No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Accumulation of Cannabinoids in Glandular Trichomes of Cannabis (Cannabaceae)
Abstract
Sessile- and capitate-stalked secretory glands are sites of cannabinoid accumulation in Cannabis (Cannabaceae). Analyses show cannabinoids to be abundant in glands isolated from bracts or leaves of pistillate plants. Cannabinoids are concentrated in the secretory cavity formed as an intrawall cavity in the outer wall of the disc cells. Specialized plastids, lipoplasts, in the disc cells synthesize lipophilic substances, such as terpenes, that migrate through the plasma membrane and into the cell wall adjacent to the secretory cavity. These substances enter the cavity as secretory vesicles. An antibody probe for THC shows it to be most abundant along the surface of vesicles, associated with fibrillar material in the cavity, in the cell wall and in the cuticle; little THC was detected in the cytoplasm of disc or other cells. The phenol, phloroglucinol, is abundant in both gland types. A working hypothesis for the site of cannabinoid synthesis is proposed, and must be examined further. Knowledge of the mechanism of cannabinoid synthesis and localization can contribute to efforts to further reduce the THC content in hemp strains for potential agricultural use in the United States and elsewhere.
... Pistillate flowers (females) are denser and produce racemoids, clustered stalks of flowers. They are often used for medicinal applications and are ideal for the fuel industry due to their increased ability to store cannabinoids in the trichomes on their buds (Finley et al., 2022;Mahlberg and Kim, 2004) and produce seeds. ...
... In contrast, staminate flowers (males) are tall and slender, with fewer leaves around the terminal (Finley et al., 2022). They bloom earlier in the cycle (Mahlberg and Kim, 2004) and are better suited for industrial fiber production (Finley et al., 2022;Salentijn et al., 2019). Generally, hemp grows to a maximum height of 4-5 m, producing broad, dark green, serrated leaves with taper-tipped ends (Salentijn et al., 2019). ...
... Department of Energy, 2019; Jun Wei et al., 2019). Also, converting oil-bearing crops/lignocellulosic biomass to biodiesel via transesterification can also be achieved utilizing non-catalytic supercritical fluids (SCF) (Bernal et al., 2012;Mahlberg and Kim, 2004). This step aims to modify pure oil's viscosity to mimic petro-diesel's viscosity, making it compatible with current engines. ...
There has been significant research into the utilization of Cannabis sativa Linn or industrial hemp for various
applications, including as a fuel alternative such as biodiesel. The advantages of this crop offer improvements in
several current environmental concerns, such as pollutant emissions, greenhouse gas (GHG) emissions, and other
drawbacks of fossil fuels. However, despite these advantages, significant enhancements in the quality of hemp
biodiesel are necessary to consider it a sustainable alternative. Currently, inorganic nanoparticle additives, such
as zinc oxide and cerium oxide, are employed within the fuel industry to enhance the quality of current market
products. Previous studies have also suggested the potential benefits of utilizing these particles for biodiesel. This
review aims to provide detailed research into defining and understanding the background information necessary
to explore the topic of hemp biodiesel enhancements further. It introduces a novel approach for modifying hemp
biodiesel as a viable fuel alternative through zinc oxide and cerium oxide nanoparticle additives.
... Cannabis planting mode in China could be divided into three main types: outdoor planting, indoor planting, and greenhouse planting. Cannabinoids are terpene phenolic compounds produced during the growth and development of cannabis and are enriched in the hairs of the female flower glands of cannabis around the flowering stage of cannabis [13][14][15][16]. They are the active chemical components in the cannabis plant and the main components of cannabis that exert pharmacological activity. ...
... Industrial cannabis fiber has several unique characteristics: it is naturally antibacterial and has excellent ultraviolet-radiation-shielding, anti-static, and heat-resistant properties. Studies have identified and resolved more than 100 cannabinoids [15,16,24]. Among them, phenolic compounds are the main research objects, including cannabinol (CBN), cannabidiol (CBD), and cannabidivarin (CBDV) [17,18]. ...
... There are three types of cannabinoids in the study: synthetic, botanical, and endogenous cannabinoids. Botanical cannabinoids Cannabinoids are terpene phenolic compounds produced during the growth and development of cannabis and are enriched in the hairs of the female flower glands of cannabis around the flowering stage of cannabis [13][14][15][16]. They are the active chemical components in the cannabis plant and the main components of cannabis that exert pharmacological activity. ...
Cannabis (Cannabis sativa L.) is an ancient cultivated plant that contains less than 0.3% tetrahydrocannabinol (THC). It is widely utilized at home and abroad and is an economic crop with great development and utilization value. There are 31 countries legalizing industrial cannabis cultivation. Cannabis fiber has been used for textile production in China for 6000 years. China is the largest producer and exporter of cannabis. China may still play a leading role in the production of cannabis fiber. China has a long history of cannabis cultivation and rich germplasm resources. Yunnan, Heilongjiang, and Jilin are three Chinese provinces where industrial cannabis can be grown legally. Cannabinoids are terpenoid phenolic compounds produced during the growth, and which development of cannabis and are found in the glandular hairs of female flowers at anthesis. They are the active chemical components in the cannabis plant and the main components of cannabis that exert pharmacological activity. At the same time, research in China on the use of cannabis in the food industry has shown that industrial cannabis oil contains 13–20% oleic acid, 40–60% omega-6 linoleic acid, and 15–30% omega-3 α-linolenic acid. At present, more than 100 cannabinoids have been identified and analyzed in China, among which phenolic compounds are the main research objects. For instance, phenolic substances represented by cannabidiol (CBD) have rich pharmacological effects. There are still relatively little research on cannabinoids, and a comprehensive introduction to research progress in this area is needed. This paper reviews domestic and foreign research progress on cannabinoids in cannabis sativa, which is expected to support cannabis-related research and development.
... These phytochemicals, in particular delta9-tetrahydrocannabinol (THC) and cannabidiol (CBD), are synthesized from pre-cursor molecules of phenolics and terpenes by specific enzymes and accumulate within stalked glandular heads of trichomes. These trichomes are found primarily on bract tissues within female inflorescences (Fairbairn 1972;Kim and Mahlberg 1997;Mahlberg and Kim 2004), while leaves and stems bear sessile (nonstalked) glandular trichomes and nonglandular hairs (Turner et al. 1980;Small 2017). The cannabis inflorescences are comprised of individual flowers clustered together in a raceme and are harvested after 7-8 weeks of development in the flowering phase of production. ...
... Considering that trichomes are the sole sites for production of these commercially and medicinally important cannabinoids, knowledge of their development over time and the potential influence of genotype and plant age on distribution, numbers and developmental features is important to ensure consistent product quality. When glandular heads mature on trichomes, the color of the resin transitions from clear (translucent) to cloudy (milky white) and progresses to amber (brown) (Mahlberg and Kim 2004;Potter 2009). Morphological changes that may be taking place in trichomes during this maturation process have not been previously investigated. ...
... The developmental regulation of epidermal cell expansion to initiate trichome stalk formation and length are also not well understood (Huchelmann et al. 2017). In Cannabis, sessile-capitate trichomes on bracts, in which the stalk cells have not expanded, are considered to be a younger developmental stage of stalked-capitate trichomes (Hammond and Mahlberg 1977;Mahlberg and Kim 2004;Livingston et al. 2020). However, not all sessile-capitate trichomes progress to become stalkedcapitate trichomes; therefore, bract tissues generally contain varying proportions of both. ...
Background:
Glandular capitate trichomes which form on bract tissues of female inflorescences of high THC-containing Cannabis sativa L. plants are important sources of terpenes and cannabinoids. The influence of plant age and cannabis genotype on capitate trichome development, morphology, and maturation has not been extensively studied. Knowledge of the various developmental changes that occur in trichomes over time and the influence of genotype and plant age on distribution, numbers, and morphological features should lead to a better understanding of cannabis quality and consistency.
Methods:
Bract tissues of two genotypes-"Moby Dick" and "Space Queen"-were examined from 3 weeks to 8 weeks of flower development using light and scanning electron microscopy. Numbers of capitate trichomes on upper and lower bract surfaces were recorded at different positions within the inflorescence. Observations on distribution, extent of stalk formation, glandular head diameter, production of resin, and extent of dehiscence and senescence were made at various time points. The effects of post-harvesting handling and drying on trichome morphology were examined in an additional five genotypes.
Results:
Two glandular trichome types-bulbous and capitate (sessile or stalked)-were observed. Capitate trichome numbers and stalk length were significantly (P = 0.05) greater in "Space Queen" compared to "Moby Dick" at 3 and 6 weeks of flower development. Significantly more stalked-capitate trichomes were present on lower compared to upper bract surfaces at 6 weeks in both genotypes, while sessile-capitate trichomes predominated at 3 weeks. Epidermal and hypodermal cells elongated to different extents during stalk formation, producing significant variation in length (from 20 to 1100 μm). Glandular heads ranged from 40 to 110 μm in diameter. Maturation of stalked-capitate glandular heads was accompanied by a brown color development, reduced UV autofluorescence, and head senescence and dehiscence. Secreted resinous material from glandular heads appeared as droplets on the cuticular surface that caused many heads to stick together or collapse. Trichome morphology was affected by the drying process.
Conclusion:
Capitate trichome numbers, development, and degree of maturation were influenced by cannabis genotype and plant age. The observations of trichome development indicate that asynchronous formation leads to different stages of trichome maturity on bracts. Trichome stalk lengths also varied between the two genotypes selected for study as well as over time. The variability in developmental stage and maturation between genotypes can potentially lead to variation in total cannabinoid levels in final product. Post-harvest handling and drying were shown to affect trichome morphology.
... Literature data classifies glands according to the color of trichome heads and cannabinoid content, corresponding to their secretory phases, in three groups: translucent (mature), yellow (aged) and brown (senescent) glands. [12] In this study three distinct physiological maturation stages of CST (capitate-stalked trichomes) were monitored: CST t-m (translucent-milky), CST y-o (yellow-orange) and CST db (dark brown) trichome heads (Figure 1-A, B, C). The results regarding capitate-stalked trichome head coloration (% of total counted capitate-stalked colored trichomes per location and time point) are presented in Table 2. Physiological maturing classification of CST head coloration was assessed based on literature data as mentioned above. ...
... The results regarding capitate-stalked trichome head coloration (% of total counted capitate-stalked colored trichomes per location and time point) are presented in Table 2. Physiological maturing classification of CST head coloration was assessed based on literature data as mentioned above. [12,13] CST t-m had highest occurrence seven days before harvest, at all sampled positions SE -7 (56.25%), CN -7 (42.99%), ...
... [17] By comparison, stalked trichomes of cannabis have a similarly shaped, slightly larger, globose head elevated several hundreds of microns above the epidermal surface by a multicellular stalk. [12,18] Our study complies to this literature data regarding trichomes classification, with difficulties in distinguishing bulbous from sessile trichomes due to zooming limitations of stereomicroscope. ...
Objectives: This study aims at determining technological maturity (ten, seven, two days prior
and at harvest day) through analyzing morphological changes of trichomes and
phytocannabinoid content in samples from cultivated medical cannabis at three plot spots
(southeast - SE, central - CN and northwest - NW) in greenhouse (with 3600 m2
). Materials and
Methods: Soil-grown medical cannabis T-492 plant flowers were sampled within a ten days’
timeline from three plot spots of 3600 m2
greenhouse. Morphology analysis was performed
using Zeiss Stemi 508 stereomicroscope and phytocannabinoids content assessment was
performed using German pharmacopoeial method for assay of cannabinoids. Results: HPLC
analysis revealed that total THC (%) in the samples was declining from SE (13.14%), all the way
through CN (12.38%) to NW (9.14%) spot. Regarding timeline, total THC (%) was the highest in
the southeast location, starting with the highest levels on the tenth day before harvest
(SE-10- 13.14%) and then decreasing on the seventh day prior to harvest (SE-7 -11.78%), on the
second day prior harvest (SE-2-11.56%) and at the harvest day (SEh
- 10.00%). The presence of
translucent-milky, yellow-orange and dark brown trichome heads was observed. An increment
of the number of yellow-orange and dark brown colored capitate-stalked trichome heads was
connected with cannabinoid content declining. Conclusion: This research gives directions
for future conceptualization of correlational studies of cannabinoid content and changes in
trichomes and purple coloration (probably from anthocyanins) in different Cannabis cultivars.
