent or clear may be considered immature, while Cannabis flowers including capitate stalked trichomes that are cloudy, white colored, or opaque may be considered fully developed, and Cannabis flowers including capitate stalked trichomes that are amber, orange, or brown colored may indicate higher cannabinol (CBN) and less tetrahydrocannabinol (THC) content in the trichomes.

[0037] In particular embodiments, capitate stalked trichomes during a stage prior to an inflorescence maturation threshold of a Cannabis plant includes capitate stalked trichomes where at least 50% to at least 90%, or at least 60% to at least 80%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, of the capitate stalked trichomes on an inflorescence are translucent. In particular embodiments, capitate stalked trichomes during a stage prior to an inflorescence maturation threshold of a Cannabis plant includes capitate stalked trichomes where less than 50% to less than 10%, or less than 40% to less than 20%, or less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less of the capitate stalked trichomes on an inflorescence are cloudy, white colored, or opaque. In particular embodiments, capitate stalked trichomes during a stage prior to an inflorescence maturation threshold of a Cannabis plant includes capitate stalked trichomes where less than 20% to less than 10%, or less than 15% to less than 5%, or less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less of the capitate stalked trichomes on an inflorescence are amber, orange, or brown colored.

[0038] Preferably, capitate stalked trichomes during a stage prior to an inflorescence maturation threshold of a Cannabis plant includes capitate stalked trichomes that are 90% translucent or more. More preferably, capitate stalked trichomes during a stage prior to an inflorescence maturation threshold of a Cannabis plant includes capitate stalked trichomes that are 95% translucent or more. Most preferably, capitate stalked trichomes during a stage prior to an inflorescence maturation threshold of a Cannabis plant includes capitate stalked trichomes that are 100% translucent.

[0039] In particular embodiments, capitate stalked trichomes during a stage prior to an inflorescence maturation threshold of a Cannabis plant includes capitate stalked trichomes that are: 100% translucent; or 95% translucent and 5% opaque and/or amber; or 90% translucent and 10% opaque and/or amber; or 85% translucent and 15% opaque and/or amber; or 80% translucent and 20% opaque and/or amber; or 75% translucent and 25% opaque and/or amber; or 70% translucent and 30% opaque and/or amber.

[0040] Translucency of capitate stalked trichomes can be determined in a number of ways. In particular embodiments, translucency can be determined by visual inspection of inflorescences by a trained person. In particular embodiments, a digital camera, a cell phone camera, or a hand held microscope can be used to visualize the color of capitate stalked trichomes. In particular embodiments, translucency can be determined by an imaging device such as described in W02020139662. The device can detect and count clear, cloudy, and amber trichomes. In particular embodiments, the imaging device can determine a ratio of amber trichomes to cloudy trichomes, clear trichomes to cloudy trichomes, clear trichomes to amber trichomes, and/or clear to cloudy to amber trichomes. Trichrome color or opaqueness may be identified as a factor that could change the quality or content of cannabinoids in trichomes. In particular embodiments, changes in trichrome color to include more amber or more brown trichomes may indicate that THC has degraded into CBN. An amount of degradation of THC to CBD may be determined by identifying from image data or spectral analysis a total percentage of all trichomes that changed color over time. For example, if 50% of trichomes changed from having an opaque white color to having an amber or brown color, a total mass of CBN in the capitate stalked trichomes may be estimated to have increased by 50%.

[0041] (ii) Cannabis and chemical composition of cannabis. “Cannabis” or “cannabis plant” refers to a genus of flowering plants in the family Cannabaceae and includes any species of the genus including Cannabis sativa, Cannabis indica, Cannabis ruderalis, and interspecific hybrids thereof. In particular embodiments, a cross between two Cannabis species creates a hybrid of those two Cannabis species. In particular embodiments, Cannabis plants include a hybrid of Cannabis sativa and Cannabis indica. In particular embodiments, Cannabis plants include a hybrid of Cannabis sativa and Cannabis ruderalis. In particular embodiments, Cannabis plants include a hybrid of Cannabis indica and Cannabis ruderalis.

[0042] Cannabis is an annual, dioecious, flowering herb. The leaves are palmately compound or digitate, with serrate leaflets. Cannabis normally has imperfect flowers, with staminate "male" and pistillate "female" flowers occurring on separate plants. It is not unusual, however, for individual plants to separately bear both male and female flowers (i.e. , have monoecious plants). Cannabis is diploid, having a chromosome complement of 2n=20, although polyploid individuals have been artificially produced and are also included herein. A cultivar includes a plant or group of plants (e.g., a variety) cultivated by humans and selected for desirable characteristics. Although some cultivars can occur in nature as plant mutations, most cultivars are developed by plant breeders, i.e., as hybrids of two plants. To propagate clones with heritable characteristics, many cultivars can be propagated vegetatively (e.g., through cuttings, grafting, or tissue culture), as opposed to propagation by seed. In particular embodiments, a variety and a cultivar can be used interchangeably.

[0043] Various types of Cannabis plants can exist within the same species, including narrow leaf and broad leaf types, as well as medicinal and nonmedicinal types. Cannabis is also classified based on cannabinoid content into 5 classes referred to as chemotypes or chemovars. Chemotype 1 (marijuana) has high amounts of THC and low amounts of CBD, with amounts of THC as high as 30%. Chemotype 2 has similar amounts of THC and CBD. Chemotype 3 (hemp) has high amounts of CBD, low amounts of THC, with amounts of CBD as high as 20%. Chemotype 4 has high amounts of cannabigerol (CBG), a precursor of THC and CBD. Chemotype 5 does not produce cannabinoids. The term “hemp” refers to a nonmedicinal strain of Cannabis sativa grown for industrial uses and includes Cannabis containing no more than 0.3% THC by dry weight.

[0044] Phytocannabinoids represent a group of C21 or C22 (for carboxylated forms) phenolic terpene compounds produced in plants. In particular embodiments, phytocannabinoids are produced in Cannabis species. More than a hundred different cannabinoids have been described in the literature, although some of them are degradation products (Radwan et al., 2009; Fischedick et al., 2010). Phytocannabinoids can be generally classified into ten subclasses (Brenneisen, 2007): cannabidiol (CBD), cannabinol (CBN), cannabinodiol (CBDN), cannabichromene (CBC), cannabigerol (CBG), cannabicyclol (CBL), cannabielsoin (CBE), cannabitriol (CBT), delta 9-tetrahydrocannabinol (A9-THC), and delta 8-tetrahydrocannabinol (D8-THC). Phytocannabinoids are biosynthesized as acids. The predominant compounds are THCA (Tetrahydrocannabinolic acid), CBDA (Cannabidiolic acid) and cannabinolic acid (CBNA), followed by cannabigerolic acid (CBGA), cannabichromogenic acid (CBCA) and cannabinodiolic acid (CBN DA).

[0045] In particular embodiments, cannabinoids of the present disclosure include tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THCA), cannabidiol (CBD), cannabidiolic acid (CBDA), cannabinol (CBN), cannabinolic acid (CBNA), CBG (cannabigerol), cannabigerolic acid (CBGA), cannabichromene (CBC), cannabichromogenic acid (CBCA), cannabicyclol (CBL), cannabivarin (CBV), tetrahydrocannabivarin (THCV), cannabidivarin (CBDV), cannabichromevarin (CBCV), cannabigerovarin (CBGV), cannabigerol monomethyl ether (CBGM), cannabielsoin (CBE), cannabicitran (CBT), and cannabinodiol (CBDL).

[0046] THCA is the main cannabinoid in the type of drug from Cannabis, while CBDA predominates in fiber. It has been reported that CBCA dominates the cannabinoid fraction of young plants and decreases with maturation (Meijer et al., 2009). Phytocannabinoid acids are not enzymatically decarboxylated in their corresponding neutral forms, such as those that occur to a greater extent within the plant; when heated after harvesting, they can be decarboxylated (Flores- Sanchez and Verpoorte, 2008). Phytocannabinoids accumulate in the secretory cavity of glandular trichomes, which occur mainly in female flowers and in most of the aerial parts of plants. They have also been detected in small quantities in other parts of plants, including seeds (Ross et al., 2000), roots (Stout et al., 2012) and pollen (Ross et al., 2005), depending on the type of Cannabis (type of tissue, age, variety, growth conditions, nutrition, humidity, light level, harvest time and storage conditions) (Khan et al., 2014).

[0047] Numerous chemicals are produced in Cannabis through secondary metabolism. They include cannabinoids, terpenes and phenolic compounds (Flores-Sanchez and Verpoorte, 2008). Although the pharmacological properties of cannabinoids have been studied extensively, the other components have also been associated with potent health-promoting properties. Research on cannabis phytochemicals, as well as the widespread therapeutic use of cannabis products, have been limited due to several reasons, including the illegality of the crop (due to its psychoactive capacity), the variability of active components, and the low abundance of some active compounds in the plant. Now more attention is paid to non-psychoactive components, which can be effective and contribute to the pharmacological power of medicine-based cannabis extracts (Russo, 2011).

[0048] (iii) In vitro propagation and cultivation of Cannabis. Cannabis cultivation is strictly regulated in many countries. In vitro cultivation of Cannabis is an advantageous way of preserving cultivars/clones (Lata et al., 2009a) with specific amounts of metabolites. Methods for multiplying C. sativa plants in vitro have been described by stimulation of axillary buds in nodal segments or induction of adventitious shoots at the tips of the shoots (Lata et al., 2009a, Wang et al., 2009b). [0049] It was shown that micropropagated plants are genetically stable; therefore, the method is appropriate and useful for the clonal multiplication of this crop (Lata et al., 2010). A protocol for hemp propagation has also been developed through synthetic seed technology. According to this procedure, axillary buds or nodal segments are encapsulated in calcium alginate beads (Lata et al., 2009b, 2011), which can then be stored and subsequently used for clonal plant propagation. It was demonstrated that this system allows the successful growth of homogeneous and genetically stable Cannabis plants even after six months of storage (Lata et al., 2011).

[0050] In vitro cultures of calluses are groups of undifferentiated cells that are actively divided and derived from plant tissue (explant), usually kept in solid medium (Pierik, 1987). The term “explant”, used herein with reference to plant tissue culture, refers to living plant tissue that is removed from the natural site of growth on a plant and placed in sterile medium (e.g., MS medium) for culture. The explant can be of any tissue type such as leaves, roots, stems, or any portion taken from a plant and used to initiate tissue culture. In particular embodiments, explants used in the methods and compositions herein are flower explants including capitate stalked trichomes during a stage prior to an inflorescence maturation threshold of a Cannabis plant. The characteristics of the callus formed from an explant, including the callus texture, compactness, friability (tendency to fall apart easily) and coloration, depends on the genotype and the age of the primary explant (Sen et al., 2014). The callus derived from the original explant can be established and maintained in a state of active growth through the transfer of its fragments to a fresh medium at regular intervals, such as every four weeks (Remotti and Loffler, 1995). The growth of the callus culture can be controlled by measuring its fresh and dry weight or volume of packed cells, as well as the determination of its growth and number of cells (Kittipongpatana et al., 1998; Mustafa et al., 2011).