... Recent work on the morphology of cannabis inflorescences found 442 inflorescence width to vary by more than a factor of 2 between the smallest and largest 443 inflorescences at harvest, indicating that flower growth probably offsets the drop in trichome gland 444 density we observed over the flowering period [26]. The reduction in density we observed across 445 all strains in the experiment has also been reported by manual counting of trichome gland heads Kim reported that capitate-stalked trichome glands, when viewed under a stereomicroscope, were 476 translucent during intense resin accumulation [10], and demonstrated that brown trichomes have a 477 lower THC content. The reduced THC content of brown trichomes has also been observed in dried 478 flowers [3]. ...
... The clear to milky phenotype transition could be due to a mixing 488 of immiscible molecules in the trichome gland head during resin accumulation [9,10]. As 489 cannabinoid precursors are secreted into the trichome gland head cavity by a disk of secretory cells 490 at the base of the gland, superior to the stipe cells [12,28], vesicles distribute cuticle building 491 molecules that enable the trichome head to continuously expand [10,29]. ...
... The clear to milky phenotype transition could be due to a mixing 488 of immiscible molecules in the trichome gland head during resin accumulation [9,10]. As 489 cannabinoid precursors are secreted into the trichome gland head cavity by a disk of secretory cells 490 at the base of the gland, superior to the stipe cells [12,28], vesicles distribute cuticle building 491 molecules that enable the trichome head to continuously expand [10,29]. The migration of waxy 492 cuticle precursors in vesicles into the gland head would result in an opaque gland head appearance 493 due to the varying refractive indices of hydrophilic and hydrophobic substances obscuring the 494 passage of light; thus, the visual transition from clear to milky likely indicates changes in amounts 495 of cuticle-forming precursors and changes in the internal contents of the head [12,30]. ...
Cannabis (Cannabis sativa L.) is cultivated by licensed producers in Canada for medicinal and recreational uses. The recent legalization of this plant in 2018 has resulted in rapid expansion of the industry, with greenhouse production representing the most common method of cultivation. Female cannabis plants produce inflorescences that contain bracts densely covered by glandular trichomes, which synthesize a range of commercially important cannabinoids (e.g., THC, CBD) as well as terpenes. Cannabinoid content and quality varies over the 8-week flowering period to such an extent that the time of harvest can significantly impact product quality. Cannabis flower maturation is accompanied by a transition in the color of trichome heads that progresses from clear to milky to brown (amber) and can be seen visually using low magnification. However, the importance of this transition as it impacts quality and describes maturity has never been investigated. To establish a relationship between trichome maturation and trichome head color changes (phenotype), we developed a novel automatic trichome gland analysis pipeline using deep learning. We first collected a macro-photography dataset based on 4 commercially grown cannabis strains, namely 'Afghan Kush', 'Green Death Bubba', 'Pink Kush', and 'White Rhino'. Images were obtained in two modalities: conventional macroscopic light photography and macroscopic UV induced fluorescence. We then implemented a pipeline where the clear-milky-brown heuristic was injected into the algorithm to quantify trichome phenotype progression during the 8-week flowering period. A series of clear, milky, and brown phenotype curves were recorded for each strain over the flowering period that were validated as indicators of trichome maturation and corresponded to previously described parameters of trichome development, such as trichome gland head diameter and stalk elongation. We also derived morphological metrics describing trichome gland geometry from deep learning segmentation predictions that profiled trichome maturation over the flowering period. We observed that mature and senescing trichomes displayed fluorescent properties that were reflected in the clear, milky, and brown phenotypes. Our method was validated by two experiments where factors affecting trichome quality and flower development were imposed and the effects were then quantified using the deep learning pipeline. Our results indicate the feasibility of automated trichome analysis as a method to evaluate the maturation of female flowers cultivated in a highly variable environment, regardless of strain. These findings have broad applicability in a growing industry in which cannabis flower quality is receiving increased circumspection for medicinal and recreational uses.
... These contributions laid the groundwork for the understanding of cannabinoid synthesis and storage in planta. Mahlberg and Kim [9] further characterized the formation of the trichome gland cuticle and proposed a working model of cannabinoid biosynthesis, hypothesizing that tetrahydrocannabinolic acid (THCA) is formed outside of plant cells in the storage cavity of the trichome gland [10]. Sirikantaramas et al. [11] confirmed this hypothesis by localizing the enzymes necessary for cannabinoid synthesis to the storage cavity, determining that cannabinoids are formed from a ring of secretory cells. ...
... The color transition of trichome heads from clear to milky to brown is often used to manually approximate the stage of maturation, with milky representing the optimal state and brown indicating overmaturation [10,13]. However, the relationship between trichome head color and cannabis flower development has not been previously investigated. ...
... Cannabis samples acquired from law enforcement and manually assessed for trichome browning on a linear scale had a lower THCA and increased cannabinolic acid (CBNA) content compared to cannabis samples with clear or milky trichomes [3]. Reduced THC content in senescent trichomes glands was also reported by Mahlberg and Kim [10]. There is currently a lack of prior reports describing trichome gland phenotype in relationship to cannabis flower development in situ in cannabis. ...
... The highest contents of cannabinoids and terpenoids are found in the glandular trichomes on cannabis bracts. The highest density of glandular hairs is found on the bract surrounding each female cannabis flower and the subtending leaflets of the female inflorescence [80,81]. ...
... Cannabinoids are produced in the sessile and stalked trichomes of C. sativa plants [80,81]. Trichomes are particularly abundant on the inflorescences of the plant, present in a lower number on leaves, petioles and stems, and absent on the roots and seeds. ...
The phytochemistry of fibre hemp (Cannabis sativa L., cv. Futura 75 and Felina 32) cultivated in Lithuania was investigated. The soil characteristics (conductivity, pH and major elements) of the cultivation field were determined. The chemical composition of hemp extracts and essential oils (EOs) from different plant parts was determined by the HPLC/DAD/TOF and GC/MS techniques. Among the major constituents, β-caryophyllene (≤46.64%) and its oxide (≤14.53%), α-pinene (≤20.25%) or α-humulene (≤11.48) were determined in EOs. Cannabidiol (CBD) was a predominant compound (≤64.56%) among the volatile constituents of the methanolic extracts of hemp leaves and inflorescences. Appreciable quantities of 2-monolinolein (11.31%), methyl eicosatetraenoate (9.70%) and γ-sitosterol (8.99%) were detected in hemp seed extracts. The octadecenyl ester of hexadecenoic acid (≤31.27%), friedelan-3-one (≤21.49%), dihydrobenzofuran (≤17.07%) and γ-sitosterol (14.03%) were major constituents of the methanolic extracts of hemp roots, collected during various growth stages. The CBD quantity was the highest in hemp flower extracts in pentane (32.73%). The amounts of cannabidiolic acid (CBDA) were up to 24.21% in hemp leaf extracts. The total content of tetrahydrocannabinol (THC) isomers was the highest in hemp flower pentane extracts (≤22.43%). The total phenolic content (TPC) varied from 187.9 to 924.7 (average means, mg/L of gallic acid equivalent (GAE)) in aqueous unshelled hemp seed and flower extracts, respectively. The TPC was determined to be up to 321.0 (mg/L GAE) in root extracts. The antioxidant activity (AA) of hemp extracts and Eos was tested by the spectrophotometric DPPH● scavenging activity method. The highest AA was recorded for hemp leaf EOs (from 15.034 to 35.036 mmol/L, TROLOX equivalent). In the case of roots, the highest AA (1.556 mmol/L, TROLOX) was found in the extracts of roots collected at the seed maturation stage. The electrochemical (cyclic and square wave voltammetry) assays correlated with the TPC. The hydrogen-peroxide-scavenging activity of extracts was independent of the TPC.
... In previous literature contributions, glandular trichomes of various morphotypes have been defined under diverse, controversial terms over time [6,9,[59][60][61]. Therefore, an update of trichome terminology would be highly desirable to redefine the gland morphotypes. ...
... They invariably co-occurred on the inflorescence axis, on the bracts and especially on bracteoles in all the investigated varieties. The second group was bulbous, with a uni-or bicellular head, a short, biseriate stalk and a two-foot cell lying at the level of the epidermis [59]. We recorded their distributions on the surfaces of all the examined plant parts. ...
New hemp (Cannabis sativa L.) strains developed by crossbreeding selected varieties represent a novel research topic worthy of attention and investigation. This study focused on the phytochemical characterization of nine hemp commercial cultivars. Hydrodistillation was performed in order to collect the essential oils (EO), and also the residual water and deterpenated biomass. The volatile fraction was analyzed by GC-FID, GC-MS, and SPME-GC-MS, revealing three main chemotypes. The polyphenolic profile was studied in the residual water and deterpenated biomass by spectrophotometric assays, and HPLC-DAD-MSn and 1H-NMR analyses. The latter were employed for quali–quantitative determination of cannabinoids in the deterpenated material in comparison with the one not subjected to hydrodistillation. In addition, the glandular and non-glandular indumentum of the nine commercial varieties was studied by means of light microscopy and scanning electron microscopy in the attempt to find a possible correlation with the phytochemical and morphological traits. The EO and residual water were found to be rich in monoterpene and sesquiterpene hydrocarbons, and flavonol glycosides, respectively, while the deterpenated material was found to be a source of neutral cannabinoids. The micromorphological survey allowed us to partly associate the phytochemistry of these varieties with the hair morphotypes. This research sheds light on the valorization of different products from the hydrodistillation of hemp varieties, namely, essential oil, residual water, and deterpenated biomass, which proved to be worthy of exploitation in industrial and health applications.
... There are three recognized species of Cannabis (e.g., Cannabis sativa, Cannabis indica and Cannabis ruderalis) and more than 700 strains (Medical News Today, 2021). Cannabis has more than 400 bioactive components, which majorly include cannabinoids (or phytocannabinoids), polyphenols, flavonoids, terpenes, terpenoids, fatty acids, oils and waxes (Ashton, 2001;Mahlberg and Kim, 2004). The significant cannabinoids, which are researched for the assessment of therapeutic activities, are known as tetrahydrocannabinol, cannabidiol, cannabinol and their carboxylic acid derivatives. ...
... However, certain solvent-free extraction processes are also available to extract cannabinoids. Cannabinoids are majorly amassed in the glandular trichome present on the leafy part of the Cannabis plant where they are protected from the outer atmosphere by the waxy layer (Mahlberg and Kim, 2004). Hence, it is comparatively easier to extract the cannabinoids from glandular trichomes by applying mechanical forces by pressing and heating without taking any organic solvents. ...
Cannabis, a genus of perennial indigenous plants is well known for its recreational and medicinal activities. Cannabis and its derivatives have potential therapeutic activities to treat epilepsy, anxiety, depression, tumors, cancer, Alzheimer's disease, Parkinson's disease, to name a few. This article reviews some recent literature on the bioactive constituents of Cannabis, commonly known as phytocannabinoids, their interactions with the different cannabinoids and non-cannabinoid receptors as well as the significances of these interactions in treating various diseases and syndromes. The biochemistry of some notable cannabinoids such as tetrahydrocannabinol, cannabidiol, cannabinol, cannabigerol, cannabichromene and their carboxylic acid derivatives is explained in the context of therapeutic activities. The medicinal features of Cannabis-derived terpenes are elucidated for treating several neuro and non-neuro disorders. Different extraction techniques to recover cannabinoids are systematically discussed. Besides the medicinal activities, the traditional and recreational utilities of Cannabis and its derivatives are presented. A brief note on the legalization of Cannabis-derived products is provided. This review provides comprehensive knowledge about the medicinal properties, recreational usage, extraction techniques, legalization and some prospects of cannabinoids and terpenes extracted from Cannabis.
... According to Mahlberg and Kim [41], cannabinoids are formed through the condensation of terpene and phenol precursors. It has been reported that phenols are transported as glycosides to the vacuole, where their aglycone component enters the cell. ...
In this work, two extraction techniques, conventional and ultrasound-assisted extraction (UAE) techniques, were employed for the extraction of natural bioactive compounds (NBCs) from the areal parts of industrial hemp (Cannabis sativa L. cv. Helena) at two harvesting stages: (i) the beginning of flowering and (ii) the full flowering of the hemp plants. In the conventional extraction, the effect of different extraction solvents on the extraction yield and the content of NBCs was examined. The extraction temperature, extraction time, and ultrasonic power were chosen for the process parameters in UAE. The highest value of the investigated responses in UAE-obtained extracts was higher compared to extract obtained with conventional extraction techniques when the same solvent was used (50% ethanol): extraction yield (17.54 compared to 15.28%), content of total phenols and total flavonoids (1.7795 compared to 1.0476 mg GAE/mL and 0.6749 compared to 0.3564 mg CE/mL, respectively) and cannabidiol (0.8752 compared to 0.4310 mg/mL). Comparing the plant material in different developmental stages, it can be concluded that hemp aerial parts at the beginning of the flowering stage represent a good source of the phenolic compound with sinapic acid and apigenin being dominant, while hemp aerial parts in the full flowering stage represent a good source of cannabinoids.
... Female flowers of cannabis (Cannabis sativa L.) possess a proliferous number of 'peltate' stalked glandular trichomes (GTs) on their surface [1][2][3]. These defensive structures develop predominantly in conjunction with female floral tissue and provide protection [4][5][6] by producing and storing large amounts of secondary metabolites, which include cannabinoids [1,7]. These metabolites are particularly interesting due to their medicinal properties, acting via the human endocannabinoid and central nervous systems [8,9]. ...
Cannabis (Cannabis sativa L.) flower glandular trichomes (GTs) are the main site of cannabinoid synthesis. Phytohormones, such as jasmonic acid (JA) and salicylic acid (SA), have been shown to increase cannabinoid content in cannabis flowers, but how this is regulated remains unknown. This study aimed to understand which biological processes in GT disc cells phytohormones control by using an in vitro assay. Live GT disc cells were isolated from a high-tetrahydrocannabinol cannabis cultivar and incubated on basal media plates supplemented with either kinetin (KIN), JA, SA, abscisic acid, ethephon, gibberellic acid, brassinolide, or sodium diethyldithiocarbamate. Quantitative proteomic analysis revealed that KIN, JA, and SA caused the greatest number of changes in the GT disc cell proteome. Surprisingly, none of the treatments concertedly increased cannabinoid content or the abundance of related biosynthetic proteins in the GT, suggesting that cannabinoid increases in previous in planta phytohormone studies are likely due to other processes, such as increased GT density. As well, KIN-, JA-, and SA-treated GTs had numerous differentially abundant proteins in common. Several were key proteins for leucoplast differentiation, cuticular wax and fatty acid metabolism, and primary metabolism regulation, denoting that cytokinin, JA, and SA signalling are likely important for coordinating cannabis GT differentiation and development.
... The glandular trichomes achieve maturation over a 7-8 week period during Cannabis flower development and presumably contain the highest content of specialized metabolites (Sutton et al., 2023). Depending on their color, hemp glandular trichomes show different secretory phases: the mature secreting gland appears translucent, while aging glands are yellow and senescing brown (Mahlberg & Kim, 2004). ...
... The key medicinal compounds produced by C. sativa are synthesised in specialised epidermal cells, glandular trichomes; the function and structure of which has been previously described [17]. As trichomes are the cell type responsible for the production of cannabinoids [18], increasing the size and/or density or altering the secondary metabolite composition of the trichomes represent potential strategies for increasing overall yield of medicinally valuable secondary metabolite end products. ...
Cannabis sativa (C. sativa L.) has garnered significant attention worldwide due to its widespread use as a pharmaceutical agent. With the increasing clinical application of C. sativa and cannabinoid therapeutics, there is strong interest in the development of superior plant varieties and optimisation of growth conditions to enhance secondary metabolite yield. Our RNA sequencing analysis revealed differential expression of hormone-related transcripts in developing C. sativa trichomes, suggesting the involvement of hormone signalling pathways in cannabinoid production. Leveraging the potency of exogenous hormones on plants, this study sought to determine if the application of cytokinin (CK), gibberellic acid (GA) and jasmonic acid (JA) modified trichome morphology and the cannabinoid profile over an 8-week period following the induction of flowering. Exogenous hormone application led to alterations in trichome morphology, with each treatment significantly reducing trichome head width by the final week of assessment. Interestingly, GA application also resulted in a significant reduction in the concentration of Δ-9-tetrahydrocannabinol (THC), Δ-9-tetrahydrocannabinolic acid (THCA), cannabidiol (CBD) and cannabidiolic acid (CBDA) by week 8 post floral induction, however, JA and CK treatment did not consistently modulate the accumulation of these cannabinoids. The minor cannabinoids, cannabidivaranic acid (CBDVA), cannabicyclolic acid (CBLA), cannabicyclol (CBL), cannabichromene (CBC), cannabigerolic acid (CBGA) and cannabigerol (CBG), were also affected by hormone treatments, with varying degrees of accumulation observed. These findings underscore the intricate interplay between phytohormones and secondary metabolite biosynthesis in C. sativa. Our study highlights the potential of hormone modulation as a strategy to enhance cannabinoid yield and offers some insights into the regulatory mechanisms governing cannabinoid biosynthesis in C. sativa trichomes.
... The pharmacologically active cannabinoids (e.g., THC/CBD) are formed when cannabis is heated to a temperature of at least 180°C resulting in 'decarboxylation'. With the use of a vaporizer, the active cannabinoids are released from the glandular trichomes in a vapour at 230°C which can then be inhaled into the lungs (Mahlberg and Kim, 2004). In Cannabis sativa, cannabinoids are biosynthesized as phytoprotectants; in fresh biomass, 95% of the THC, CBD, and CBC exist as their acidic parents: tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), and cannabichromenic acid (CBCA). ...
Cannabis is now one of the most thoroughly studied and analyzed plant materials. More than 100 cannabinoids have been isolated and identified in cannabis along with the primary psychoactive component, Δ9-tetrahydrocannabinol (Δ9-THC). In addition to Δ9-THC, there are other components of cannabis that are medically beneficial. For example, cannabidiol (CBD) and cannabigerol (CBG) can moderate or influence the psychoactive effects of Δ9-THC. The raw cannabis plant consists of cannabinoids in their acidic form. When someone states that cannabinoids are in their "acidic form", they are referring to the chemical structure of the compound itself. A cannabinoid in its acidic form has a carboxyl group (-COOH) attached. While tetrahydrocannabinolic acid (THCA) is the non-psychoactive precursor to THC, it does not bind to the CB1 and CB2 receptors. Instead, it binds with other cannabinoids receptors in the endocannabinoid system. When THC is not decarboxylated, it is THCA. Although THCA possesses therapeutic effects, like anti-inflammatory and neuroprotective qualities, it is not in its most beneficial or psychoactive form. Decarboxylation is a chemical reaction that removes a carboxyl group (-COOH) and releases carbon dioxide (CO2). The two main catalysts for decarboxylation to occur are heat and time. High CBD strains tend to decarboxylate a bit slower than those with high THC content. Decarboxylate high CBD strains by baking them for 15-20 minutes at 149°C and decarboxylate high THC strains by baking them for 10-18 minutes at the same temperature (149°C) in the oven. Full decarboxylation may require more time to occur. It is important to keep tight temperature control applying cannabis to various technological applications. While heat is needed to decarboxylate the acids into the active form of cannabinoids our bodies can use, extreme temperatures can destroy many of the important plant materials that contribute to positive health outcomes, like terpenes.
... An ethephon dose of 840 µM m − 1 resulted in a lower CBG concentration than the 1240 µM m − 1 dose, indicating an inconsistent CBG response to the different ethephon doses. Cannabinoids are synthesized from terpene and phenol precursor geranyl pyrophosphate (Mahlberg and Eun, 2004). There are studies demonstrating a close relationship between cannabinoid and terpene concentrations in cannabis. ...
In pharmaceutical cannabis (Cannabis sativa L.) crops sex distribution plays a pivotal role in influencing the yield and quality of inflorescences. Male plants or inflorescences do not produce sufficient concentrations of key bioactive compounds, including cannabinoids and terpenes; moreover, pollination significantly reduces the quality of female inflorescences. Existing studies suggest that ethephon may be effective in feminizing hemp crops and altering their canna-binoid content. However, comprehensive research is essential to fully understand ethephon's specific effects on hemp. This study, incorporating a two-year field trial, was conducted to assess the effects of ethylene-releasing ethephon on sex expression and the concentrations of main cannabinoids in monoecious, dioecious, cannabi-diolic, and cannabigerolic hemp varieties. Aqueous ethephon solutions of 420, 840, 1240, and 1660 µM m − 1 were administered biweekly via foliar spraying during the flowering period. The trial incorporated three varieties of hemp: Felina 32 (monoecious, cannabidiol dominant chemotype), Santhica 27 (monoecious, cannabigerol dominant chemotype), and Kompolti (dioecious, cannabidiol dominant chemotype). All treatments significantly (p < 0.05) increased the number of fully female phenotypes and reduced the number of monoecious phenotypes in monoecious varieties Felina 32 and Santhica 27. Almost all but a few phenotypes turned into 100% female flower phenotypes. However, these treatments did not produce feminizing effects on either 100% male phenotypes in monoecious varieties or male plants in the dioecious variety Kompolti. Almost all concentrations of ethephon significantly (p < 0.05) altered the content of dominant cannabinoids in the inflorescences of Felina 32, Kompolti, and Santhica 27 varieties. However, the effect of the 840 µM treatment on the Santhica 27 variety was not statistically significant. The alteration in cannabinoid production did not correlate with the increased number of female plants. All treatments significantly (p < 0.05) decreased the height of plants in all varieties.
... The trichomes are usually invisible to the naked eye and are divided into three types based on their structural appearances on SEM, such as capitate-sessile, capitate-stalked, and bulbous (Hammond and Mahlberg, 1973). In this study, the visualized trichomes had large globular heads attached above the epidermis on the large stalks and are referred to as capitate-stalked trichomes (or stalked trichomes) (Livingston et al., 2020;Mahlberg and Kim, 2004;Tanney et al., 2021). SEM analysis (Fig. 7) showed that the general structure of cannabis inflorescences was significantly impacted by different drying methods and conditions. ...
Conventional cannabis drying is time-consuming and energy intensive. A quick and dependable drying of cannabis is essential to ensure high-quality products to meet increasing demands. This study explored a combined microwave and infrared (MI) drying on cannabis comparing with control environmental (CE) drying. MI was very efficient with a short drying time of 16-200 min, high moisture diffusivity of 7.95×10 − 09-8.70×10 − 08 m 2 /s, and low energy consumption of 390.49-1611.42 kJ. The Page and Modified Page drying models fitted well to describe and predict the MI drying characteristics of cannabis. Microstructural images identified shrinkage in glandular trichomes of cannabis, whereas colorimetric assessment resulted in alteration of color attributes due to MI drying. It also facilitated the conversion of acidic cannabinoids to their neutral forms by decreasing tetra-hydrocannabinolic acid (THCA) in g/g of dry matter from 20.15% to 7.57% and increasing tetrahydrocannabinol (THC) from 6.31% to 16.65% that insignificantly (p≥0.05) affected the total THC level. MI drying resulted in a total terpenes concentration (%g/g of dry matters) of 0.541-0.730, insignificantly lower than CE drying (0.768). Overall, the study highlights MI as a rapid and energy-efficient drying for obtaining high quality cannabis, particularly for medicinal applications.
... The shift from a transparent (translucent) initial state to an opaque (milky white) appearance, and, eventually, to an amber (brown) shade, indicates the synthesis and accumulation of cannabinoids and other resinous compounds within the glandular trichomes. This alteration in resin color could signify the maturation and chemical transformation of secondary metabolites, including cannabinoids and terpenes, within the trichomes [13,15,16]. These compounds play crucial roles in the biological and therapeutic properties of cannabis [13]. ...
This study extensively characterizes the morphological characteristics, including the leaf morphology, plant structure, flower development, and trichome features throughout the entire life cycle of Cannabis sativa L. cv. White Widow. The developmental responses to photoperiodic variations were investigated from germination to mature plant senescence. The leaf morphology showed a progression of complexity, beginning with serrations in the 1st true leaves, until the emergence of nine leaflets in the 6th true leaves, followed by a distinct shift to eight, then seven leaflets with the 14th and 15th true leaves, respectively. Thereafter, the leaf complexity decreased, culminating in the emergence of a single leaflet from the 25th node. The leaf area peaked with the 12th leaves, which coincided with a change from opposite to alternate phyllotaxy. The stipule development at nodes 5 and 6 signified the vegetative phase, followed by bract and solitary flower development emerging in nodes 7-12, signifying the reproductive phase. The subsequent induction of short-day photoperiod triggered the formation of apical inflorescence. Mature flowers displayed abundant glandular trichomes on perigonal bracts, with stigma color changing from whitish-yellow to reddish-brown. A pronounced increase in trichome density was evident, particularly on the abaxial bract surface, following the onset of flowering. The trichomes exhibited simultaneous growth in stalk length and glandular head diameter and pronounced shifts in color. Hermaphroditism occurred well after the general harvest date. This comprehensive study documents the intricate photoperiod-driven morphological changes throughout the complete lifecycle of Cannabis sativa L. cv. White Widow. The developmental responses characterized provide valuable insights for industrial and research applications.