[0051] When the callus is suspended in liquid growth medium, its cells disperse producing a cell suspension culture characterized by faster and more uniform growth (Mustafa et al., 2011). Researchers have studied callus cultures from explants of different organs of C. sativa (roots, hypocotyls, epicotyls, cotyledons, petioles, immature floral leaves and buttons) (Loh et al., 1983, Braut-Boucher et al., 1985, Fisse and Andres, 1985). In addition, callogenic masses could be derived from seed explants of numerous hemp varieties, such as Carmagnola, Fibranova, Uniko and Kompolti (Mandolino and Ranalli, 1999); Uniko-B, Kompolti Anka and Felina-34 (Feeney and Punja, 2003); Sileia, Fibriman-24, Novosadska, Juso-15 and Fedrina-74 (Slusarkiewicz-Jarzina et al., 2005); Carmagnola (Pacifico et al., 2008); and Beniko, Silesia and Bialobrzeskie (Wielgus et al., 2008). [0052] Although there are many publications that describe in vitro studies of other medicinal plants, there are however no reports of scientific records on Cannabis cultures in cell suspension, established for the extraction of secondary metabolites, for example cannabinoids.

[0053] In contrast, Itokawa et al. (1977) examined the components of Cannabis callus cultures induced by different explants (roots, hypocotyls, seedlings, leaves, and male and female flowers) by culturing them in MS agar medium supplemented with 0.1-0.01 mg/L of kinetina (KIN) and 1.0 mg/L of 2,4-Dichlorophenoxyacetic acid (2,4-D), where the cannabinoids were not detected, but other compounds, such as methyl palmitate, methyl oleate, methyl stearate, 5a-ergostan-3-one, 5a-stigmastan-3-one, campesterol, stigmasterol, p-sitosterol, 5a-stigmast-22-in-3-one as well as A5.24(28) unsaturated sterols and fatty acid esters were detected.

[0054] Hartsei et al. (1983) reported the biotransformation of CBD to cannabielsoin (CBE) in cell cultures of C. sativa and Saccharum officinarum grown in 1.5% DM agar medium containing the B5 medium vitamins, with 3 mg/L of 2.4.5 trichlorophenoxyacetic acid (2,4,5-T) as the only hormone. The suspensions were shaken in an orbital shaker (100 rpm) at 27°C, with a daily photoperiod of 8 hours. The authors also found that incubation of the cultures with olivetol resulted in the generation of an unidentified cannabinoid characterized by a molecular ion of m/z 210 in the mass spectrometry analysis.

[0055] Braemer et al. (1987) investigated the bioconversion of flavonoids in their glycosides in suspension cultures of C. sativa. The cells were grown in B5 medium supplemented with 0.5 mg/L of KIN and 1 mg/L of 2,4-D in a rotary shaker (120 rpm), with a photoperiod of 16 hours a day at 25°C. The results showed that quercetin was completely transformed into quercetin 3-O- glycoside, quercetin 3-O-diglucoside, isorhamnetin 3-O-glucoside, and isorhamnetin 3-O- diglucoside, while apigenin was converted into apigenin 7-O-glucoside and 7-O-glucuronide and vitexin.

[0056] At the same time, the group studied the biotransformation of cannabinoids in cannabis crops obtained from leaf explant calluses. Cell suspensions were cultured under the conditions mentioned above; however, they remained in total darkness. Subcultures were transferred to new medium every three weeks. In fact, CBD was converted into joined CBE ((R) - and (S) - cannabielsoin) and THC into cannabicoumaronon (CBC), as determined by gas-liquid chromatography. Unfortunately, quantitative analysis was not feasible due to insufficient accumulation of the compounds produced (Braemer and Paris, 1987).

[0057] Flores-Sanchez et al. (2009) studied the influence of biotic and abiotic elicitors on the production of cannabinoids in C. sativa cultures. Leaf-derived callus cell suspensions were obtained and grown in basal MS medium supplied with specific B5 vitamins, 1mg/L 2,4-D and 1mg/L KIN on an orbital shaker (110 rpm), under 1000-1700 light intensity at 25°C. Subcultures were transferred to new medium every two weeks. The authors reported that cannabinoid biosynthesis was not stimulated or induced by biotic and abiotic elicitors. They also noticed low levels of expression of the THCA synthase gene.

[0058] Other elicitation studies focused on the influence of jasmonic acid and pectin on the metabolism of two C. sativa cell lines. Applied nuclear magnetic resonance imaging (NMR) and multivariate data analysis resulted in the detection of tyrosol, a natural phenolic antioxidant (Pec et al., 2010).

[0059] There are other protocols described for the induction of calluses from the hypocotyl/cotyledon and cells in suspension growing in their optimal state of cell growth proliferation from Papaver bracteatum and Celosia cristata Linn, for the production of thebaine, and betacyanins and betaxanthins, respectively, or other secondary metabolites to be scaled for mass production of compounds of interest in bioreactors (Farjaminezhad et al., 2013; Lystvan et al., 2018).

[0060] The culture of plant cells in suspension is a tool that allows study of various aspects of the culture, such as its metabolic, physiological and biochemical behavior, as well as control and optimization of the conditions of cultivation for biomass production, or the production of secondary metabolites using different elicitors (Moscatiello et al., 2013). This strategy requires an initial process that includes the formation of friable callus and subsequently the establishment of cell suspensions. For this, it is important to know the cell growth kinetics and their behavior in these systems, in order to estimate the required subculture time and the days in which the active growth of the cell culture occurs (Trejo-Tapia et al., 2007).

[0061] At present, few industrial processes are known for the production of secondary metabolites using this culture strategy; some of the successful processes have involved obtaining: paclitaxel, an anticancer drug obtained from cell suspensions of species of the genus Taxus up to volumes of 75,000 liters; shikonin by cellular suspensions of Lithospermum erythrorhizon', berberine from cellular suspensions of Coptis japonica, and extracts of the Panax ginseng plant in reactors of up to 25,000 liters (Arias et al., 2009).

[0062] (iv) Therapeutic potential in the market. In the pharmaceutical industry, cannabinoids (including CBD, THC and CBN) have become increasingly important as valuable starting compounds for the development of new drugs. A few examples are described as follows.

[0063] In Canada, Sativex® (oral spray: 27 mg/ml A9-THC and 25 mg/ml CBD; GW Pharmaceuticals) was approved to treat muscle pain and stiffness in multiple sclerosis and cancer patients (Whittle and Guy, 2004; Whittle, 2007), under medical prescription, and for clinical trials, and has been used in countries including the United States, England, Germany and Spain, while Cannador® oral capsules reduces tremor related to multiple sclerosis (Holdcroft et al., 2006; Rahn and Hohmann, 2009). Bedrocan® (18% A9-THC and < 1 % CBD), Bedrobinol® (11% A9-THC and < 1 % CBD), and Bediol® (6% A9-THC and < 1% CBD) are dried preparations of bleeding flower buds for medicinal uses.

[0064] In addition, several synthetic cannabinoid-based drugs have been approved to relieve nausea and vomiting associated with cancer chemotherapy (Marinol®, Dronabinol, Solvay Pharmaceuticals; and Cesamet®, Nabilone, Valeant Pharmaceuticals International) (Stott and Guy, 2004). Several new cannabinoid-based products that are currently being developed worldwide are expected to be introduced in the market in the near future.

[0065] (v) Methods to obtain callus and callus cell suspension producing cannabinoids. The present compositions and methods differ from and improve on the compositions and methods of the art. The following processes can be performed to grow Cannabis seed to Cannabis plants, obtaining explants of flowers including capitate stalked trichomes during a stage prior to an inflorescence maturation threshold of a Cannabis plant, culturing the flower explants to develop into callus that produces cannabinoids, and culturing the cannabinoid-producing callus as a cell suspension.

[0066] Cannabis seeds are first germinated and the sprouts are transplanted into planters for growth. In particular embodiments, Cannabis seeds are germinated on filter paper in a humid chamber for 7 to 8 days, and then the sprouts are placed in small planters to reach a height of fifteen cm. Plants of 15 cm can then be transplanted to another pot to continue the vegetative and flowering process in a substrate of perlite and vermiculite, although other specific growth substrates may be used. In particular embodiments, a substrate composition of soil: perlite: vermiculite (2:1 :1) is used to grow Cannabis plants.

[0067] The growth conditions for Cannabis plants include a temperature of 23°C and a photoperiod of sixteen hours light/eight hours darkness in the vegetation stage, then twelve hours light/twelve hours dark in the flowering stage. Light conditions for growth can include illuminance of 2000-3000 lux (lumens/m²). In particular embodiments, growth conditions for Cannabis plants can include a temperature of 20°C, 21°C, 22°C, 23°C, 24°C, and 25°C. In particular embodiments, growth conditions for Cannabis plants can include a temperature of 21 °C to 22°C. Organic fertilizer can optionally be applied once or twice or more as preferred to accelerate the flowering phase. For example, organic fertilizer may be dissolved in an irrigation solution.

[0068] Plant growth media or plant tissue culture media described herein includes sterile liquid, semi-solid, or solid media containing nutrients and other ingredients. Tissue culture described herein refers to the growth of tissues or cells separate from the plant. This is typically facilitated via use of a liquid, semi-solid, or solid growth medium, such as broth or agar.

[0069] The physical state of the medium can vary by the incorporation of one or more gelling agents. Any gelling agent known in the art that is suitable for use in plant tissue culture media can be used. Agar is most commonly used for this purpose. Examples of such agars include Agar Type A, E or M and Bacto™ Agar. Other exemplary gelling agents include carrageenan, gellan gum (commercially available as PhytaGel™, Gelrite®, and Gelzan™), alginic acid and its salts, and agarose. Blends of these agents, such as two or more of agar, carrageenan, gellan gum, agarose and alginic acid or a salt thereof also can be used. In particular embodiments, no gelling agent or very little gelling agent is used for a liquid medium.

[0070] In particular embodiments, the medium includes minimum nutrition necessary for plant growth, such as amino acids; macroelements including nitrogen (nitrates), potassium, phosphorous (phosphates), magnesium, and sulphur (sulphates); microelements including aluminum, boron, chlorine (chloride), chromium, cobalt, copper, iodine, iron, lead, manganese, molybdenum, silicon, sodium, titanium, vanadium, and zinc; and undefined media components such as casein hydrolysates or yeast extracts.

[0071] The medium can include a carbon source, such as a sugar. Exemplary sugars include sucrose, glucose, maltose, galactose and sorbitol or combinations thereof.

[0072] In particular embodiments, culture medium is prepared as described in the Examples, based on medium of Murashige and Skoog (Murashige T & Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-97, 1962; referred to herein as “MS”) and Plant Agar (Phytotechnology Laboratories, LLC, Kansas, US). In particular embodiments, MS basal medium includes: ammonium nitrate, calcium chloride, magnesium sulphate, potassium nitrate, potassium phosphate monobasic, boric acid, cobalt chloride hexahydrate, copper sulfate pentahydrate, ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate, ferrous sulfate heptahydrate, manganese sulfate monohydrate, molybdic acid (sodium salt), potassium iodide, and zinc sulphate heptahydrate. In particular embodiments, the ammonium nitrate is at a concentration of 1650 mg/L, the calcium chloride is at a concentration of 332.2 mg/L, the magnesium sulphate is at a concentration of 180.69 mg/L, the potassium nitrate is at a concentration of 1900 mg/L, the potassium phosphate monobasic is at a concentration of 170 mg/L, the boric acid is at a concentration of 6.2 mg/L, the cobalt chloride hexahydrate is at a concentration of 0.025 mg/L, the copper sulfate pentahydrate is at a concentration of 0.025 mg/L, the EDTA disodium salt dihydrate is at a concentration of 37.3 mg/L, the ferrous sulfate heptahydrate is at a concentration of 27.8 mg/L, the manganese sulfate monohydrate is at a concentration of 16.9 mg/L, the molybdic acid (sodium salt) is at a concentration of 0.213 mg/L, the potassium iodide is at a concentration of 0.83 mg/L, and the zinc sulphate heptahydrate is at a concentration of 8.6 mg/L.