... Female cannabis inflorescences are the primary source of cannabinoids and terpenes (Ohlsson et al., 1971), with stalked glandular trichomes on the surface of floral organs and bracts being the key secretory structures (Mahlberg and Eun, 2004;Livingston et al., 2020). Recent work has used a deep learning pipeline to identify stages of trichome development based on their age-based transition through clear-milky-brown phenotypes, providing a sophisticated tool for cannabis product investigation (Sutton et al., 2023). ...
Cannabis sativa remains under heavy legal restriction around the globe that prevents extensive investigations into agricultural applications for improving its development. This work investigates the potential of specific plant growth-promoting rhizobacteria (PGPR) to improve Cannabis cannabinoid yield through increased trichome densities on floral organs, and to determine if sub-optimal environmental conditions would affect the outcomes of PGPR presence by altering plant development and cannabinoid profiles. Here, Pseudomonas sp. or Bacillus sp. were applied to the root system either separately or in a consortium to determine the effect of this bacterial treatment on the density of stalked glandular trichomes. Further, a low nutrient regime was applied for the first half of plant development to determine if an environmental stressor interacts with the effects of the microbial treatments on stalked trichome densities. Following 8 weeks of flower development, trichome density on calyces and bracts of inflorescences were determined using microscopy. Our findings unexpectedly indicate that recommended nutrient levels were linked to a decreasing trend in trichome densities with PGPR inoculations, but a low nutrient regime coupled with PGPR treatment increased them. Cannabinoid content is partially consistent with these results, in that a low nutrient regime increased the abundance of key cannabinoids compared to recommended regimes, with Bacillus sp. inoculation linked to the greatest number of significant changes between the two nutrient regimes. Overall, this work provides insight into how PGPR presence affects Cannabis stalked trichome development and cannabinoid profiles, and how environmental stressors can affect, and even enhance, trichome densities and influence major cannabinoid production, thereby pointing towards avenues for reducing the reliance on synthetic fertilizers during plant production without compromising yield.
... Drinic et al. [51], described the extraction of adult plants with 50 % ethanol provides the best performance for important substances in a group of cannabinoid compounds (5.21 as mg of catechin equivalent per g dry weight hemp (mg CE/g dw)). Terpenes and phenols condense to become cannabinoids in the glands that cover the plant's above-ground portion [52]. The extraction in this experiment was done with ethanol solvent which highly influenced the total flavonoid content. ...
This study investigated the formulation of a product prototype RMUTI-SKC by using the legal part of cannabis plants (leaves). Five product prototypes were composed of cannabis (leaves), stevia and mint. The herbs were added to improve the taste, smell and physical appearance properties of the product prototype which was yellowish-green. The analysis of the chemical properties found no heavy metals (Cd, As, and Pb) were observed (based on THAI community product standard, and demonstrated a moderate antioxidant activity, total phenolic content and total flavonoid content. The GC-MS profile of crude extracts analysis revealed the phytochemicals based on pharmacological action of essential oil, flavoring substance, anti-inflammatory, antioxidant, anti-bacterial, antidiabetic, antifungal and anti-cancer, respectively. The experimental results showed the pharmacological properties of the product prototype which can be a guideline for applying cannabis plants to other products. Further study on pathogen testing and product aging should be carried out to satisfy the quality of production. HIGHLIGHTS The formulation of a product prototype RMUTI-SKC by using the legal part of cannabis plants (leaves). The analysis of the chemical properties found no heavy metals (Cd, As and Pb) were observed (based on THAI community product standard, and demonstrated a moderate antioxidant activity, total phenolic content and total flavonoid content. GRAPHICAL ABSTRACT
... The copyright holder for this preprint (which this version posted April 18, 2023. ; https://doi.org/10.1101/2023.04.17.537043 doi: bioRxiv preprint on female plants, contain high densities of structures known as glandular trichomes within which 60 cannabinoids are produced (Mahlberg and Kim, 2004). At least 150 cannabinoids have been 61 discovered with potentially different medicinal or psychoactive functions (Hanuš et al., 2016), of 62 which cannabidiol (CBD), THC, and cannabigerol (CBG) are produced in the greatest quantities. ...
Hemp ( Cannabis sativa L.) is a primarily short-day crop grown for grain, fiber, and secondary metabolites. Although terminal flowering of most non-domesticated hemp is regarded as photoperiod sensitive, limited germplasm is available for the development of day-neutral hemp. Day-neutral plants experience flower maturation independently of the short-day photoperiod cue which is typically triggered by photoperiods of less than 14 hours. The day-neutral trait is the subject of increased commercial interest for the purpose of breeding varieties with accelerated flowering time or cultivars that do not require labor- and cost-intensive light deprivation production systems. Genetic markers for the photoperiodic response would help breeders make early selection in the design of suitable cultivars for specific environments and cultivation calendars. Limited refereed information has been produced regarding the genetic regulation of photoperiodism in C. sativa . A population of 318 F2 individuals segregating for day-neutral flowering was developed, phenotyped, and genotyped. Genome-wide association analysis identified markers associated with the day-neutral trait indicating that a single recessive gene controlling photoperiod sensitivity is located within a large region of Chromosome 1. Flanking region sequence data of linked SNP markers was used in the development and validation of a TaqMan-based qPCR assay for the day-neutral trait. A genetic linkage map was produced, and QTL mapping identified two additional markers on Chromosome 1. Candidate genes, TARGET OF EARLY ACTIVATION TAGGED (TOE)/APETALA2 (AP2) , and PSEUDO-RESPONSE REGULATOR 3 (PRR3) , may work together to impact phase transition and photoperiodic flowering respectively and have key domains that are disrupted in day-neutral plants.
... Considerable literature describes the sites of cannabinoid biosynthesis in Cannabis plants [32][33][34][35]. Cannabinoids and terpenes accumulate in the glandular trichomes of the plants; in particular, female flowers show the highest density of glandular trichomes. ...
Cannabis (Cannabis sativa L.) is widely cultivated and studied for its psychoactive and medicinal properties. As the major cannabinoids are present in acidic forms in Cannabis plants, non-enzymatic processes, such as decarboxylation, are crucial for their conversion to neutral active cannabinoid forms. Herein, we detected the levels of cannabidivarin (CBDV), cannabidiol (CBD), cannabichromene (CBC), and Δ9-tetrahydrocannabinol (Δ9-THC) in the leaves and vegetative shoots of five commercial Cannabis cultivars using a combination of relatively simple extraction, decarboxylation, and high-performance liquid chromatography analyses. The CBDV, CBC, and Δ9-THC levels were 6.3–114.9, 34.4–187.2, and 57.6–407.4 μg/g, respectively, and the CBD levels were the highest, ranging between 1.2–8.9 μg/g in leaf and vegetative shoot tissues of Cannabis cultivars. Additionally, correlations were observed between cannabinoid accumulation and transcription levels of genes encoding key enzymes for cannabinoid biosynthesis, including CsCBGAS, CsCBDAS, CsCBCAS, and CsTHCAS. These data suggest that the high accumulation of cannabinoids, such as CBC, Δ9-THC, and CBD, might be derived from the transcriptional regulation of CsCBGAS and CsCBDAS in Cannabis plants.
... In C. sativa, PCs and most terpenes accumulate primarily in the secretory cavity of capitate stalked glandular trichomes (Mahlberg and Eun, 2004;Sirikantaramas et al., 2004). Trichomes are plant hairs of epidermal origin that cover the leaves, bracts, stems and floral organs and are the first line of defence against a/biotic stresses acting as both a physical and chemical barrier. ...
Cannabis sativa L. has been known for at least 2000 years as a source of important, medically significant specialised metabolites and several bio-active molecules have been enriched from multiple chemotypes. However, due to the many levels of complexity in both the commercial cultivation of cannabis and extraction of its specialised metabolites, several heterologous production approaches are being pursued in parallel. In this review, we outline the recent achievements in engineering strategies used for heterologous production of cannabinoids, terpenes and flavonoids along with their strength and weakness. We provide an overview of the specialised metabolism pathway in C. sativa and a comprehensive list of the specialised metabolites produced along with their medicinal significance. We highlight cannabinoid-like molecules produced by other species. We discuss the key biosynthetic enzymes and their heterologous production using various hosts such as microbial and eukaryotic systems. A brief discussion on complementary production strategies using co-culturing and cell-free systems is described. Various approaches to optimise specialised metabolite production through co-expression, enzyme engineering and pathway engineering are discussed. We derive insights from recent advances in metabolic engineering of hosts with improved precursor supply and suggest their application for the production of C. sativa speciality metabolites. We present a collation of non-conventional hosts with speciality traits that can improve the feasibility of commercial heterologous production of cannabis-based specialised metabolites. We provide a perspective of emerging research in synthetic biology, allied analytical techniques and plant heterologous platforms as focus areas for heterologous production of cannabis specialised metabolites in the future.
... There are five types of trichomes on Cannabis sativa L., three of them are glandular trichomes, namely capitate-stalked, capitate-sessile, and bulbous trichomes (Dayanandan and Kaufman, 1976;Happyana et al., 2013). The glandular trichomes are as the main production and storage site to terpenes, and cannabinoids which is famous for their psychoactive and therapeutic effects (Kim and Mahlberg, 1991;Mahlberg and Kim, 2004). Two types of trichomes are described on the leaves of tobacco, the short type may secret hydrophilic nicotine, while the long type produces a resin containing diterpene cembratrienediol (Meyberg et al., 1991;Amme et al., 2005). ...
Trichomes, which are classified as glandular or non-glandular, are hair-like epidermal structures that are present on aerial parts of most plant species. Glandular secretory trichomes (GSTs) have the capacity to secrete and store specialized metabolites, which are widely used as natural pesticides, food additives, fragrance ingredients or pharmaceuticals. Isolating individual trichomes is an essential way for identifying trichome-specific gene functions and discovering novel metabolites. However, the isolation of trichomes is difficult and time-consuming. Here, we report a method to isolate the GSTs from leaf epidermis dispense with fixation using laser capture microdissection (LCM). In this study, 150 GSTs were captured efficiently from Artemisia annua leaves and enriched for artemisinin measurement. UPLC analysis of microdissected samples indicated specific accumulation of secondary metabolites could be detected from a small number of GSTs. In addition, qRT-PCR revealed that the GST-specific structural genes involved in artemisinin biosynthesis pathway were highly expressed in GSTs. Taken together, we developed an efficient method to collect comparatively pure GSTs from unfixed leaved, so that the metabolites were relatively obtained intact. This method can be implemented in metabolomics research of purely specific plant cell populations and has the potential to discover novel secondary metabolites.
... It is challenging to probe how the cannabis trichomes produce and secrete these metabolites since live-cell imaging approaches are limited by the lack of genetic transformation methods for cannabis and by the high intrinsic fluorescence of trichome cells. 2 Early electron microscopy studies on cannabis trichome cells described a number of distinctive features, such as plastids with paracrystalline cores and lipidic inclusions throughout the glandular cells, which was interpreted as an exosome-like mechanism of specialized metabolite trafficking. [3][4][5] To obtain highresolution structural data and to address the concern that lipids are poorly preserved with conventional chemical fixation, we cryo-fixed cannabis glandular trichomes to immobilize both cellular structures and the metabolites in situ and imaged with transmission electron microscopy (TEM). ...
For centuries, humans have cultivated cannabis for the pharmacological properties that result from consuming its specialized metabolites, primarily cannabinoids and terpenoids. Today, cannabis is a multi-billion-dollar industry whose existence rests on the biological activity of tiny cell clusters, called glandular trichomes, found mainly on flowers. Cannabinoids are toxic to cannabis cells,¹ and how the trichome cells can produce and secrete massive quantities of lipophilic metabolites is not known.¹ To address this gap in knowledge, we investigated cannabis glandular trichomes using ultra-rapid cryofixation, quantitative electron microscopy, and immuno-gold labeling of cannabinoid pathway enzymes. We demonstrate that the metabolically active cells in cannabis form a “supercell,” with extensive cytoplasmic bridges across the cell walls and a polar distribution of organelles adjacent to the apical surface where metabolites are secreted. The predicted metabolic role of the non-photosynthetic plastids is supported by unusual membrane arrays in the plastids and the localization of the start of the cannabinoid/terpene pathway in the stroma of the plastids. Abundant membrane contact sites connected plastid paracrystalline cores with the plastid envelope, plastid with endoplasmic reticulum (ER), and ER with plasma membrane. The final step of cannabinoid biosynthesis, catalyzed by tetrahydrocannabinolic acid synthase (THCAS), was localized in the cell-surface wall facing the extracellular storage cavity. We propose a new model of how the cannabis cells can support abundant metabolite production, with emphasis on the key role of membrane contact sites and extracellular THCA biosynthesis. This new model can inform synthetic biology approaches for cannabinoid production in yeast or cell cultures.
... However, they are generally called capitate-sessile, capitate-stalked, and bulbous types. Usually, the stalked type trichome is dominant along the leaf veins, and the sessile type is dominant in the non-vein parts [23]. The stalk type has more cannabinoids and terpenes than the sessile type [24]. ...
Cannabis breeders are combining several genes to develop economically valuable fiber, seed, and medicinal hemp. This study analyzed the characteristics and selection of traits based on cannabidiol production of medicinal cannabis lines successfully grown under artificial light and nutrient solution cultivation conditions in smart farm conditions. Sixteen female plants were selected by seeding medical hemp F1 hybrid specimens obtained by randomly crossing Cherry Wine and native hemp from each country. The F1 generation was treated with 12 h light to induce flower differentiation. CBD production peaked on day 50 of the treatment, and this was selected as the harvesting day. All F1 hybrids were separated by leaf and inflorescence after collecting morphological data, and fresh and dry weights were measured. The CBD production of leaf and inflorescence per cubic meter was calculated. The CW21-5 line produced a total of 53.002 ± 0.228 g of CBD per cubic meter, the highest CBD producer. In addition, heatmap correlation analysis showed that most morphological data were not related to cannabinoid content. Principal Component Analysis (PCA) and Self-Organizing Map (SOM) analysis showed that CW21-5 is an arbitrary line that does not cluster with other lines, and the reason for its excellent CBD yield per cubic meter is that it has a narrow plant diameter and a high CBD content at the same time.
... Cannabinoids biosynthesis is primarily localised on the capitate-stalked trichomes mainly found on the flower and sugar leaf surface of the pistillate plant [9]. Disk-like structures formed on the trichome head synthesizes and secretes the cannabinoids which are then accumulated in the fibrillar matrix, the subcuticular wall, and the cuticle [9,10]. Shortly after the recoveries of the cannabinoids, the cannabinoid receptors (CB1 and CB2) were identified and many endogenous ligands for the cannabinoid receptors were also recognised [11]. ...
Phytocannabinoids are isoprenylated resorcinyl polyketides produced mostly in glandular trichomes of Cannabis sativa L. These discoveries led to the identification of cannabinoid receptors, which modulate psychotropic and pharmacological reactions and are found primarily in the human central nervous system. As a result of the biogenetic process, aliphatic ketide phytocannabinoids are exclusively found in the cannabis species and have a limited natural distribution, whereas phenethyl-type phytocannabinoids are present in higher plants, liverworts, and fungi. The development of cannabinomics has uncovered evidence of new sources containing various phytocannabinoid derivatives. Phytocannabinoids have been isolated as artifacts from their carboxylated forms (pre-cannabinoids or acidic cannabinoids) from plant sources. In this review, the overview of the phytocannabinoid biosynthesis is presented. Different non-cannabis plant sources are described either from those belonging to the angiosperm species and bryophytes, together with their metabolomic structures. Lastly, we discuss the legal framework for the ingestion of these biological materials which currently receive the attention as a legal high.
... Male plants are more advantageous for industrial uses due to their production of a finer fiber (Salentijn et al., 2019). Although CBD is present in both male and female plants, most are found in resin glands on trichomes of the female flower buds (Mahlberg and Kim, 2004). The terpenophenolic compounds are secreted from head cells of trichome glands, specifically from the capitate-stalked glandular hairs (Happyana et al., 2013). ...
Forensic laboratories are required to have analytical tools to confidently differentiate illegal substances such as marijuana from legal products (i.e., industrial hemp). The Achilles heel of industrial hemp is its association with marijuana. Industrial hemp from the Cannabis sativa L. plant is reported to be one of the strongest natural multipurpose fibers on earth. The Cannabis plant is a vigorous annual crop broadly separated into two classes: industrial hemp and marijuana. Up until the eighteenth century, hemp was one of the major fibers in the United States. The decline of its cultivation and applications is largely due to burgeoning manufacture of synthetic fibers. Traditional composite materials such as concrete, fiberglass insulation, and lumber are environmentally unfavorable. Industrial hemp exhibits environmental sustainability, low maintenance, and high local and national economic impacts. The 2018 Farm Bill made way for the legalization of hemp by categorizing it as an ordinary agricultural commodity. Unlike marijuana, hemp contains less than 0.3% of the cannabinoid, Δ9-tetrahydrocannabinol, the psychoactive compound which gives users psychotropic effects and confers illegality in some locations. On the other hand, industrial hemp contains cannabidiol found in the resinous flower of Cannabis and is purported to have multiple advantageous uses. There is a paucity of investigations of the identity, microbial diversity, and biochemical characterizations of industrial hemp. This review provides background on important topics regarding hemp and the quantification of total tetrahydrocannabinol in hemp products. It will also serve as an overview of emergent microbiological studies regarding hemp inflorescences. Further, we examine challenges in using forensic analytical methodologies tasked to distinguish legal fiber-type material from illegal drug-types.
... However, the cost of electricity limits production because it is significantly higher than the other production costs (Mills 2012). Although the synthesis cannabinoids is largely genetically controlled (Mahlberg and Kim 2004), environmental factors can influence the final cannabinoid concentrations . ...
Hemp is a crop that in recent years has received renewed attention and been cultivated in numerous countries after having been abandoned by many during the twentieth century. This ‘rebirth’ is due to numerous factors: its favorable agronomical characteristics, its image of being a sustainable crop, and the plasticity of the products it can provide. However, due to its absence for a long time, there is a lack of expert knowledge on cultivating hemp. There is a lack of scientific knowledge regarding the specificities of its biology, and the strong interaction between genotype and environment remains a limiting factor of hemp cultivation, affecting both the yield and quality of the biomass produced. In this chapter, we have discussed the ins and outs of the cultivation of hemp through a scientific prism to address the principal factors, environmental and genotypic, that drive the agronomical characteristics of a hemp crop. Thereafter, we have focussed on the best crop management practices for optimizing hemp cultivation in terms of yield and quality parameters of the different fractions of the biomass that hemp can provide.KeywordsAgronomyCrop managementCultivationEcophysiologyIndustrial hemp
... However, the cost of electricity limits production because it is significantly higher than the other (Mills 2012). Although the synthesis cannabinoids is largely genetically controlled (Mahlberg and Kim 2004), environmental factors can influence the final cannabinoid concentrations (Campbell et al. 2019). ...
Some African countries have decriminalized cannabis production for medicinal purposes. This has resulted in the commercial cultivation of the once illegal crop from hidden areas to either indoor or outdoor gardens. Cannabis health and socio-economic effects have been widely researched while ignoring its environmental impacts on commercial-scale cultivation. The extensive production methods have both negative and positive impacts on the environment. It was established that though cannabis production has been legalized in a few African countries and thus grown extensively, it is still illegal in most countries and cultivated in hidden fields in forests and other public lands. Indoor cannabis cultivation involves manipulating light, humidity, temperature, and other factors to optimal levels. Cannabis cultivation has beneficial effects (soil improvement, bio-economy development, soil moisture maintenance, control of weeds emergence, organic matter accumulation) and adverse effects (loss and fragmentation of habitats, grading and burying of streams; sedimentation, eutrophication, water contamination, and greenhouse gas emissions) on the environment. Cannabis is a high-value crop that can improve national economies through exports and the livelihoods of individual farmers at the local level, despite the adverse impacts it may have on the environment. Most studies on the environmental effects of cannabis cultivation have been conducted in Europe and North America. There is thus still a gap in this aspect in Africa. Given the double-edged effects of cannabis cultivation, it is pertinent that scientists address the environmental effects of cannabis cultivation in Africa, and design strategies to minimize the risks associated with its cultivation, and inform the development of regulations for the growing cannabis industry in Africa. This review focuses on cultivation methods, physiological factors for growth, and the effects of cannabis growing on the environment. Also, the review deals with how to increase the yields and quality of different varieties of cannabis.KeywordsAfricaCultivationCannabisEnvironment impactPhysiological factors
... Previously, three types of glandular trichomes on cannabis flowers were described -referred to as capitate-sessile, capitatestalked, and bulbous -based on structural assessments by scanning electron microscopy (Hammond and Mahlberg, 1973). The trichomes were differentiated based on their morphology, where bulbous trichomes were small and low, sessile trichomes were comprised of a globular head on a very short stalk, and stalked trichomes had a larger globular head on a long stalk; of the three trichome types, stalked trichomes produce the greatest amount of cannabinoids (Hammond and Mahlberg, 1973;Mahlberg and Kim, 2004;Livingston et al., 2020). Unfortunately, this non-specific differentiation between trichome types led to misidentification of trichomes due to the similar appearance of sessile and stalked morphs (Dayanandan and Kaufman, 1976;Livingston et al., 2020). ...
Cannabis has been legalized for recreational use in several countries and medical use is authorized in an expanding list of countries; markets are growing internationally, causing an increase in demand for high quality products with well-defined properties. The key compounds of Cannabis plants are cannabinoids, which are produced by stalked glandular trichomes located on female flowers. These trichomes produce resin that contains cannabinoids, such as tetrahydrocannabinolic acid and cannabidiolic acid, and an array of other secondary metabolites of varying degrees of commercial interest. While growers tend to focus on improving whole flower yields, our understanding of the “goldmines” of the plant – the trichomes – is limited despite their being the true source of revenue for a multi-billion-dollar industry. This review aims to provide an overview of our current understanding of cannabis glandular trichomes and their metabolite products in order to identify current gaps in knowledge and to outline future research directions.
... Another report revealed that THC attenuates the immune response of host in murine lung cancer model (LC12) and mouse mammary carcinoma (4T1), by enhancing IL-10 and TGF-β promoting tumour growth. However, LC12 tumor growth in immune deficient mice remained unaltered after treatment with THC illustrating the fact that THC induced immunosuppression depends on the host immunosuppressive networks [32]. Increase in cancer cell growth in THC treated groups due to the suppression of host immune response has raised queries on the medicinal use of THC in cancer patients. ...
Cannabinoids are the major chemical constituents of the plant Cannabis sativa L. and are known to exhibit a wide range of pharmacological effects viz., psychotropic, analgesic, anticancer, antiinflammatory, antidiabetic, anticonvulsive, antibacterial and antifungal etc. The use of cannabis, cannabinoids and their products is restricted in several countries due to the high risk of misuse. Recently, cannabinoids have regained the interest of the researchers due to their therapeutic applications. Ever since the discovery of the cannabinoids, most of the studies carried out on the evaluation of their biological activities were limited to only preclinical levels. The quality of the preclinical data still remains only low to moderate, thus, leaving behind an uncertainty in their use for therapeutic applications. Problems associated with the solubility, stability and bioavailability of the cannabinoid drugs are also a major concern in the quality of the study. Nanoparticle based drug delivery system could be a potential method to increase the reliability of the data. While considering the immense pharmacological properties of the cannabinoids, there is an urgency to perform intensive clinical trials and to know their mechanism of action in various disease conditions, evaluate their efficacy and safety, and register them as drug candidates. This review highlights the chemistry, types and biological activities of the cannabinoids such as THC, CBD and CBN in focus with their anticancer activity, neuroprotective effect and nanoformulating the cannabinoid drugs.
... Tetrahydrocannabinolic acid synthase (THCA synthase) and cannabidiolic acid synthase (CBDA synthase) catalyse the oxidative cyclization of cannabigerolic acid (CBGA), which results in the production of THCA and CBDA, respectively ( Fig. 1). It was suggested that cannabinoid synthases follow the secretory pathway and that the final products may be biosynthesized in the hydrophobic exudates of glandular trichomes [2,3]. In this study we tested the activity of cannabinoid synthases accumulated in the exudates of glandular trichomes in the organic phase. ...
... In recent decades, high essential oil hemp cultivars have been selected for high cannabinoid secondary metabolites, led by cannabidiol (CBD) varieties; with evolving interest in varieties bred for higher levels of other cannabinoids (canabigerol, canachromine, etc) [1,2]. Maximum essential oil production occurs within unpollinated flowers of dioecious, female Cannabis concentrated within glandular trichomes [3,4]. THC and CBD are the two most abundant cannabinoids of the over 200 known phytocannabinoids [5,6]. ...