[0073] In particular embodiments, MS medium can include vitamins (Product ID: M519 from Phytotechnology Laboratories, Lenexa, KS, USA). In particular embodiments, the vitamins can include: myo-inositol, nicotinic acid, pyridoxine hydrochloride, and thiamine hydrochloride. In particular embodiments, the myo-inositol is at a concentration of 100 mg/L, the nicotinic acid is at a concentration of 0.5 mg/L, the pyridoxine hydrochloride is at a concentration of 0.5 mg/L, and the thiamine hydrochloride is at a concentration of 0.1 mg/L.

[0074] In particular embodiments, MS medium can include amino acids. In particular embodiments, the amino acid can include glycine. In particular embodiments, the glycine is at a concentration of 2.0 mg/L.

[0075] One of skill in the art can select any appropriate media that supports growth of callus and callus cell suspension disclosed herein. Examples of other media include: Woody Plant Basal Salt Mixture (Lloyd and McCown (1980) Int. Plant Propagators’ Soc. Proc. 30:421-427; e.g., L154 from Phytotechnology Laboratories, KS, USA); Driver and Kuniyuki Walnut Basal Salt Mixture (Driver and Kuniyuki (1984) HortScience 19(4): 507-509; e.g., D190 from Phytotechnology Laboratories, KS, USA); Gamborg’s B-5 Basal Salt Mixture (B5; Gamborg et al. (1968) Exp Cell Res 50:150-158; e.g., G398 from Phytotechnology Laboratories, KS, USA); and BABI Basal Salt Mixture (Greenway et al. (2012) In Vitro Cellular & Developmental Biology-Plant, 48(4):403-410; e.g., B1471 from Phytotechnology Laboratories, KS, USA).

[0076] In particular embodiments, auxin hormones (e.g., NAA/1 -naphthaleneacetic acid) (Phytotechnology Laboratories, LLC, Kansas, US) and cytokinins (e.g., 6- benzylaminopurine/BAP) (Phytotechnology Laboratories, LLC, Kansas, US) are added to the culture medium for a final concentration of 0.5-2.0 mg/L and 0.5-1.0 mg/L, respectively, in the medium. This hormone-supplemented medium can be placed in sterile petri dishes, 50 mL per dish, and allowed to gel under sterile conditions.

[0077] Auxin is a plant hormone important for plant body development including plant growth, cell elongation, and cell differentiation. Auxins are compounds with an aromatic ring and a carboxylic acid group. In particular embodiments, auxins or auxin derivatives useful in the present disclosure include indole-3-acetic acid (IAA), 2-phenylacetic acid (PAA), 4-chloroindole-3-acetic acid (4-CI- IAA), indole-3-butyric acid (IBA), indole-3-propionic acid (IPA), indole-3-acetaldoxime, indole-3- acetamide (IAM), indole-3-ethanol, indole-3-pyruvate, glucose-bound auxin, 1 -naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), or a combination thereof. IAA, 4-CI-IAA, PAA, I BA, and I PA are naturally produced in plants, with IAA being the most abundant and most potent auxin in plants. In particular embodiments, the auxin hormone useful in the present disclosure is NAA, a synthetic auxin analog. In particular embodiments, auxin hormone is at a concentration of 0.5 mg/L to 2.0 mg/L, or 0.5 mg/L to 1.5 mg/L in the medium. In particular embodiments, auxin hormone is at a concentration of 0.5 mg/L to 1.0 mg/L in the medium. In particular embodiments, auxin hormone is at a concentration of 0.5 mg/L, 0.6 mg/L, 0.7 mg/L, 0.8 mg/L, 0.9 mg/L, 1.0 mg/L, 1.1 mg/L, 1.2 mg/L, 1.3 mg/L, 1.4 mg/L, 1.5 mg/L, 1.6 mg/L, 1.7 mg/L, 1.8 mg/L, 1.9 mg/L, or 2.0 mg/L in the medium. In particular embodiments, NAA is at a concentration of 0.5 mg/L to 2.0 mg/L in the medium. In particular embodiments, NAA is at a concentration of 1 mg/L in the medium.

[0078] Cytokinins are plant hormones that promote cell division in plant roots and shoots. Two types of cytokinins exist: adenine-type cytokinins include kinetin, zeatin, and 6-benzylaminopurine (BAP); and phenylurea-type cytokinins include diphenylurea and thidiazyron (TDZ). In particular embodiments, the cytokinin or cytokinin derivative useful in the present disclosure include kinetin, cis-zeatin, trans-zeatin, BAP, di hydroxyzeatin, N6-(D2-isopentenyl) adenine, ribosilzeatin, N6- (D2-isopentenyl) adenosine, 2-methylthio-cis-ribosylzeatin, cis-ribosylzeatin, trans-ribosylzeatin, 2-methylthio-trans-ribosylzeatin, ribosylzeatin-5-monosphosphate, N6-methylaminopurine, N6- dimethylaminopurine, 2'-deoxyzeatinriboside, 4-hydroxy-3-methyl-trans-2-butenylaminopurine, ortho-topolin riboside, meta-topolin, ortho-methyl topolin, meta-methyl topolin, or a combination thereof. In particular embodiments, the cytokinin is BAP. In particular embodiments, the cytokinin is at a concentration of 0.5 mg/L to 1 .0 mg/L, or 0.5 mg/L to 0.8 mg/L in the medium. In particular embodiments, the cytokinin is at a concentration of 0.5 mg/L, 0.6 mg/L, 0.7 mg/L, 0.8 mg/L, 0.9 mg/L, or 1.0 mg/L in the medium. In particular embodiments, BAP is at a concentration of 0.5 mg/L to 1.0 mg/L in the medium. In particular embodiments, BAP is at a concentration of 0.5 mg/L in the medium.

[0079] As noted above, the flower is a preferred source for cannabinoid extraction. Cannabinoids are synthesized and stored in hair-like epidermal protrusions called glandular trichomes. In particular embodiments, capitate stalked trichomes synthesize and store a large amount of cannabinoids as compared to capitate sessile or bulbous trichomes. Capitate stalked trichomes are densely concentrated in bracts and flowers of cannabis plants.

[0080] To fully utilize the cannabinoid potential of the flower structures, female flowers may be in an optimal state for explant extraction at five to six months after sowing the seeds, depending on the cannabis variety and its agronomic management. In particular embodiments, female flowers including capitate stalked trichomes are used for explant extraction at five to six months after sowing the seeds. In particular embodiments, male flowers including capitate stalked trichomes are used for explant extraction five to six months after sowing the seeds.

[0081] The preparation of petri dishes with solid culture medium, the preparation of liquid culture medium, the preparation of flower explants for culture, the preparation of calluses for subculture, and/or the preparation of a callus cell suspension are carried out under sterile conditions, e.g., in a laminar flow hood, previously sterilized with technical alcohol and UV light. Explants of flowers of one to two cm² are cut and placed into petri dishes containing solid culture medium as described herein, and the cultures are grown in the dark at 21°C-22°C for four weeks for callus formation. Flowers of Cannabis plants can be recognized by one of skill in the art (Spitzer-Rimon et al. (2019) Frontiers in Plant Science 10:350) and flowers can be obtained from a Cannabis plant for the purpose of culturing as explants for callus formation. For example, inflorescences with >90% translucent capitate stalked trichomes can be excised from apical and axillary shoots of Cannabis plants of a given variety. Bracts and leaves can be removed, and the flower explants can be surface-sterilized with ethanol, washed with water, sterilized with bleach, then washed again in water. The sterilized flower explants can then be blotted dry on, e.g., filter paper, before placing on solid culture medium for culturing to form callus.

[0082] “In the dark” refers to growth in a suitable location, such as a plant growth chamber, without application of light. In particular embodiments, “in the dark” refers to growth in a suitable location, such as a plant growth chamber, and can include illuminance of 0 to 1000 lux, or 0 to 500 lux, or 0 to 250 lux, or 0 to 100 lux, or less than 1000 lux, less than 900 lux, less than 800 lux, less than 700 lux, less than 600 lux, less than 500 lux, less than 400 lux, less than 300 lux, less than 200 lux, less than 100 lux, less than 90 lux, less than 80 lux, less than 70 lux, less than 60 lux, less than 50 lux, less than 40 lux, less than 30 lux, less than 20 lux, less than 10 lux, less than 9 lux, less than 8 lux, less than 7 lux, less than 6 lux, less than 5 lux, less than 4 lux, less than 3 lux, less than 2 lux, less than 1 lux, or 0 lux. Growth in the dark of callus or callus cell suspension can include wrapping a dish or flask containing the callus or callus cell suspension, respectively, with foil to further minimize exposure to light and placing it in a growth chamber without application of light. In particular embodiments, the cultures are grown in the dark at 21°C-22°C for two weeks or more, and up to eight to ten weeks for callus formation. In particular embodiments, the cultures are grown in the dark at 21°C-22°C for two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, or ten weeks for callus formation. In particular embodiments, a callus refers to an unorganized mass of cells that has been formed in vitro by treatment of plant tissue (e.g., flower explants) with plant growth regulators. In particular embodiments, the growth regulators include an auxin hormone and a cytokinin. [0083] In particular embodiments, the formed calluses are subcultured every fifteen days to fresh culture medium supplemented with the same hormone concentrations as used for callus formation from flower explants. Subculturing refers to transfer of a portion of a callus or a portion of a callus cell suspension to new media to continue culture of the callus or callus cell suspension. In particular embodiments, subculturing allows further growth of the callus or callus cell culture. For maintenance of the cultures, the calluses can be subcultured every four weeks, as described in the Examples. In particular embodiments, callus can be subcultured every 15 days, every 16 days, every 17 days, every 18 days, every 19 days, every 20 days, every 21 days, every 22 days, every 23 days, every 24 days, every 25 days, every 26 days, every 27 days, every 28 days, every 29 days, every 30 days, every 31 days, every 32 days, every 33 days, every 34 days, or every 35 days. In particular embodiments, callus can be sub-cultured every 3 weeks, every 4 weeks, or every 5 weeks.

[0084] Subculturing a callus can include the following. A grown callus is removed from the culture plate, using sterile tweezers, and deposited inside a Petri dish with culture medium and fresh hormones. The callus may be cut into two or more parts, depending on the size of the cell mass, using a scalpel and sterile forceps. Nine to twelve units (callus parts) are placed per dish, and then the dish is sealed with a parafilm sheet. In particular embodiments, subculturing calluses can remove corns that could have been contaminated in the first crop. The plates are incubated in a plant growth chamber in darkness at 21°C-22°C. In particular embodiments, the culture medium is MS supplemented with at least one auxin hormone and at least one cytokinin. In particular embodiments, the auxin hormone is NAA at a concentration of 0.5 mg/L to 2.0 mg/L. In particular embodiments, the auxin hormone is NAA at a concentration of 1 mg/L. In particular embodiments, the cytokinin is BAP at a concentration of 0.5 mg/L to 1.0 mg/L. In particular embodiments, the cytokinin is BAP at a concentration of 0.5 mg/L.

[0085] Frequency of callus induction can be calculated as follows: Frequency of callus induction (%) = (Number of explants induced callus/Total number of explants inoculated) x 100. Callus can be characterized based on morphology and structure by methods known in the art, such as light microscopy, scanning electron microscopy (SEM), and histology. For example, for SEM, callus are fixed in solvents (e.g., formaldehyde, ethanol, acetic acid), dehydrated through an ethyl alcohol series, dried, and coated with gold for observation. As another example, for histology, callus can be fixed in solvents (e.g., formaldehyde, ethanol, acetic acid), dehydrated through an ethyl alcohol series, embedded in paraffin wax, sectioned, and stained with hematoxylin for observation. In particular embodiments, callus formed by the methods of the present disclosure are white, friable, and/or exhibit induced buds. In particular embodiments, callus produced by the methods of the present disclosure are brown and friable, are green/white and friable, are green/white and compact, and/or are white and friable. In particular embodiments, callus produced by the methods of the present disclosure are compact.

[0086] After harvest, the callus is removed as described in the Examples, and the extracts are analyzed by high performance liquid chromatography (HPLC) for cannabinoid production. The particular cannabinoid(s) produced is a function of the original seed variety, but the amount produced is a function of crop management and the timing of harvest of the trichomes present in the inflorescences. When growing a high CBD strain, for example, the resulting formed callus will produce high purity CBD. Callus can be prepared as follows for analysis by HPLC, following a modified protocol from LINODC (United Nations Office on Drugs and Crime), “Recommended methods for the identification and analysis of Cannabis and Cannabis products”, New York, 2009. Dry samples of callus are dried at 100°C for three hours for decarboxylation, then are ground into a fine powder in a mortar. An amount of powder (e.g., 200 mg) are weighed per sample and placed in a vial for extraction with methanol: chloroform solvent mixture 9:1 (v/v). The sample is placed in a sonicator (e.g., for thirty min), with warm water. The extract is filtered in an amber vial and diluted in the same solvent solution. The solvents in the sample are evaporated by placing the sample in a rotary evaporator, and the sample is then diluted in acetonitrile: water 5:5 (v/v). [0087] Cannabinoids can be produced in a callus cell suspension. Instead of subculturing callus formed from flower explants including capitate stalked trichomes during a stage prior to the inflorescence maturation threshold of a Cannabis plant, the formed callus is suspended and cultured in liquid growth medium with the same hormones and concentrations as used in the production of callus (e.g., culture medium such as MS including at least one auxin and at least one cytokinin, as described herein). In particular embodiments, white and friable portions of callus are used for callus cell suspension culture. In particular embodiments, the culture medium for callus cell suspension includes MS medium including 0.5-2 mg/L of NAA and 0.5-1 mg/L of BAP. [0088] A callus cell suspension can be cultured for a period of time in the dark. In particular embodiments, a callus cell suspension is cultured for 28 days at 21°C to 23°C in the dark. In particular embodiments, a callus cell suspension is cultured in the dark at 21°C-23°C for two weeks or more, and up to eight to ten weeks. In particular embodiments, a callus cell suspension is cultured in the dark at 21°C-23°C for two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, or more. In particular embodiments, a callus cell suspension is cultured in the dark at 21°C-23°C for a period of time (e.g., two weeks to ten weeks) and a portion of the callus cell suspension culture (i.e., a volume of the callus cell suspension culture) is transferred to fresh culture medium to continue culture. In particular embodiments, the callus cell suspension subculture can be cultured in the dark at 21°C-23°C for another one week, another two weeks, another three weeks, another four weeks, another five weeks, another six weeks, another seven weeks, another eight weeks, another nine weeks, another ten weeks, or more. In particular embodiments, a callus cell suspension culture can be subcultured one or more times.

[0089] In particular embodiments, cells of a callus cell suspension are harvested after four weeks, lyophilized for two days, decarboxylated, and analyzed by a method known to one of skill in the art. In particular embodiments, cannabinoid production from callus and callus cell suspension can be analyzed by methods including immunoassays, ion mobility spectrometry (IMS), thin-layer chromatography (TLC), gas chromatography-flame ionization detection (GC-FID), gas chromatography-mass spectrometry (GC-MS), HPLC, HPLC-mass spectrometry, or a combination thereof. Some of these methods are described in, for example, LINODC (United Nations Office on Drugs and Crime), “Recommended methods for the identification and analysis of Cannabis and Cannabis products”, New York, 2009.

[0090] In particular embodiments, a callus line established by the methods disclosed herein refers to callus that produces cannabinoids consistently and is formed from culturing flower explants including capitate stalked trichomes during a stage prior to an inflorescence maturation threshold of a Cannabis plant. In particular embodiments, a callus line established by the methods disclosed herein include callus that has been cultured for one month. In particular embodiments, a callus line established by the methods disclosed herein include callus that has been cultured for one month or more. In particular embodiments, a callus line established by the methods disclosed herein include callus that has been subcultured for one month, for two months, for three months, or more. In particular embodiments, a callus line established by the methods disclosed herein include callus that has not been subcultured.

[0091] In particular embodiments, a callus line established by the methods disclosed herein refers to a callus cell suspension that produces cannabinoids consistently and is derived from culturing in liquid medium callus obtained from culturing flower explants of a Cannabis plant, and wherein the flower explants include capitate stalked trichomes during a stage prior to an inflorescence maturation threshold of the Cannabis plant. In particular embodiments, a callus line includes a callus cell suspension that has been cultured for one month. In particular embodiments, a callus line includes a callus cell suspension that has been cultured for one month, for two months, for three months, or more. In particular embodiments, a callus line includes a callus cell suspension that has been subcultured. In particular embodiments, a callus line includes a callus cell suspension that has not been subcultured. [0092] Consistent production of cannabinoids can include obtaining values of cannabinoid levels after repeated measurements (e.g., measuring 2, 3, 4 times, or more times) that are not statistically significantly different. A measure is not statistically significantly different if the difference is within a level that would be expected to occur based on chance alone. In contrast, a statistically significant difference or increase is one that is greater than what would be expected to occur by chance alone. Statistical significance or lack thereof can be determined by any of various methods well-known in the art. Examples of commonly used measures of statistical significance include the t-test and the p-value. The p-value represents the probability of obtaining a given result equivalent to a particular datapoint, where the datapoint is the result of random chance alone. In particular embodiments, a result is often considered significant (not random chance) at a p-value less than or equal to 0.05.

[0093] In particular embodiments, callus produced by the methods described herein produces CBD at a yield of 1 mg/kg to 50 mg/kg or at a yield of 1 mg/kg to 20 mg/kg, where mg/kg refers to mg CBD per kg dry weight of callus. In particular embodiments, callus produced by the methods described herein produces CBD at a yield of at least 1 mg/kg, at least 2 mg/kg, at least 3 mg/kg, at least 4 mg/kg, at least 5 mg/kg, at least 6 mg/kg, at least 7 mg/kg, at least 8 mg/kg, at least 9 mg/kg, at least 10 mg/kg, at least 11 mg/kg, at least 12 mg/kg, at least 13 mg/kg, at least 14 mg/kg, at least 15 mg/kg, at least 16 mg/kg, at least 17 mg/kg, at least 18 mg/kg, at least 19 mg/kg, at least 20 mg/kg, or more. In particular embodiments, dry weight of callus refers to the weight (e.g., in kg) of callus after the callus has been lyophilized and a constant weight is achieved. In particular embodiments, dry weight of callus refers to the weight (e.g., in kg) of callus after the callus has been dried in an oven at 60°C and a constant weight is achieved.

[0094] In particular embodiments, a callus cell suspension produced by the methods described herein produces CBD at a yield of 10 mg/kg to 150 mg/kg or at a yield of 13 mg/kg to 100 mg/kg, where mg/kg refers to mg CBD per kg dry weight of callus. In particular embodiments, a callus cell suspension produced by the methods described herein produces CBD at a yield of at least 13 mg/kg, at least 14 mg/kg, at least 15 mg/kg, at least 16 mg/kg, at least 17 mg/kg, at least 18 mg/kg, at least 19 mg/kg, at least 20 mg/kg, at least 25 mg/kg, at least 30 mg/kg, at least 35 mg/kg, at least 40 mg/kg, at least 45 mg/kg, at least 50 mg/kg, at least 55 mg/kg, at least 60 mg/kg, at least 65 mg/kg, at least 70 mg/kg, at least 75 mg/kg, at least 80 mg/kg, at least 85 mg/kg, at least 90 mg/kg, at least 95 mg/kg, at least 100 mg/kg, or more.

[0095] The Examples provide detailed descriptions of cannabinoid production from both callus and cell suspensions, using as starting materials several varieties of Cannabis plants. The varieties were chosen for their varied cannabinoid content. This allowed comparison of the cannabinoid content between the mother plants and the callus and cell suspensions. The varieties also differ in terms of their rate of callus induction.

[0096] Following callus culture and subculture, the cannabinoids were extracted and analyzed by HPLC; these results showed a further difference in the varieties, in terms of CBD, THC, and CBN content. The data is presented in the Examples.

[0097] For cannabinoids from cell suspensions, as illustrated in FIGs. 6A-6C, the Examples describe growing cells from a friable callus in culture flasks containing the desired medium, with addition of hormones as indicated. The cell suspension is further subcultured, then analyzed for cannabinoid content.

[0098] In conclusion, this disclosure provides support for new methods of production of cannabinoids (including CBD, CBN and THC) from callus and callus cell suspensions, specifically formed from capitate stalked trichomes during a stage prior to an inflorescence maturation threshold of a Cannabis plant. These methods can be broadened and expanded to include additional Cannabis species, and allow production of individual cannabinoids on a commercial scale suitable for pharmaceutical use.

[0099] The Exemplary Embodiments and Examples below are included to demonstrate particular embodiments of the disclosure. Those of ordinary skill in the art should recognize in light of the present disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the disclosure. [0100] (vi) Exemplary Embodiments.

1. A method for producing cannabinoid-yielding callus derived from a Cannabis plant, the method including:

(a) placing flower explants of a Cannabis plant on a solid culture medium including at least one auxin hormone and at least one cytokinin; and

(b) culturing the flower explants in the dark for two to ten weeks to produce cannabinoid-yielding callus, wherein the flower explants include capitate stalked trichomes during a stage prior to an inflorescence maturation threshold of the Cannabis plant.

2. The method of embodiment 1 wherein the culturing in the dark is for four weeks.

3. The method of embodiment 1 or 2, further including

(c) subculturing the callus on the solid culture medium including the at least one auxin hormone and the at least one cytokinin.

4. The method of embodiment 3, further including repeating step (c) at least every three weeks, at least every four weeks, or at least every five weeks. 5. The method of embodiment 4, wherein the repeating step (c) is every four weeks.

6. The method of any of embodiments 1-5, wherein the Cannabis plant is a species selected from the group consisting of Cannabis sativa, Cannabis indica and Cannabis ruderalis.