Five essential oil hemp (Cannabis sativa L.) cultivars (Cherry Blossom, Cherry Blossom (Tuan), Berry Blossom, Cherry Wine, and Cherry Blossom × Trump) were treated with six fertigation treatments to quantify the effects of synthetic fertilizer rates and irrigation electrical conductivity on plant growth, biomass accumulation, and cannabinoid profiles. Irrigation water was injected with a commercial 20-20-20 fertilizer at rates of 0, 50, 150, 300, 450, and 600 ppm nitrogen equating to 0.33 (control), 0.54, 0.96, 1.59, 2.22, and 2.85 dS m⁻¹, respectively. Plants were grown under artificial lighting (18 hr) to maintain vegetative growth for eight weeks, followed by an eight-week flowering period. High linear relationship between chlorophyll concentrations and SPAD-502 measurements validated the utilization of SPAD meters to rapidly identify nutrient deficiency in essential oil hemp. Cultivars expressed significant variation in plant height and cannabinoid profiles (% dry mass), in concurrence with limited biomass and cannabinoid (g per plant) yield variation. Cherry Blossom was the best performing cultivar and Cherry Wine was the least productive. Variation in plant growth, biomass, and cannabinoid concentrations were affected to a greater extent by fertilizer rates. Optimal fertilizer rates were observed at 50 ppm N, while increased fertilizer rates significantly reduced plant growth, biomass accumulation, and cannabinoid concentrations. Increased fertilizer rates (> 300 ppm N) resulted in compliant THC levels (< 0.3%), although when coupled with biomass reductions resulted in minimal cannabinoid yields. Additionally, CBD concentration demonstrated higher sensitivity to increased fertilizer rates (> 300 ppm N) compared to THC and CBG (> 450 ppm N). The results of this study can serve as a guide when using fertigation methods on essential oil hemp cultivars; although results may differ with cultivar selection, environmental conditions, and management practices.
... To overcome the toxic effect of the plant's own metabolites, these chemicals accumulate to very high levels in specific tissues, organs, cells, and organelles. For example, several cyanogenic plants accumulate nearly 25-50% of cyanogens in the seedlings only, and in Cannabis spp. up to 25% of cannabinoids accumulate in specific glandular trichomes (Adewusi, 1990;Mahlberg and Kim, 2004;Livingston et al., 2020). Conversely, some defensive metabolites are specifically biosynthesized only when required. ...
Plants are unsurpassed biochemists that synthesize a plethora of molecules in response to everchanging environment. The majority of these molecules considered as specialized metabolites, effectively protect the plant against pathogens and herbivores. However, this defense most likely comes at a high expense, leading to reduction of growth (known as the 'growth-defense tradeoff'). Plants employ several strategies to reduce the high metabolic costs associated with chemical defense. Production of specialized metabolites is tightly regulated by a network of transcription factors facilitating its fine-tuning in time and space. Multifunctionality of specialized metabolites, their effective recycling system by re-using carbon, nitrogen and sulphur, thus, re-introducing them back to the primary metabolite pool allows further cost reduction. Spatial separation of biosynthetic enzymes and their substrates, sequestration of potentially toxic substances and conversion to less toxic metabolite forms are plant's solutions to avoid the detrimental effects of metabolites they produce as well as reduce production costs. Constant fitness pressure from herbivores, pathogens and abiotic stressors lead to honing of specialized metabolite biosynthesis reactions to be timely, efficient and metabolic cost-effective. In this review we assess the costs of specialized metabolites production for chemical defense and the different plant mechanisms to reduce the price of of such metabolic activity in terms of self-toxicity and growth.
Cannabis sativa L. is an ancient crop whose agricultural adoption has been interrupted to prevent the use of marijuana as psychoactive drug. Nevertheless, hemp – the Cannabis sativa type with low concentrations of intoxicating Δ9-tetrahydrocannabinoid – is experiencing resurged interest thanks to loosened cultivation restrictions and its potential as multipurpose bio-based crop. In fact, hemp has valuable applications, including production of medicines from its non-intoxicating cannabinoids, food, medical, and industrial uses of its seed oil rich in poly-unsaturated fatty acids, and production of fibers for textiles and industry from its stems. Recently, several hemp genomic and genetic resources have been developed, allowing for a significant expansion of the genetic knowledge on major hemp traits, as cannabinoids, oil, and fibers synthesis, and regulation of flowering and sex determination. Still, hemp is an under-improved crop, whose advancement will depend on the ability to expand and collectively use the novel resources available in light of the fast advancements in bioinformatics and plant phenotyping technologies. This review discusses on the current genetic and genomic knowledge on the most important hemp traits, and provides a perspective on how to further expand such knowledge and tackle hemp improvement with the most up-to-date tools for plant and hemp research.
Studies with secretory cavity contents and air-dried inflorescence extracts of the CBD-rich hemp strain, Cannabis sativa cv. ‘Cherry Wine’, were conducted to compare the decarboxylation rates of acidic cannabinoids between two groups. The secretory cavity contents acquired from the capitate-stalked glandular trichomes by glass microcapillaries, and inflorescence samples air-dried for 15 days of storage in darkness at room temperature were analysed by high-pressure liquid chromatography. The ratio of acidic cannabinoids to the total cannabinoids was ranging from 0.5% to 2.4% lower in the air-dried inflorescence samples compared to the secretory cavity samples as follows. In the secretory cavity content, the percentage of acidic cannabinoids to the total cannabinoids was measured as 86.4% cannabidiolic acid (CBDA), 6.5% tetrahydrocannabinolic acid (THCA), 4.3% cannabichromenic acid (CBCA), 1.4% cannabigerolic acid (CBGA), and 0.6% cannabidivarinic acid (CBDVA), respectively. In the air-dried inflorescence, however, the acidic cannabinoids were detected with 84% CBDA, 4.8% THCA, 3.3% CBCA, 0.8% CBGA, and 0.3% Δ⁹-tetrahydrocannabivarinic acid (Δ⁹-THCVA), respectively. The ratio of cannabidiol (CBD) to cannabidiolic acid (CBDA) was close to 1:99 (w/w) in secretory cavity contents, however, it was roughly 1:20 (w/w) in the air-dried inflorescence. In addition, Δ⁹-tetrahydrocannabivarin (Δ⁹-THCV) and Δ⁹-tetrahydrocannabivarinic acid (Δ⁹-THCVA) were only detected in the air-dried inflorescence sample, and the ratio of Δ⁹-THCV to Δ⁹-THCVA was about 1:20 (w/w). Besides, cannabidivarinic acid (CBDVA) was only observed in the secretory cavity content.
For millennia, various cultures have utilized cannabis for food, textile fiber, ethno-medicines, and pharmacotherapy, owing to its medicinal potential and psychotropic effects. An in-depth exploration of its historical,
chemical, and therapeutic dimensions provides context for its contemporary understanding. The criminalization
of cannabis in many countries was influenced by the presence of psychoactive cannabinoids; however, scientific
advances and growing public awareness have renewed interest in cannabis-related products, especially for
medical use. Described as a ’treasure trove,’ cannabis produces a diverse array of cannabinoids and noncannabinoid compounds. Recent research focuses on cannabinoids for treating conditions such as anxiety,
depression, chronic pain, Alzheimer’s, Parkinson’s, and epilepsy. Additionally, secondary metabolites like
phenolic compounds, terpenes, and terpenoids are increasingly recognized for their therapeutic effects and their
synergistic role with cannabinoids. These compounds show potential in treating neuro and non-neuro disorders,
and studies suggest their promise as antitumoral agents. This comprehensive review integrates historical,
chemical, and therapeutic perspectives on cannabis, highlighting contemporary research and its vast potential in
medicine.
Introducing soybean cultivars resistant to 2,4-D and dicamba allowed for postemergence applications of these herbicides. These herbicides pose a high risk for off-target movement, and the potential influence on crops such as hemp is unknown. Two studies were conducted from 2020 through 2022 in controlled environments to evaluate hemp response to rates simulating off-target events of 2,4-D and dicamba. The objectives of this study were to: 1) determine the effects of herbicide (2,4-D and dicamba) and rate (1x to 1/100,000x labeled rate) on visible injury, height, and branching, and 2) determine the effect of 2,4-D rate (1x to 1/100,000x labeled rate) on visible injury, height, branching, and reproductive parameters. Herbicides were applied in the early vegetative stage, and evaluations took place 14 and 28 days after treatment (DAT) and at trial termination (42 DAT in the greenhouse trial and at harvest in the growth chamber trial). In the greenhouse trial, 2,4-D and dicamba at the 1x rate and the 1/10x rate caused 68, 78, and 20% injury 28 DAT, respectively. At the time of trial termination 42 DAT, plants treated with 1x rates of 2,4-D and dicamba or 1/10x dicamba were 19, 25, and 9 cm shorter than the nontreated control, respectively. At trial termination, simulated off-target rates of 2,4-D and dicamba did not influence branching or plant weight. In the growth chamber study, the 1x and 1/10x rates of 2,4-D caused 82% and 2% injury 28 DAT, respectively. Plant height, fresh weight, and cannabidiol (CBD) levels of plants treated with simulated off-target rates of 2,4-D were not different from the nontreated control. These studies suggest that hemp grown for CBD exposed to off-target rates of 2,4-D or dicamba in early vegetative stages may not have distinguishable effects 42 DAT or at harvest.
In recent years, there have been significant growth and interest in cannabinoid-based drugs for a wide range of medical conditions, some of which are neurogenic diseases, pain control, and seizures. As there is an increased demand for cannabinoid-based drugs, it is necessary to adapt biotechnological techniques to develop new traits for the sophisticated and selective breeding of Cannabis plants aimed for cannabinoid production.
Despite Biotech companies aspiring to replace cannabis plants with heterologous hosts, genome editing for precision cannabis breeding is yet to be embraced. The availability of genome-editing technologies might herald a new dawn in breeding yielding new varieties with improved profiles of bioactive cannabinoids and terpenes. In this review, we highlight novel breeding approaches such as marker-assisted selection (MAS), mutation breeding, micropropagation, transgenic breeding, and CRISPR/Cas-based editing techniques aimed for enhanced cannabinoid production.
Trema, a genus of the popularly known Cannabaceae, has recently been the subject of cannabinoid bioprospection. T. micrantha is a tree with pharmacological potential widely used in folk medicine. It has two types of glandular trichomes, bulbous and filiform, spread throughout the plant body. Considering the proximity of this species to Cannabis sativa and Trema orientalis, species containing cannabinoids, the glandular trichomes of T. micrantha are also expected to be related to the secretion of these compounds. Thus, this study aims to detail the morphology of secretory trichomes during the synthesis, storing and release of metabolites in T. micrantha. We tested the proposition that they could be a putative type of cannabinoid-secreting gland. Pistillate and staminate flowers and leaves were collected and processed for ontogenic, histochemical, and ultrastructural analyses. Both types of glandular trichomes originate from a protodermal cell. They are putative cannabinoid-secreting sites because: (1) terpene-phenols and, more specifically, cannabinoids were detected in situ; (2) their secretory subcellular apparatus is consistent with that found in C. sativa: modified plastids, polyribosomes, an extensive rough endoplasmic reticulum, and a moniliform smooth endoplasmic reticulum. Plastids and smooth endoplasmic reticulum are involved in the synthesis of terpenes, while the rough endoplasmic reticulum acts in the phenolic synthesis. These substances cross the plasma membrane by exocytosis and are released outside the trichome through cuticle pores. The study of the cell biology of the putative cannabinoid glands can promote the advancement of prospecting for natural products in plants.
Cannabichromene (CBC, 1a) occurs in Cannabis (Cannabis sativa) as a scalemate having a composition that is strain-dependent in terms of both enantiomeric excess and enantiomeric dominance. In the present work, the chirality of CBC (1a), a noncrystalline compound, was shown not to be significantly affected by standard conditions of isolation and purification, and enantiomeric self-disproportionation effects were minimized by carrying out the chiral analysis on crude fractions rather than on purified products. A genetic basis for the different enantiomeric state of CBC in Cannabis therefore seems to exist, implying that the chirality status of natural CBC (1a) in the plant is associated with the differential expression of CBCA-synthase isoforms and/or of associated directing proteins with antipodal enantiospecificity. The biological profile of both enantiomers of CBC should therefore be investigated independently to assess the contribution of this compound to the activity of Cannabis preparations.