7. The method of any of embodiments 1-5, wherein the Cannabis plant is a hybrid of Cannabis sativa and Cannabis indica.

8. The method of any of embodiments 1-7, wherein the solid culture medium includes Murashige and Skoog (MS) medium and agar.

9. The method of embodiment 8, wherein the MS medium includes ammonium nitrate, calcium chloride, magnesium sulphate, potassium nitrate, potassium phosphate monobasic, boric acid, cobalt chloride hexahydrate, copper sulfate pentahydrate, ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate, ferrous sulfate heptahydrate, manganese sulfate monohydrate, molybdic acid (sodium salt), potassium iodide, and zinc sulphate heptahydrate.

10. The method of embodiment 9, wherein the ammonium nitrate is at a concentration of 1650 mg/L, the calcium chloride is at a concentration of 332.2 mg/L, the magnesium sulphate is at a concentration of 180.69 mg/L, the potassium nitrate is at a concentration of 1900 mg/L, the potassium phosphate monobasic is at a concentration of 170 mg/L, the boric acid is at a concentration of 6.2 mg/L, the cobalt chloride hexahydrate is at a concentration of 0.025 mg/L, the copper sulfate pentahydrate is at a concentration of 0.025 mg/L, the EDTA disodium salt dihydrate is at a concentration of 37.3 mg/L, the ferrous sulfate heptahydrate is at a concentration of 27.8 mg/L, the manganese sulfate monohydrate is at a concentration of 16.9 mg/L, the molybdic acid (sodium salt) is at a concentration of 0.213 mg/L, the potassium iodide is at a concentration of 0.83 mg/L, and the zinc sulphate heptahydrate is at a concentration of 8.6 mg/L.

11 . The method of any of embodiments 8-10, wherein the MS medium includes the vitamins: myo-inositol, nicotinic acid, pyridoxine hydrochloride, and thiamine hydrochloride.

12. The method of embodiment 11 , wherein the myo-inositol is at a concentration of 100 mg/L, the nicotinic acid is at a concentration of 0.5 mg/L, the pyridoxine hydrochloride is at a concentration of 0.5 mg/L, and the thiamine hydrochloride is at a concentration of 0.1 mg/L.

13. The method of any of embodiments 8-12, wherein the MS medium includes the amino acid glycine.

14. The method of embodiment 13, wherein the glycine is at a concentration of 2.0 mg/L.

15. The method of any of embodiments 1-14, wherein the at least one auxin hormone is 1- naphthaleneacetic acid (NAA) in a concentration range of 0.5-2.0 mg/L.

16. The method of any of embodiments 1-15, wherein the at least one cytokinin is 6- benzylaminopurine (BAP) in a concentration range of 0.5-1.0 mg/L. 17. The method of any of embodiments 1-16, wherein the method further includes measuring cannabinoid levels.

18. The method of embodiment 17, wherein the measuring is by an immunoassay, ion mobility spectrometry (IMS), thin-layer chromatography (TLC), gas chromatography-flame ionization detection (GC-FID), gas chromatography-mass spectrometry (GC-MS), HPLC, HPLC-mass spectrometry, or a combination thereof.

19. The method of embodiment 17 or 18, wherein the cannabinoid levels include CBD at a yield of at least 5.8 mg CBD/kg dry weight callus.

20. A cannabinoid-yielding callus line produced by the method of any of embodiments 1-19.

21. The cannabinoid-yielding callus line of embodiment 20, wherein the CBD yield is at least 5.8 mg CBD/kg dry weight callus in one month of culture.

22. The cannabinoid-yielding callus line of embodiment 20 or 21 , wherein the cannabinoid- yielding callus line is produced from a Cannabis plant of a species selected from the group consisting of Cannabis sativa, Cannabis indica and Cannabis ruderalis.

23. The cannabinoid-yielding callus line of embodiment 20 or 21 , wherein the cannabinoid- yielding callus line is produced from a Cannabis plant that is a hybrid of Cannabis sativa and Cannabis indica.

24. The cannabinoid-yielding callus line of any of embodiments 20-23, wherein the cannabinoid-yielding callus line includes tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), cannabinol (CBN), or a combination thereof.

25. The cannabinoid-yielding callus line of any of embodiments 20-23, wherein the cannabinoid-yielding callus line does not include THC and/or CBN.

26. A method for producing a cannabinoid-rich callus cell suspension, said method including:

(a) suspending callus in liquid culture medium including at least one auxin hormone and at least one cytokinin; and

(b) culturing the culture in the dark for two to ten weeks, wherein the callus is obtained from culturing flower explants of a Cannabis plant, and wherein the flower explants include capitate stalked trichomes during a stage prior to an inflorescence maturation threshold of the Cannabis plant.

27. The method of embodiment 26, wherein the culturing the culture in the dark is for four weeks.

28. The method of embodiment 26 or 27, wherein the Cannabis plant is a species selected from the group consisting of Cannabis sativa, Cannabis indica and Cannabis ruderalis.

29. The method of embodiment 26 or 27, wherein the Cannabis plant is a hybrid of Cannabis sativa and Cannabis indica.

30. The method of any of embodiments 26-29, wherein the at least one auxin hormone is 1- naphthaleneacetic acid (NAA) in a concentration range of 0.5-2.0 mg/L.

31. The method of any of embodiments 26-30, wherein the at least one cytokinin is 6- benzylaminopurine (BAP) in a concentration range of 0.5-1.0 mg/L.

32. The method of any of embodiments 26-31 , wherein the callus was formed on solid culture medium including at least one auxin hormone and at least one cytokinin.

33. The method of any of embodiments 26-32, wherein the flower explants were cultured in the dark for four weeks to obtain the callus.

34. The method of any of embodiments 26-33, wherein the liquid culture medium includes Murashige and Skoog (MS) medium.

35. The method of embodiment 34, wherein the MS medium includes ammonium nitrate, calcium chloride, magnesium sulphate, potassium nitrate, potassium phosphate monobasic, boric acid, cobalt chloride hexahydrate, copper sulfate pentahydrate, ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate, ferrous sulfate heptahydrate, manganese sulfate monohydrate, molybdic acid (sodium salt), potassium iodide, and zinc sulphate heptahydrate.

36. The method of embodiment 35, wherein the ammonium nitrate is at a concentration of 1650 mg/L, the calcium chloride is at a concentration of 332.2 mg/L, the magnesium sulphate is at a concentration of 180.69 mg/L, the potassium nitrate is at a concentration of 1900 mg/L, the potassium phosphate monobasic is at a concentration of 170 mg/L, the boric acid is at a concentration of 6.2 mg/L, the cobalt chloride hexahydrate is at a concentration of 0.025 mg/L, the copper sulfate pentahydrate is at a concentration of 0.025 mg/L, the EDTA disodium salt dihydrate is at a concentration of 37.3 mg/L, the ferrous sulfate heptahydrate is at a concentration of 27.8 mg/L, the manganese sulfate monohydrate is at a concentration of 16.9 mg/L, the molybdic acid (sodium salt) is at a concentration of 0.213 mg/L, the potassium iodide is at a concentration of 0.83 mg/L, and the zinc sulphate heptahydrate is at a concentration of 8.6 mg/L.

37. The method of any of embodiments 34-36, wherein the MS medium includes the vitamins: myo-inositol, nicotinic acid, pyridoxine hydrochloride, and thiamine hydrochloride.

38. The method of embodiment 37, wherein the myo-inositol is at a concentration of 100 mg/L, the nicotinic acid is at a concentration of 0.5 mg/L, the pyridoxine hydrochloride is at a concentration of 0.5 mg/L, and the thiamine hydrochloride is at a concentration of 0.1 mg/L.

39. The method of any of embodiments 34-38, wherein the MS medium includes the amino acid glycine.

40. The method of embodiment 39, wherein the glycine is at a concentration of 2.0 mg/L. 41. The method of any of embodiments 34-40, wherein the method further includes measuring cannabinoid levels.

42. The method of embodiment 41 , wherein the measuring is by an immunoassay, ion mobility spectrometry (IMS), thin-layer chromatography (TLC), gas chromatography-flame ionization detection (GC-FID), gas chromatography-mass spectrometry (GC-MS), HPLC, HPLC-mass spectrometry, or a combination thereof.

43. The method of embodiment 41 or 42, wherein the cannabinoid levels include CBD at a yield of 13 mg CBD/kg dry weight callus to 100 mg CBD/kg dry weight callus.

44. A cannabinoid-yielding callus cell suspension produced by the method of any of embodiments 26-43.

45. The cannabinoid-yielding callus cell suspension of embodiment 44, wherein the CBD yield is at least 13 mg CBD/kg dry weight callus in one month of culture.

46. The cannabinoid-yielding callus cell suspension of embodiment 44 or 45, wherein the cannabinoid-yielding callus cell suspension is produced from a Cannabis plant of a species selected from the group consisting of Cannabis sativa, Cannabis indica and Cannabis ruderalis.

47. The cannabinoid-yielding callus cell suspension of embodiment 44 or 45, wherein the cannabinoid-yielding callus cell suspension is produced from a Cannabis plant that is a hybrid of Cannabis sativa and Cannabis indica.

48. The cannabinoid-yielding callus cell suspension of any of embodiments 44-47, wherein the cannabinoid-yielding callus cell suspension includes tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), cannabinol (CBN), or a combination thereof.

[0101] (vii) Experimental Examples.

[0102] Example 1. The following materials and methods were used for the studies described in Examples 2 and 3.

[0103] Plant material. Three Cannabis varieties (CB Dutch Treat, Swiss Dream CBD, and Mataro Blue CBD) were selected according to their CBD content and established in an indoor tent. The varieties have the characteristics as shown in Table 1.

[0104] Table 1 : Characteristics of Cannabis varieties used in Examples.

CB Dutch Treat Swiss Dream CBD Mataro Blue CBD

Type of seed Feminized Feminized Feminized

Percent Hybrid Sativa Hybrid Indica Dominant Hybrid Indica crossing Dominant Dominant

80%/20% 75%/25% Genetics Azura Haze x Mainly Indica Black Domina x

Amnesia Haze Mazari-Sharif x Blue

Monster

CBD content CBD: 7.16% CBD: 6.42% CBD: 0.10%

THC: 4.57% THC: 0.14% THC: 0.31%

CBN:0.03% CBN:0.88% CBN: 4.31%

[0105] Fifteen seeds per variety were germinated, and the first batch was placed to germinate two seeds per variety. The seeds were germinated in vitro after one week the sprouts were placed in small planters to reach a height of 15 cm and then transplanted to their final pot, where they continued their vegetation and flowering process (substrate composition 2:1 :1 ; soil, perlite, and vermiculite). The mother plants were grown under controlled conditions at a temperature of 23°C and photoperiod 16 hours light/8 hours dark in the vegetation stage and 12 hours light/12 hours dark in the flowering stage. Two fertilizations were made based on organic products to accelerate their flowering period and obtain a better inflorescence performance.

[0106] In vitro culture conditions. Preparation of culture media: To make 1 L of solid culture medium, 4.43 g of Murashige and Skoog 1972 (MS) medium were weighed with vitamins (M519) (Phytotechnology Laboratories, Lenexa, KS, USA). (M519 contains glycine (free base), 2 mg/L; myo-inositol, 100 mg/L; nicotinic acid (free acid) 0.5mg/L; pyridoxine*HCI, 0.5 mg/L; and thiamine*HCI, 0.1 mg/L.)