Introduction:
In recent years, industrial production of Cannabis sativa has increased due to increased demand of medicinal products based on the plant. In these medicinal products, it is mainly the contents of cannabinoids like THCA and CBDA which are of interest, but also the flavonoids of C. sativa have pharmaceutical interest.
Objectives:
The primary aim is to study the distribution of the different cannabinoids in leaves of C. sativa and specifically to which extent they are located on the trichomes found on the surface of C. sativa leaves. Desorption electrospray ionization (DESI) and matrix assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI) provide non-targeted imaging of numerous compounds in the same experiment. Therefore, the distribution of flavonoids is also mapped in the same experiments.
Material and methods:
Fan leaves from C. sativa were imaged in the lateral dimension using direct DESI-MSI as well as indirect DESI-MSI via a porous PTFE surface using pixel sizes of 150-200 μm. For cross sections of sugar leaves, MALDI-MSI was performed at 20 μm pixel size.
Results:
From indirect DESI-MSI experiments, a connection was made between the cannabinoid CBGA and capitate-stalked trichomes. Other cannabinoids like THCA/CBDA (isomers, which are not resolved in an MSI experiment) were also detected in the capitate-stalked trichomes, but in addition to this also in the small glandular trichomes. MALDI-MSI experiments on cross sections of sugar leaves confirmed that the cannabinoids were not an integral part of the leaf tissue itself, but originated from the trichomes on the surface of the leaf.
Conclusion:
The study provides visual evidence that the cannabinoids are produced and accumulated in the trichomes of C. sativa leaves.
Cannabis glandular trichomes produce and store an abundance of lipidic specialised metabolites (e.g. cannabinoids and terpenes) that are consumed by humans for medicinal and recreational purposes. Due to a lack of genetic resources and inherent autofluorescence of cannabis glandular trichomes, our knowledge of cannabinoid trafficking and secretion is limited to transmission electron microscopy (TEM). Advances in cryofixation methods has resulted in ultrastructural observations closer to the 'natural state' of the living cell, and recent reports of cryofixed cannabis trichome ultrastructure challenge the long-standing model of cannabinoid trafficking proposed by ultrastructural reports using chemically fixed samples. Here, we compare the ultrastructural morphology of cannabis glandular trichomes preserved using conventional chemical fixation and ultrarapid cryofixation. We show that chemical fixation results in amorphous metabolite inclusions surrounding the organelles of glandular trichomes that were not present in cryofixed samples. Vacuolar morphology in cryofixed samples exhibited homogenous electron density, while chemically fixed samples contained a flocculent electron dense periphery and electron lucent lumen. In contrast to the apparent advantages of cryopreservation, fine details of cell wall fibre orientation could be observed in chemically fixed glandular trichomes that were not seen in cryofixed samples. Our data suggest that chemical fixation results in intracellular artefacts that impact the interpretation of lipid production and trafficking, while enabling greater detail of extracellular polysaccharide organisation.
This study was undertaken to compare cannabinoid levels and yields in floral extracts from unpollinated and artificially pollinated industrial hemp (Cannabis sativa L. cv. Finola) flowers grown under identical growth chamber conditions. Of the 16 cannabinoids analyzed using high performance liquid chromatography (HPLC), the levels of 10, cannabichromene (CBC), cannabichromenic acid (CBCA), cannabidivarin (CBDV), cannabigerol (CBG), cannabicyclol (CBL), cannabinol (CBN) cannabinolic acid (CBNA), ∆⁸-tetrahydrocannabinol (∆⁸⁻THC), tetrahydrocannabivarin (THCV) and tetrahydrocannabivarinic acid (THCVA) were near or below the limit of quantification. Total ∆⁹-tetrahydrocannabinol (THC), was present at concentrations below the legal limit of 0.3% (w/w). The level of cannabidiol (CBD) in extracts from pollinated flowers was the same as that from unpollinated flowers, but cannabidiolic acid (CBDA) and cannabidivarinic acid (CBDVA) levels were not. This suggested that, although pollination changes the pool sizes of the precursors in the metabolic pathway leading to CBD production, cannabinoid levels in floral extracts from the Finola cultivar, were reduced but not eliminated, by pollination of hemp flowers compared with levels in floral extracts from unpollinated flowers.
Medicinal cannabis (Cannabis sativa L.) is a growing agro-industrial sector with the
end product required to be free of pesticides. C. sativa is covered by specialized hairs
called trichomes, namely, two types of non-glandular and three types of glandular
trichomes. Despite the great importance of biological pest control in medicinal
cannabis little is known about the impact of cannabis trichomes on natural enemies´
mobility and their interaction with prey. In the current study, by employing a video
recording set-up, we determined the mobility of Aphidoletes aphidimyza and
Chrysoperla carnea larvae and their interaction with the aphid pests Phorodon
cannabis and Aphis gossypii on C. sativa leaf disks and inflorescences. As benchmark,
we tested the same parameters on sweet pepper leaf disks, a benign host plant for
both predators, and without trichomes. It was found that A. aphidimyza females readily
oviposited on P. cannabis colonies developing on C. sativa plants. On C. sativa leaves,
covered by non-glandular trichomes, the larvae of both predators were able to move
and prey upon aphids. On C. sativa inflorescences, where glandular trichomes
prevailed, the larvae of A. aphidimyza were generally inactive, while the C. carnea
larvae were still able to move and interact with prey albeit to a lesser degree compared
to the cannabis leaves. We suggest that the use of A. aphidimyza and C. carnea in
augmentative biological control programs in medicinal cannabis should depend on the
crop´s growth stage; the former should be employed during the vegetative and the
latter during vegetative and flowering crop stages.
Cannabis sativa is most prominent for its psychoactive secondary compound tetrahydrocannabinol, or THC. However, THC is only one of many phytocannabinoids found in this (in)famous medicinal plant. The stepwise legalization of Cannabis in many countries has opened opportunities for its medicinal and commercial use, sparking scientific interest in the genetics and biochemistry of phytocannabinoid synthesis. Advances in plant biology and genomics help to accelerate research in the Cannabis field, which is still lagging behind other comparable high-value crops. Here, we discuss the intriguing genetics and evolutionary history of phytocannabinoid synthases, and also show that an increased understanding of Cannabis developmental genetics and morphology are of critical importance to leverage the full potential of phytocannabinoid production.
Secondary metabolites are known to play a role in the plant's defense system, which can be triggered by biotic or abiotic stress. Cannabis (Cannabis sativa L.) plants and mainly their female flowers, have a variety of bioactive metabolites, predominantly cannabinoids and terpenes, which are synthesized and secreted by the trichomes. Many studies have examined their chemistry and bioactive effects; however, there is insufficient information on the effect of biotic stress on the presence of secondary metabolites in cannabis. The present study examined the effect of a well-known cannabis pest, Tetranychus urticae, on the occurrence and concentration of cannabinoids and terpenes in cannabis leaves and flowers. Six cannabis plants were infested with T. urticae mites (treatment group), and six plants were used as the control group. Cannabinoids and terpenes were analyzed and quantified by liquid chromatograph mass spectrometer and gas chromatograph mass spectrometer, respectively. The contents of several cannabinoids and terpenes increased significantly in the leaves of the treatment group of plants in their late vegetative phase as the mite population increased, compared with the control group. Significantly increased content of almost all terpenes, and the cannabinoids; Δ⁹-tetrahydrocannabinol, cannabichromene, and cannabigerol, was also seen in mature flowers of the treatment group plants, compared with the control group. Thus, cannabis plant infestation has an impact on its secondary metabolites, cannabinoids and terpenes, reflected by an overall increase in these compounds.
The identification of cannabis chemotypes at an early stage of a plant's growth, which is long before anthesis, has been intensively pursued in order to control the on-target selection of the cultivar type at the beginning of cultivation, so as to avoid economic and legal drawbacks. However, this issue has been systematically addressed by only few and relatively recent studies of analytical chemistry, possibly because result validations require long-term monitoring of the content and ratio of cannabinoids and terpenes in a great number of plant specimens suitably selected and grown. Here, we review the procedures, the chromatographic techniques and the statistics used in topical investigations during the past thirteen years. Through heterogeneous and not easily comparable approaches, they prove the feasibility of chemotypes safe determination within the first month of a plant's life.
We identified a unique enzyme that catalyzes the oxidocyclization of cannabigerolic acid to cannabidiolic acid (CBDA) in Cannabis sativa L. (CBDA strain). The enzyme, named CBDA synthase, was purified to apparent homogeneity by a four-step procedure: ammonium sulfate precipitation followed by chromatography on DEAE-cellulose, phenyl-Sepharose CL-4B, and hydroxylapatite. The active enzyme consists of a single polypeptide with a molecular mass of 74 kDa and a pI of 6.1. The NH2-terminal amino acid sequence of CBDA synthase is similar to that of Delta1-tetrahydrocannabinolic-acid synthase. CBDA synthase does not require coenzymes, molecular oxygen, hydrogen peroxide, and metal ion cofactors for the oxidocyclization reaction. These results indicate that CBDA synthase is neither an oxygenase nor a peroxidase and that the enzymatic cyclization does not proceed via oxygenated intermediates. CBDA synthase catalyzes the formation of CBDA from cannabinerolic acid as well as cannabigerolic acid, although the kcat for the former (0.03 s-1) is lower than that for the latter (0.19 s-1). Therefore, we conclude that CBDA is predominantly biosynthesized from cannabigerolic acid rather than cannabinerolic acid.
Plastids in lipophilic glandular trichomes of chemically fixed (CF) and high pressure cryofixed-cryosubstituted (HPC-CS) bracteal tissues of Cannabis were examined by transmission electron microscopy. In CF preparations, plastids in disc cells prior to secretory cavity formation possessed several lobed and dilated thylakoid-like features. In glands with secretory cavities, thylakoid-like features aggregated to form reticulate bodies that distended regions of the elongated plastids. Electron-gray inclusions evident on the plastid surface appeared continuous with the reticulate body. Inclusions of similar electron density also appeared in the cell cytoplasm, along the plasma membrane, between the plasma membrane and cell wall facing the cavity, and in the secretory cavity in both CF and HPC-CS preparations. The bilayer structure of membranes of the plastid envelope was evident in HPC-CS but not in CF preparations. In HPC-CS preparations, secretions were evident on the plastid surface and were continuous with those in the plastid through pores in the envelope. This study supports an interpretation that these specialized plastids, lipoplasts, synthesize secretions that are transported through the plasma membrane and cell wall to subsequently accumulate in the secretory cavity.
A plastid vesicle preparation isolated from exocarpium of young Citrofortunella mitis (calamondin) fruits was able to synthesise monoterpene hydrocarbons when incubated with isopentenyl pyrophosphate. The electron-microscope comparison between this organelle fraction and the various plastid classes present in the peel tissues has shown the structural identity between these plastid vesicles and the leucoplasts of the epithelial cells lining the secretory pockets. The monoterpene biosynthesis required the presence of dimethylallyl pyrophosphate, Mn(2+) or Mg(2+) and was increased by addition of 2-mercaptoethanol. Evidence is provided that the leucoplast vesicles act as a complete system in which occur all the successive steps involved in monoterpene hydrocarbon elaboration from isopentenyl pyrophosphate.
Malonic acid, mevalonic acid, geraniol and nerol were incorporated into tetrahydrocannabinolic acid and cannabichromenic acid in Cannabis sativa. The pathway from cannabigerolic acid to tetrahydrocannabinolic acid via cannabidiolic acid was established by feeding labelled cannabinoid acids. Cannabichromenic acid was shown to be formed on a side pathway from cannabigerolic acid.
Formation of secretory vesicles in the noncellular secretory cavity of glandular trichomes of Cannabis sativa L. was examined by transmission electron microscopy. Two patterns of vesicle formation occurred during gland morphogenesis. 1) During initial phases of cavity formation small hyaline areas arose in the wall near the plasma membrane of the disc cell. Hyaline areas of elongated shape and different sizes were distributed throughout the wall and adjacent to the secretory cavity. Hyaline areas increased in size, some possibly fusing with others. These hyaline areas, possessing a membrane, moved into the cavity where they formed vesicles. As membraned vesicles they developed a more or less round shape and their contents became electron-dense. 2) During development of the secretory cavity and when abundant secretions were present in the disc cells, these secretions passed through the wall to accumulate as membraned vesicles of different sizes in the cavity. As secretions emerged from the wall, a membrane of wall origin delimited the secretory material from cavity contents. Vesicles released from the wall migrated in the secretory cavity and contacted the sheath where their contents permeated into the subcuticular wall as large or diffused quantities of secretions. In the subcuticular wall these secretions migrated to the wall-cuticle interface where they contributed to structural thickening of the cuticle. This study demonstrates that the secretory process in glands of Cannabis involves not only secretion of materials from the disc cell, but that the disc cell somehow packages these secretions into membraned vesicles outside the cell wall prior to deposition into the secretory cavity for subsequent structural development of the sheath.