[0107] The powder was poured into a 1 L bottle and 30 g of sucrose was added. Distilled water was added until the final volume of 1 L was completed, and the pH of the medium was adjusted between 5.6-5.8 with 1 M KOH. Then, 7 g of Plant Agar (Phytotechnology Laboratories, LLC, Kansas, USA) for plants were added to the solution, which was finally autoclaved for 20 min at 120°C.

[0108] Preparation of culture plates with fresh medium. The preparation of petri dishes with fresh solid culture medium was carried out in a laminar flow hood, previously sterilized with technical alcohol and UV light for fifteen min. The freshly autoclaved, or melted, culture medium was cooled to 50°C, before adding the volume of hormones necessary to achieve the desired final concentration. For example, the desired final range for auxin is 0.5-2.0 mg/L, and for cytokinin, 0.5-1.0 mg/L. 50 mL of the basal medium with hormones were added for each sterile petri dish. Finally, the medium was allowed to gel under the laminar flow hood, with the lids of the plates partially open.

[0109] Callus induction and maintenance. The calluses were obtained in vitro from explants extracted from female flowers of the mother plants, which were in their optimal state after six months from sowing. Explants of different sizes between 1-2 cm² were cut and placed in the MS culture medium inside the supplemented Petri dish as described above. The cultures were grown in the dark at 22°C for four weeks. Callus induction was determined after four weeks of culture.

[0110] Subsequently, the calluses were sub-cultured every fifteen days to fresh MS culture medium supplemented with the same hormonal concentrations, to avoid the oxidation of the calluses.

[0111] Calluses were photographed under the Leyca EZBM50 stereoscopic magnifying glass.

[0112] Subculture of callus cultures. For the maintenance of the cultures in the following example and subsequent examples, the calluses are sub-cultured every four weeks. To maintain the established treatment, the following callus subculture protocol is used, which is developed in a laminar flow hood, previously sterilized with technical alcohol and UV light for fifteen min.

[0113] A grown callus is removed from the culture plate, using sterile tweezers, and deposited inside a Petri dish with medium and fresh hormones. The callus may be cut into two or more parts, according to the size of the cell mass, using a scalpel and sterile forceps. Nine to twelve units are placed per plate and then it is sealed with a parafilm sheet. This step also takes advantage of removing corns that could have been contaminated in the first crop. The plates are incubated in a plant growth chamber in darkness at 21 °C.

[0114] High performance liquid chromatography with Diode-Array Detection (HPLC-DAD) analysis of the content of cannabinoids (CBD, THC and CBN). Sample preparation (Modified methodology of LINODC, 2009): Dry samples of calluses were dried at 100°C for three hours for decarboxylation, then they were ground in a mortar until a fine powder was obtained. 200 mg of powder were weighed per sample and placed in a vial for extraction with 10 mL of methanol/chloroform solvent mixture (v/v: 9/1). The sample was placed in a sonicator for thirty minutes, with warm water. The extract was filtered in an amber vial and diluted in the same solvent solution prepared above.

[0115] The sample was taken to evaporate the solvents in a rotary evaporator and diluted in acetonitrile/water (v/v: 5/5).

[0116] Example 2. Production of cannabinoids from Cannabis callus.

[0117] As described above, the plants were obtained by germinating seeds of three varieties of Cannabis, all germinated after eight days on filter paper in a humid chamber. The root elongation and the development of the first cotyledons were from the tenth day of cultivation. The germination percentage for all varieties was 50%.

[0118] The planting and germination process of the different varieties of Cannabis are shown in the photographs of FIGs. 1A-1G. FIG. 1A shows the germination of cannabis seeds; FIG. 1 B shows a Cannabis seedling of one month transplanted to a definitive pot; and FIG. 1C shows the vegetative stage of Cannabis plants with high CBD content at two months of cultivation.

[0119] FIGs. 1 D and 1 E show, respectively, the start and end of the flowering stage under controlled conditions in an indoor marquee exclusively for Cannabis. FIG. 1 F shows the harvest of Cannabis inflorescences of Swiss Dream Variety, and FIG. 1G shows capitate stalked trichomes during a stage prior to an inflorescence maturation threshold of a Cannabis plant. The inflorescences of these same plants were used to obtain the explants for callus induction on MS culture medium and hormonal combination.

[0120] During the first and second week of cultivation the explants remained green and fresh, and at the third week the explants began to dedifferentiate as shown in FIGs. 2A-2D; in the Swiss Dream variety a faster dedifferentiation process was observed. In the fourth week, the transformation to callus mass was evident in all varieties, some more friable callus and others more compact depending on the variety, as shown in FIGs. 5A-5C, and in Table 2. The percentage of callus induction was as follows: CB Dutch Treat: 92%; Mataro Blue: 27%; and Swiss Dream: 76% (FIG. 3).

[0121] Table 2: Characteristics of induced callus according to Cannabis cultivation.

Cultivar Average fresh Average dry Color Consistency weight (g) (FW) weight (g) (DW)

Mataro Blue 1.0981 0.0610 Brown Friable

Swiss Dream 1.5565 0.0864 Green/White Friable/Compact

CB Dutch Treat 1.7043 0.0997 Green/White Friable

[0122] The cannabis strain that had a high callus induction was CB Dutch Treat with 92% after four weeks of cultivation, some of them being friable and others compact (FIGs. 5A-5C), and even many calluses had small buds originating from the cells of friable corns (somatic embryos) (FIG. 4).

[0123] The calluses of the three varieties were harvested and extracted using a modified LINODC 2009 method and analyzed by HPLC to detect the production of CBD and other cannabinoids, with the Swiss Dream variety (which accumulates a higher CBD content in the callogenic mass cells) obtaining an average of 1.1 ± 0.05 mg/Kg of CBD; Mataro Blue callus however did not produce CBD.

[0124] Chromatograms of the decarboxylated and methanol-treated extracts of mother plants, callus and cell suspensions showed the peaks coinciding with the corresponding retention times of CBD (6.42 min), CBN (9.45 min) and THC (11.77 min) confirming the presence and quantifying their composition of each sample. The contents of the CBD, THC, and CBN are shown in Tables 3-8 below, corresponding to different sample numbers.

[0125] Analysis technique: All analyses were performed according to the United Nations UNODC, “Recommended methods for the identification and analysis of Cannabis and Cannabis products”, 2009.

[0126] Table 3. Mother plant Mataro Blue, Code A-316, 0.104 g:

[0127] Table 4. Callus Mataro Blue, Code A-321, 0.115 g:

[0128] Table 5. Mother plant Swiss Dream, Code A-318, 0.116 g:

[0129] Table 6. Callus Swiss Dream, Code A-331 , 0.129 g:

[0130] Table 7. Mother plant CB Dutch Treat, Code A-441 , 6.0 g:

[0131] Table 8. Callus CB Dutch Treat, Code A-502, 1.18 g:

[0132] Other cannabinoids that also have applications in the pharmaceutical and therapeutic area were produced by this technique, which is useful for formulating different combinations of these compounds that have multiple properties against various diseases.

[0133] Table 9: Cannabinoid content in the calluses of different varieties of Cannabis sp. analyzed by HPLC-DAD.

Cannabinoid Content (mg/Kg Dry Weight (DW))

Matrix Variety CBD CBN THC

Mother Plant CB Dutch Treat 71.6±0.09 0.3±0.07 45.7±0.56

Callus CB Dutch Treat 5.8±0.04 ND ND

Mother Plant Mataro Blue 1.0±0.00 43.1±0.01 3.1±0.56

Callus Mataro Blue ND 1.4±0.01 1.1±0.01

Mother Plant Swiss Dream 64.2±0.07 8.8±0.02 1.4±0.02

Callus Swiss Dream 1.6±0.02 0.4±0.01 1.0±0.01

[0134] Callus were harvested at four weeks of growth, dried, decarboxylated, extracted with methanol and analyzed by HPLC-DAD. (Data are mean of two independent replicates ±SD). The mother plants were used as a control. ND, not detected.

[0135] In summary, this Example provides a protocol for the production of calluses and extraction of the active ingredients CBD, THC and CBN from the calluses, and the method is capable of being scaled up for production of commercially viable amounts of pharmaceutical Cannabis sp. products.

[0136] Example 3. Production of cannabinoids from Cannabis callus cell suspensions.

[0137] As an alternative to obtaining cannabinoids from callus as described in Example 2, cannabinoids can be extracted from cell suspension and sub-suspensions as described in this Example. An advantage of this method is that by suspending a callus in liquid growth medium, its cells disperse and produce a cell suspension culture characterized by faster and more uniform growth. Cell suspensions also provide a convenient method of scaling up production in larger volumes, in which the cells retain consistent exposure to medium and nutrients during growth. [0138] The plant growth and callus culture using CB Dutch Treat Variety was carried out as described above for Example 2. Then, instead of subculturing the callus, the callus was suspended in growth medium MS adding the same hormones and concentrations used in the production of calluses. Cells were harvested after four weeks, lyophilized for two days, decarboxylated and analyzed by HPLC-DAD. Standard deviation was calculated from two experiments.

[0139] Detailed methods for Example 3: White and friable portions of callus of the CB Dutch Treat variety of four weeks of growth were subcultured in fresh medium, obtaining a homogeneous callus line with repetitive measurements of cannabinoids, which was used for the establishment of cultures in cell suspension in the presence of 0.5-2 mg/L of NAA and 0.5-1 mg/L of BAP.

[0140] FIGs.6A and 6B show the image of the flasks with the cell suspensions with 28 days of growth under light and no light conditions; and FIG. 6C shows the measurement of cells in suspension of 48.085 pm in their exponential phase where they were observed healthy, large, some in aggregates and others individual; these cultures were suitable for metabolite production experiments.

[0141] The lyophilized cell suspension cultures were subjected to HPLC-DAD analysis for the presence of cannabinoids. The yield of the suspensions was 10%. The value of the total CBD content was 13.1 mg/Kg of dry weight (DW); this value was calculated from the amount of CBD- A found through the formula CBD = CBD-A x 0.877 + CBD; in the same way, the THC present was calculated THC = THC-A x 0.877 + THC, resulting in smaller amounts. The amount of CBN present was 6 mg/Kg dry weight (DW) (Table 10).

[0142] Table 10: Content of cannabinoids in the cell suspension culture of CB Dutch Treat Variety.

Cannabinoid Content (mg/Kg Dry Weight (DW))

Treatments CBD-A CBD THC-A THC CBN Incubation

Condition

Cell 15.0 13.1 1.0 0.80 6.0 Dark /23°C suspension

[0143] In conclusion for Example 3, an efficient cell suspension culture was established from friable cannabinoid-producing calli in vitro from Cannabis inflorescences (Trichomes) for the production of compounds CBD, THC, CBDA, THC-A and CBN. These results confirm the potential of Cannabis in vitro plant cell culture systems as a drug bio-factory using the present cannabinoids as active ingredients.

[0144] (viii) Closing Paragraphs. Each embodiment disclosed herein can comprise, consist essentially of, or consist of its particular stated element, step, ingredient, or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of’ limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment. A material effect would cause a statistically significant reduction in formation of Cannabis callus or callus cell suspension producing cannabinoids.

[0145] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.

[0146] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0147] The terms “a,” “an,” “the”, and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0148] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0149] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0150] Furthermore, references have been made to patents, printed publications, journal articles and other written text throughout this specification (referenced materials herein). Each of the referenced materials is individually incorporated herein by reference in their entirety for their referenced teaching.

[0151] It is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described. [0152] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and/or examples making apparent to those skilled in the art how the several forms of the invention can be embodied in practice.

[0153] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the example(s) or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3rd Edition or a dictionary known to those of ordinary skill in the art, such as the Biochemistry and Molecular Biology of Plants, 2nd Edition (Eds. Bob B. Buchanan, Wilhelm Gruissem, Russell L. Jones, John wiley & sons, 2015).

[0154] REFERENCES

Andre CM, Hausman J-Fand Guerriero G (2016) Cannabis sativa’. The Plant of the Thousand and One Molecules. Front. Plant Sci. 7:19.

Arias, M, et al., (2009). Aspectos ingenieriles del cultivo in vitro de celulas vegetales para la production de metabolites secundarios. DYNA, 76(157), 109-121.

Braemer R, and Paris M (1987). Biotransformation of cannabinoids by a cell suspension culture of Cannabis sativa L. Plant Cell Rep 6 (2): 150-152.

Braut-Boucher F, Paillard N, Phytotherapie PMe (1985) Detection of volatile products in tissue cultures of Cannabis sativa L. strains. Plantes Medicinales et Phytotherapie (1985), 19(1), 22-8. Brenneisen, R. (2007). Chemistry and analysis of phytocannabinoids and other cannabis constituents, in Marijuana and the Cannabinoids Forensic Science and Medicine, ed. M. El Sohly(NY: Humana Press), 17-49.

Dai, X., Wang, G., Yang, D. S., Tang, Y., Broun, P., and Marks, M. D. (2010). TrichOME: a comparative omics database for plant trichomes. Plant Physiol. 152, 44-54.

Farjaminezhad Reza, et al. (2013). Establishment and optimization of cell growth in suspension culture of Papaver bracteatum: a biotechnology approach for thebaine production Turk J Biol 37: 689-697

Fassio A, et al. (2013). llnidad de Comunicacion y Transferencia de Tecnologia de INIA, Editores, Canamo (Cannabis sativa L). Uruguay; 96.

Feeney, M., and Punja, Z. K. (2003). Tissue culture and Agrobacterium-mediated transformation of hemp (Cannabis sativa L.).ln vitro Cell. Dev. Biol. Plant 39, 578-585.

Fischedick, J.T., et al. (2010). Metabolic fingerprinting of Cannabis sativa L., cannabinoids and terpenoids for chemotaxonomic and drug standardization purposes. Phytochemistry 71 , 2058- 2073.

Fisse, J., and Andres, J. (1985). Organogenesis and biosynthesis in an in vitro culture of Cannabis sativa L. Boletin da Academia Galega de Ciencias 4, 57-67.

Flores-Sanchez, I. J. and Verpoorte, R. (2008). Secondary metabolism in Cannabis. Phytochem.Rev.7, 615-639.

Flores-Sanchez, I. J., Pec, J., Fei, J., Choi, Y. H., Dusek, J., & Verpoorte, R. (2009). Elicitation studies in cell suspension cultures of Cannabis sativa L. Journal of biotechnology, 143(2), 157- 168.

Furuya, T. et al. (1973). Isolation of saponins and saponigens from callus tissue of Panax ginseng. Chem. & Pharm. Bull. (Tokyo) 21 :98-101.

Happyana, N., Agnolet, S., Muntendam, R., Van Dam, A., Schneider, B., and Kayser, O. (2013). Analysis of cannabinoids in laser-microdissected trichomes of medicinal Cannabis sativa using LCMS and cryogenic NMR. Phytochemistry 87, 51-59.

Hartsei, S. C., Loh, W. T., & Robertson, L. W. (1983). Biotransformation of cannabidiol to cannabielsoin by suspension cultures of Cannabis sativa and Saccharum officinarum. Planta medica, 48(05), 17-19.

Holdcroft A, et al. (2006). A multicenter dose-escalation study of the analgesic and adverse effects of an oral Cannabis extract (Cannador) for postoperative pain management. Anesthesiology 104 (5): 10401046.

Itokawa H, et al. (1977). Biotransformation of cannabinoid precursors and related alcohols by suspension cultures of callus induced from Cannabis sativa L. Chem Pharm Bull 25 (8): 1941- 1946.

Khan, B., et al. (2014). Antibacterial properties of hemp and other natural fibre plants: a review. Bioresources 9, 3642-3659

Kittipongpatana N, et al. (1998). Production of solasodine by hairy root, callus, and cell suspension cultures of Solanum aviculare Forst. Plant Cell Tiss Org 52 (3):133-143.

Lata, H., et al. (2010). High frequency plant regeneration from leaf derived callus of high 9- tetrahydrocannabinol yielding Cannabis sativa L. PlantaMed. 76, 1629-1633.

Lata, H., et al. (2009a). Thidiazuron induced high frequency direct shoot organogenesis of Cannabis sativa L. In vitro Cell. Dev. Biol. Plant 45,12-19.

Lata, H., et al. (2009b). Propagation through alginate encapsulation of axillary buds of Cannabis sativa L. - an important medicinal plant. Physiol. Mol. Biol. Plants15, 79-86.

Lata, H., et al. (2011). Molecular analysis of genetic fidelity in Cannabis sativa L. plants grown from synthetic (encapsulated) seeds following in vitro storage. Biotechnol.Lett.33, 2503-2508.

Li, G. et al. (1999). Analysis of ephedrine in ephedra callus by acetonitrile modified capillary zone electrophoresis. Taianta 48:1023-1029.

Loh, W.H.T., et al. (1983). Tissue culture of Cannabis sativa L. and in vitro biotransformation of phenolics. Zeitschrift fuer PflanzenphysiologieW (5):395-400.

Lystvan, K, et al. (2018). Study on Betalains in Celosia cristata Linn. Callus Culture and Identification of New Malonylated Amaranthins J. Agric. Food Chem., 66, 3870-3879.

Mahlberg, P. G., and Kim, E. S. (2004). Accumulation of cannabinoids in the secretory cavity of Cannabis. J. Industr. Hemp 9, 15-36.

Mandolino G, and Ranalli P (eds) (1999). Advances in biotechnological approaches for hemp breeding and industry. Advances in hemp research. Haworth, New York.

Meijer, E.P.M., et al. (2009). The inheritance of chemical phenotype in Cannabis sativa L. (Ill): variation in cannabichromene proportion. Euphytical 65,293-311.

Moscatiello, R., et al. (2013). Plant Cell Suspension Cultures. En Maathuis, F. J. M. (Ed.). Plant Mineral Nutrients: Methods in Molecular Biology. Vol. 953. Springer.

Mustafa NR, et al. (2011). Initiation, growth and cryopreservation of plant cell suspension cultures.

Nature Protocols 6 (6):715742.

Pacifico, D.,et al. (2008). Time course of cannabinoid accumulation and chemotype development during the growth of Cannabis sativa L. Euphytica 160, 231-240.

Pec, J., et al. (2010). Metabolic analysis of elicited cells suspension cultures of Cannabis sativa L. by (1)H-NMR spectroscopy. Biotechnol. Lett. 32,935-941.

Pierik RLM (1987). In vitro culture of higher plants. Martinus Nijhoff Dordrecht, the Netherlands.

Popova EV, et al. (2015). A simple method for cryopreservation of Ginkgo biloba callus. Plant Cell Tissue Organ Cult. 97:337-43.

Radwan, M.M., et al. (2009). Biologically active cannabinoids from high-potency Cannabis sativa.

J. Nat. Prod. 72, 906-911.

Rahn EJ, and Hohmann AG (2009). Cannabinoids as pharmacotherapies for neuropathic pain:

From the bench to the bedside. Neurotherapeutics 6 (4):713-737

Remotti PC, and Loffler HJM (1995). Callus induction and plant regeneration from Gladiolus. Plant Cell Tiss Org 42 (2):171-178. Ross, S. A., et al. (2005). Flavonoid glycosides and cannabinoids from the pollen of Cannabis sativa L. Phytochem. Anal. 16,45-48.

Ross, S. A., et al. (2000). GC-MS analysis of the total 9-THC content of both drug- and fiber-type Cannabis seeds. J. Anal. Toxicol. 4, 715-717.

Russo, E. B. (2011). Taming THC: potential cannabis synergy and phytocannabinoid-terpenoid entourage effects. Br. J. Pharmacol. 163, 1344-1364.

Sen MK, et al. (2014). In vitro callus induction and plantlet regeneration of Achyranthes aspera L., a high value medicinal plant. Asian Pac J Trop Biomed 4 (1):40-46.

Sirikantaramas, S., Taura, F., Tanaka, Y., Ishikawa, Y., Morimoto, S., and Shoyama, Y. (2005). Tetrahydrocannabinolic acid synthase, the enzyme controlling marijuana psychoactivity, is secreted into the storage cavity of the glandular trichomes. Plant Cell Physiol. 46, 1578-1582.

Slusarkiewicz-Jarzina, A. II. R. E. L. I. A., Ponitka, A., & Kaczmarek, Z. (2005). Influence of cultivar, explant source and plant growth regulator on callus induction and plant regeneration of Cannabis sativa L. Acta Biol. Crac. Ser. Bot, 47, 145-151.

Stott CG, and Guy GW (2004). Cannabinoids for the pharmaceutical industry. Euphytica 140 (1- 2):83-93.

Stout, J. M., et al. (2012). The hexanoyl-CoA precursor for cannabinoid biosynthesis is formed by an acyl-activating enzymein Cannabis sativa trichomes. PlantJ.71 , 353-365.

Trejo, G., et al. (2005). Monoterpenoid Oxindole Alkaloid Production by Uncaria tomentosa (Willd) D.C. Cell Suspension Cultures in a Stirred Tank Bioreactor. Biotechnology Progress, 21(3), 786- 792.

Trejo-Tapia, G. and Rodriguez-Monroy, M. (2007). La agregacion celular en la producton de metabolites secundarios en cultivos vegetales in vitro. Interciencia, 32(10), 669-674.

LINODC (United Nations Office on Drugs and Crime), “Recommended methods for the identification and analysis of Cannabis and Cannabis products”, New York, 2009.

Wang, R., et al. (2009). A micropropagation system for cloning of hemp (Cannabis sativa L.) by shoot tipculture. Pak. J. Bot.41 , 603-608.

Whittle BA (2007). Cannabis as a medicine (benefits and risks associated with phytomedicines). J Pharm Pharmacol 59:A69-A69.

Whittle BA, and Guy GW (2004). Development of Cannabis based medicines: risk, benefit and serendipity. In: Guy GW, Robson PJ, Whittle BA (eds) The Medicinal Uses of Cannabis and Cannabinoids. Pharmaceutical Press, London, pp 427-463.

Wielgus K, et al. (2008). Estimation of Cannabis sativa L. Tissue Culture Conditions Essential for Callus Induction and Plant Regeneration. J Nat Fib 5 (3):199-207.