Formation of the cuticle from components of the secretory cavity and subcuticular wall was studied by transmission electron microscopy of glandular trichomes of Cannabis prepared by high pressure cryofixation-cryosubstitution. Secretory vesicles in the secretory cavity resembled those localized in the subcuticular wall as well as the vesicle-related material associated with the irregular inner surface of the cuticle and appeared to provide precursors for thickening of the cuticle. Some contiguous vesicles in the secretory cavity and subcuticular wall lacked a surface feature at their point of contact, supporting an interpretation of vesicle fusion. Fibrillar matrix from the secretory cavity contributed fibrillar matrix to the subcuticular wall, and persisted as residual fibrillar matrix associated with secretory materials coalesced to the thickened inner surface of the cuticle. Elongated fibrils arranged in uniformly spaced parallel pairs contributed to the organization of fibrillar matrix in the subcuticular wall. Striae were evident in the outer portion of the cuticle, and appeared to represent sites of degraded residual fibrillar matrix associated with secretory materials coalesced to the inner cuticular surface. This study supports an interpretation that contents of secretory vesicles From the secretory cavity contribute to formation of glandular cuticle.
Development of the secretory cavity and formation of the subcuticular wall of glandular trichomes in Cannabis sativa L. was examined by transmission electron microscopy. The secretory cavity originated at the wall-cuticle interface in the peripheral wall of the discoid secretory cells. During the presecretory phase in development of the glandular trichome, the peripheral wall of the disc cells became laminated into a dense inner zone adjacent to the plasma membrane and a less dense outer zone subjacent to the cuticle. Loosening of wall matrix in the outer zone initiated a secretory cavity among fibrous wall materials. Membrane-bound hyaline areas, compressed in shape, arose in the wall matrix. They appeared first in the outer and subsequently in the inner zone of the wall. The membrane of the vesicles, and associated dense particles attached to the membrane, arose from the wall matrix. Hyaline areas, often with a conspicuous electron-dense content, were released into the secretory cavity where they formed rounded secretory vesicles. Fibrous wall material released from the surface of the disc cells became distributed throughout the secretory cavity among the numerous secretory vesicles. This wall material was incorporated into the developing subcuticular wall that increased five-fold in thickness during enlargement of the secretory cavity. The presence of a subcuticular wall in the cavity of Cannabis trichomes, as contrasted to the absence of this wall in described trichomes of other plants, supports a polyphyletic interpretation of the evolution of the secretory cavity in glandular trichomes among angiosperms.
The dermal sheath of glandular trichomes of Cannabis sativa L., consisting of cuticle and a subcuticular wall, was examined by transmission electron microscopy. Cuticle thickened selectively on the outer wall of disc cells of each trichome prior to formation of the secretory cavity, whereas thickening was less evident on the dermal cells of the bract. Membraned secretory vesicles that differ in size and appearance in the secretory cavity were the source of precursors for synthesis of cuticle. Vesicle contents, released following the degradation of the vesicle membrane upon contact with the subcuticular wall, contributed to both structured and amorphous phases of cuticle development. The structured phase was represented by deposition and thickening of cuticle at the subcuticular wall-cuticle interface to form a thickened cuticle. In the amorphous phase precursors permeated the cuticle in a liquid state, as shown by fusion of cuticles and wax layers between contiguous glands, and may have contributed to growth in surface area of the expanding sheath. Disc cells are interpreted to control growth of secretory cavity by secretion of membraned vesicles into the cavity. The thickened cuticle, which increased eightfold in thickness during enlargement of the gland, provided structural strength for the extensive surface area of the dermal sheath. The gland of Cannabis in which vesicle contents contribute to the growth in thickness and surface area of the cuticle of the sheath is interpreted to represent a phylogenetically derived state as contrasted to secretory glands possessing only cuticle and lacking a complement of secretory vesicles.
A B S T R A C T The relationship between glandular trichomes and cannabinoid content in Cannabis sativa L. was investigated. Three strains of Cannabis, which are annuals, were selected for either a drug, a non-drug, or a fiber trait and then cloned to provide genetically uniform material for analyses over several years. The distribution of the number and type of glands was determined for several organs of different ages including the bract and its subtending monoleaflet leaf and the compound leaf on pistillate plants. Quantitation of glands on these structures was integrated with gas chromatographic analyses of organ cannabinoid profiles. A negative correlation was found between cannabinoid content and gland number for each of the three clones. Isolated heads of the capitate-stalked glands also were analyzed for cannabinoid content and found to vary in relation to clone and gland age. These studies indicate that cannabinoids may occur in plant cells other than glandular trichomes. The results of these studies emphasize the need for stringent sampling procedures in micromorphological studies on trichome distribution and analytical determinations of cannabinoid content in Cannabis.
Cannabinoid levels of individual mature glandular trichomes from two clones and two strains of Cannabis sativa L., which included both drug and fiber phenotypes, were investigated by gas-liquid chromatographic analyses. Capitate-stalked glands were selectively harvested from vein and nonvein areas of pistillate bracts while capitate-sessile glands were harvested from these areas of leaves. The qualitative cannabinoid profile characteristic of the strain or clone was maintained in the individual capitate-stalked glands while the quantitative cannabinoid profiles varied with each strain or clone and between vein and nonvein areas as well. Capitate-sessile glands were found to contain conspic uously lower levels of cannabinoids than capitate-stalked glands. This study emphasizes that glands of Cannabis represent a dynamic system within the cannabinoid synthesizing activities of this plant. GLANDULAR trichomes are prominent features on the shoot system of Cannabis saliva L. Sev eral studies using histochemical and analytical procedures
Three distinct types of glandular hairs of increasing morphological complexity which occur on flowering tops of Cannabis sativa L. (marihuana) are described from scanning electron microscopy. These gland types-termed bulbous, capitate-sessile, and capitate-stalked, described from pistillate plants-occur in greatest abundance on the outer surface of bracts ensheathing the ovary. Bulbous and capitate-sessile glands, which arise at an early stage in bract development, are scattered over the bract surface. Mature bulbous glands have a small swollen head on a short stalk, whereas capitate-sessile glands have a large globular head attached directly to the bract surface. Because of their numbers and large size, capitate-sessile glands are the most conspicuous gland type during the early phase of bract development. Capitate-stalked glands, which have a large globular head on a tall, multicellular stalk, differentiate during subsequent bract development. These stalked glands arise first along the bracteal veins and then over the entire bract surface. A voluminous, fluid secretory product accumulates in the glandular head of all three types. These glands are believed to be a primary site of localization of the marihuana hallucinogen, tetrahydrocannabinol.
The capitate-sessile and capitate-stalked glands of the glandular secretory system in Cannabis, which are interpreted as lipophilic type glandular hairs, were studied from floral bracts of pistillate plants. These glands develop a flattened multicellular disc of secretory cells, which with the extruded secretory product forms the gland head and the auxiliary cells which support the gland head. The secretory product accumulates beneath a sheath derived from separation of the outer wall surface of the cellular disc. The ultrastructure of secretory cells in pre-secretory stages is characterized by a dense ground plasm, transitory lipid bodies and fibrillar material, and well developed endoplasmic reticulum. Dictyosomes and dictyosome-derived secretory vesicles are present, but never abundant. Secretory stages of gland development are characterized by abundant mitochondria and leucoplasts and by a large vacuolar system. Production of the secretory product is associated with plastids which increase in number and structural complexity. The plastids develop a paracrystalline body which nearly fills the mature plastid. Material interpreted as a secretion appears at the surface of plastids, migrates, and accumulates along the cell surface adjoining the secretory cavity. Extrusion of the material into the secretory cavity occurs directly through the plasma membrane-cell wall barrier.
Individual plant organs from different geographical strains of Cannabis saliva L. were analyzed for their cannabinoid content by gas-liquid chromatography. Analyses showed that different plant parts from each strain varied quantitatively in their cannabinoid content. However, each plant part possessed a cannabinoid profile which characterized the chemical phenotype of that strain. Accumulation of a specific cannabinoid in high quantities that was uncharacteristic of that strain was found. Factors such as maturity of plant organ, sex of the plant, location of the plant organ on the plant and sampling procedures influenced the accumulation of cannabinoids. Pollen grains and seeds (intact or crushed) were found to lack detectable levels of cannabinoids. Based on these results, precautions that should be taken when accumulating data on the chemical phenotype of a Cannabis plant are discussed.
Phloroglucinol β-d-glucoside was identified from shoot laticifer exudate of Cannabis sativa (marihuana) by TLC. Isolation of the aglycone from acid-heat hydrolysis or emulsin treatment yielded the free phenol, phloroglucinol (1,3,5-trihydroxybenzene), as identified by GC-mass and 1H NMR spectrometry. Phloroglucinol also was identified by TLC as a prominent component in glandular trichomes.
Gas—liquid chromatographic and high-performance liquid chromatographic analyses on the effects of leaf treatment as well as the conditions for cannabinoid extraction were examined in two clones of Cannabis sativa L. Cannabinoid extracts of dried leaves, when analyzed by gas—liquid chromatography, showed no significant quantitative or qualitative differences regardless of drying procedure or temperature and duration of extraction investigated. Comparable high-performance liquid chromatographic analyses, however, indicated that while extraction temperature did not influence the cannabinoid profile, drying conditions had a significant effect. High ratios of acid to neutral forms were derived only from extracts of leaves dried at 37°C as compared to 60°C. Fresh, non-dried leaf material also yielded high ratios of acid to neutral forms, but the duration of extraction was found to affect cannabinoid yield significantly. Longer extractions of fresh leaves resulted in lower amounts of cannabinoids extracted. This study determined optimal procedures for analyzing fresh plant materials.
Delta 9-tetrahydrocannabinol (THC) localization in glandular trichomes and bracteal tissues of Cannabis, prepared by high pressure cryofixation-cryosubstitution, was examined with a monoclonal antibody-colloidal gold probe by electron microscopy (EM). The antibody detected THC in the outer wall of disc cells during the presecretory cavity phase of gland development. Upon formation of the secretory cavity, the immunolabel detected THC in the disc cell wall facing the cavity as well as the subcuticular wall and cuticle throughout development of the secretory cavity. THC was detected in the fibrillar matrix associated with the disc cell and with this matrix in the secretory cavity. The antibody identified THC on the surface of secretory vesicles, but not in the secretory vesicles. Gold label also was localized in the anticlinal walls between adjacent disc cells and in the wall of dermal and mesophyll cells of the bract. Grains were absent or detected only occasionally in the cytoplasm of disc or other cells of the bract. No THC was detected in controls. These results indicate THC to be a natural product secreted particularly from disc cells and accumulated in the cell wall, the fibrillar matrix and surface feature of vesicles in the secretory cavity, the subcuticular wall, and the cuticle of glandular trichomes. THC, among other chemicals, accumulated in the cuticle may serve as a plant recognition signal to other organisms in the environment.
Cannabichromenic acid synthase was purified to apparent homogeneity by sequential column chromatography including DEAE-cellulose, phenyl-Sepharose CL-4B, and hydroxylapatite. The enzyme catalysed the oxidocyclization of cannabigerolic acid and cannabinerolic acid to cannabichromenic acid. The K(m) values for both substrates were in the same order of magnitude although the Vmax value for the former was higher than that for the latter. These results suggested that cannabichromenic acid is predominantly formed from cannabigerolic acid rather than cannabinerolic acid. The enzyme required neither molecular oxygen nor hydrogen peroxide, indicating that the cannabichromenic acid synthase reaction proceeds through direct dehydrogenation without hydroxylation.
Early development of the secretory cavity of chemically fixed peltate glands in Humulus lupulus L. showed secretions with different densities, light, gray and dark, in the cytoplasm of disc cells and in the periplasmic space adjacent to the developing secretory cavity. Secretions were detected in the disc cell wall and subsequently in the developing secretory cavity under the subcuticular wall of the sheath. Light and gray secretions in the cavity possessed a membrane-like surface feature. Secretions were in contact with the irregular inner surface of the cuticle. Secretions contributed to the thickening of the cuticle, whereas the membrane-like surface feature contributed to a network of Cannabis striae distributed throughout the cuticle. This study supports an early development and organization of the secretory cavity in H. lupulus, parallel to those in Cannabis, and may represent common features for lipophilic glands in angiosperms.