Claims

CLAIMS The invention claimed is:

1. A method for producing cannabinoid-yielding callus derived from a Cannabis plant, the method comprising:

(a) placing flower explants of a Cannabis plant on solid culture medium comprising at least one auxin hormone and at least one cytokinin;

(b) culturing the flower explants in the dark for four weeks to form callus; and

(c) sub-culturing the callus on the solid culture medium comprising the at least one auxin hormone and the at least one cytokinin for four weeks to produce cannabinoid-yielding callus, wherein the flower explants comprise capitate stalked trichomes during a stage prior to an inflorescence maturation threshold of the Cannabis plant, and wherein the cannabinoid-yielding callus comprises at least 5.8 mg cannabidiol (CBD)/kg dry weight callus.

2. The method of claim 1 , wherein the Cannabis plant is a species selected from the group consisting of Cannabis sativa, Cannabis indica, or Cannabis ruderalis.

3. The method of claim 1 , wherein the Cannabis plant is a hybrid of Cannabis sativa and Cannabis indica.

4. The method of claim 1 , wherein the solid culture medium comprises Murashige and Skoog medium and agar.

5. The method of claim 1 , wherein the at least one auxin hormone is 1 -naphthaleneacetic acid (NAA) in a concentration range of 0.5-2.0 mg/L.

6. The method of claim 1 , wherein the at least one cytokinin is 6-benzylaminopurine (BAP) in a concentration range of 0.5-1.0 mg/L.

7. A method for producing cannabinoid-yielding callus derived from a Cannabis plant, the method comprising:

(a) placing flower explants of a Cannabis plant on a solid culture medium comprising at least one auxin hormone and at least one cytokinin; and

(b) culturing the flower explants in the dark for two to ten weeks to produce cannabinoid-yielding callus, wherein the flower explants comprise capitate stalked trichomes during a stage prior to an inflorescence maturation threshold of the Cannabis plant.

8. The method of claim 7 wherein the culturing in the dark is for four weeks.

9. The method of claim 7, further comprising

39 (c) sub-culturing the callus on the solid culture medium comprising the at least one auxin hormone and the at least one cytokinin.

10. The method of claim 9, further comprising repeating step (c) at least every three weeks, at least every four weeks, or at least every five weeks.

11 . The method of claim 10, wherein the repeating step (c) is every four weeks.

12. The method of claim 7, wherein the Cannabis plant is a species selected from the group consisting of Cannabis sativa, Cannabis indica and Cannabis ruderalis.

13. The method of claim 7, wherein the Cannabis plant is a hybrid of Cannabis sativa and Cannabis indica.

14. The method of claim 7, wherein the solid culture medium comprises Murashige and Skoog (MS) medium and agar.

15. The method of claim 14, wherein the MS medium comprises ammonium nitrate, calcium chloride, magnesium sulphate, potassium nitrate, potassium phosphate monobasic, boric acid, cobalt chloride hexahydrate, copper sulfate pentahydrate, ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate, ferrous sulfate heptahydrate, manganese sulfate monohydrate, molybdic acid (sodium salt), potassium iodide, and zinc sulphate heptahydrate.

16. The method of claim 15, wherein the ammonium nitrate is at a concentration of 1650 mg/L, the calcium chloride is at a concentration of 332.2 mg/L, the magnesium sulphate is at a concentration of 180.69 mg/L, the potassium nitrate is at a concentration of 1900 mg/L, the potassium phosphate monobasic is at a concentration of 170 mg/L, the boric acid is at a concentration of 6.2 mg/L, the cobalt chloride hexahydrate is at a concentration of 0.025 mg/L, the copper sulfate pentahydrate is at a concentration of 0.025 mg/L, the EDTA disodium salt dihydrate is at a concentration of 37.3 mg/L, the ferrous sulfate heptahydrate is at a concentration of 27.8 mg/L, the manganese sulfate monohydrate is at a concentration of 16.9 mg/L, the molybdic acid (sodium salt) is at a concentration of 0.213 mg/L, the potassium iodide is at a concentration of 0.83 mg/L, and the zinc sulphate heptahydrate is at a concentration of 8.6 mg/L.

17. The method of claim 14, wherein the MS medium comprises the vitamins: myo-inositol, nicotinic acid, pyridoxine hydrochloride, and thiamine hydrochloride.

18. The method of claim 17, wherein the myo-inositol is at a concentration of 100 mg/L, the nicotinic acid is at a concentration of 0.5 mg/L, the pyridoxine hydrochloride is at a concentration of 0.5 mg/L, and the thiamine hydrochloride is at a concentration of 0.1 mg/L.

19. The method of claim 14, wherein the MS medium comprises the amino acid glycine.

20. The method of claim 19, wherein the glycine is at a concentration of 2.0 mg/L.

21. The method of claim 7, wherein the at least one auxin hormone is 1 -naphthaleneacetic

40 acid (NAA) in a concentration range of 0.5-2.0 mg/L.

22. The method of claim 7, wherein the at least one cytokinin is 6-benzylaminopurine (BAP) in a concentration range of 0.5-1.0 mg/L.

23. The method of claim 7, wherein the method further comprises measuring cannabinoid levels.

24. The method of claim 23, wherein the measuring is by an immunoassay, ion mobility spectrometry (IMS), thin-layer chromatography (TLC), gas chromatography-flame ionization detection (GC-FID), gas chromatography-mass spectrometry (GC-MS), HPLC, HPLC-mass spectrometry, or a combination thereof.

25. The method of claim 23, wherein the cannabinoid levels comprise CBD at a yield of at least 5.8 mg CBD/kg dry weight callus.

26. A cannabinoid-yielding callus line produced by the method of claim 7.

27. The cannabinoid-yielding callus line of claim 26, wherein the CBD yield is at least 5.8 mg CBD/kg dry weight callus in one month of culture.

28. The cannabinoid-yielding callus line of claim 26, wherein the cannabinoid-yielding callus line is produced from a Cannabis plant of a species selected from the group consisting of Cannabis sativa, Cannabis indica and Cannabis ruderalis.

29. The cannabinoid-yielding callus line of claim 26, wherein the cannabinoid-yielding callus line is produced from a Cannabis plant that is a hybrid of Cannabis sativa and Cannabis indica.

30. The cannabinoid-yielding callus line of claim 26, wherein the cannabinoid-yielding callus line comprises tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), cannabinol (CBN), or a combination thereof.

31. The cannabinoid-yielding callus line of claim 26, wherein the cannabinoid-yielding callus line does not comprise THC and/or CBN.

32. A method for producing a cannabinoid-rich callus cell suspension, said method comprising:

(a) suspending callus in liquid culture medium comprising at least one auxin hormone and at least one cytokinin; and

(b) culturing the culture in the dark for two to ten weeks, wherein the callus is obtained from culturing flower explants of a Cannabis plant, and wherein the flower explants comprise capitate stalked trichomes during a stage prior to an inflorescence maturation threshold of the Cannabis plant.

33. The method of claim 32, wherein the culturing the culture in the dark is for four weeks.

34. The method of claim 32, wherein the Cannabis plant is a species selected from the group

41 consisting of Cannabis sativa, Cannabis indica and Cannabis ruderalis.

35. The method of claim 32, wherein the Cannabis plant is a hybrid of Cannabis sativa and Cannabis indica.

36. The method of claim 32, wherein the at least one auxin hormone is 1 -naphthaleneacetic acid (NAA) in a concentration range of 0.5-2.0 mg/L.

37. The method of claim 32, wherein the at least one cytokinin is 6-benzylaminopurine (BAP) in a concentration range of 0.5-1 .0 mg/L.

38. The method of claim 32, wherein the callus was formed on solid culture medium comprising at least one auxin hormone and at least one cytokinin.

39. The method of claim 32, wherein the flower explants were cultured in the dark for four weeks to obtain the callus.

40. The method of claim 32, wherein the liquid culture medium comprises Murashige and Skoog (MS) medium.

41. The method of claim 40, wherein the MS medium comprises ammonium nitrate, calcium chloride, magnesium sulphate, potassium nitrate, potassium phosphate monobasic, boric acid, cobalt chloride hexahydrate, copper sulfate pentahydrate, ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate, ferrous sulfate heptahydrate, manganese sulfate monohydrate, molybdic acid (sodium salt), potassium iodide, and zinc sulphate heptahydrate.

42. The method of claim 41 , wherein the ammonium nitrate is at a concentration of 1650 mg/L, the calcium chloride is at a concentration of 332.2 mg/L, the magnesium sulphate is at a concentration of 180.69 mg/L, the potassium nitrate is at a concentration of 1900 mg/L, the potassium phosphate monobasic is at a concentration of 170 mg/L, the boric acid is at a concentration of 6.2 mg/L, the cobalt chloride hexahydrate is at a concentration of 0.025 mg/L, the copper sulfate pentahydrate is at a concentration of 0.025 mg/L, the EDTA disodium salt dihydrate is at a concentration of 37.3 mg/L, the ferrous sulfate heptahydrate is at a concentration of 27.8 mg/L, the manganese sulfate monohydrate is at a concentration of 16.9 mg/L, the molybdic acid (sodium salt) is at a concentration of 0.213 mg/L, the potassium iodide is at a concentration of 0.83 mg/L, and the zinc sulphate heptahydrate is at a concentration of 8.6 mg/L.

43. The method of claim 40, wherein the MS medium comprises the vitamins: myo-inositol, nicotinic acid, pyridoxine hydrochloride, and thiamine hydrochloride.

44. The method of claim 43, wherein the myo-inositol is at a concentration of 100 mg/L, the nicotinic acid is at a concentration of 0.5 mg/L, the pyridoxine hydrochloride is at a concentration of 0.5 mg/L, and the thiamine hydrochloride is at a concentration of 0.1 mg/L.

45. The method of claim 40, wherein the MS medium comprises the amino acid glycine.

46. The method of claim 45, wherein the glycine is at a concentration of 2.0 mg/L.

47. The method of claim 32, wherein the method further comprises measuring cannabinoid levels.

48. The method of claim 47, wherein the measuring is by an immunoassay, ion mobility spectrometry (IMS), thin-layer chromatography (TLC), gas chromatography-flame ionization detection (GC-FID), gas chromatography-mass spectrometry (GC-MS), HPLC, HPLC-mass spectrometry, or a combination thereof.

49. The method of claim 47, wherein the cannabinoid levels comprise CBD at a yield of 13 mg CBD/kg dry weight callus to 100 mg CBD/kg dry weight callus.

50. A cannabinoid-yielding callus cell suspension produced by the method of claim 32.

51. The cannabinoid-yielding callus cell suspension of claim 50, wherein the CBD yield is at least 13 mg CBD/kg dry weight callus in one month of culture.

52. The cannabinoid-yielding callus cell suspension of claim 50, wherein the cannabinoid- yielding callus cell suspension is produced from a Cannabis plant of a species selected from the group consisting of Cannabis sativa, Cannabis indica and Cannabis ruderalis.

53. The cannabinoid-yielding callus cell suspension of claim 50, wherein the cannabinoid- yielding callus cell suspension is produced from a Cannabis plant that is a hybrid of Cannabis sativa and Cannabis indica.

54. The cannabinoid-yielding callus cell suspension of claim 50, wherein the cannabinoid- yielding callus cell suspension comprises tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), cannabinol (CBN), or a combination thereof.