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Cannabis sativa L. is an important herbaceous species originating from Central Asia, which has been used in folk medicine and as a source of textile fibre since the dawn of times. This fast-growing plant has recently seen a resurgence of interest because of its multi-purpose applications: it is indeed a treasure trove of phytochemicals and a rich source of both cellulosic and woody fibres. Equally highly interested in this plant are the pharmaceutical and construction sectors, since its metabolites show potent bioactivities on human health and its outer and inner stem tissues can be used to make bioplastics and concrete-like material, respectively. In this review, the rich spectrum of hemp phytochemicals is discussed by putting a special emphasis on molecules of industrial interest, including cannabinoids, terpenoids and phenolic compounds, and their biosynthetic routes. Cannabinoids represent the most studied group of compounds, mainly due to their wide range of pharmaceutical effects in humans, including psychotropic activities. The therapeutic and commercial interests of some terpenoids and phenolic compounds, and in particular stilbenoids and lignans, are also highlighted in view of the most recent literature data. Biotechnological avenues to enhance the production and bioactivity of hemp secondary metabolites are proposed by discussing the power of plant genetic engineering and tissue culture. In particular two systems are reviewed, i.e. cell suspension and hairy root cultures. Additionally, an entire section is devoted to hemp trichomes, in the light of their importance as phytochemical factories. Ultimately, prospects on the benefits linked to the use of the -omics technologies, such as metabolomics and transcriptomics to speed up the identification and the large-scale production of lead agents from bioengineered Cannabis cell culture, are presented.
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published: 04 February 2016
doi: 10.3389/fpls.2016.00019
Edited by:
Eugenio Benvenuto,
ENEA, Italian National Agency for New
Technologies, Energy and Sustainable
Economic Development, Italy
Reviewed by:
Biswapriya Biswavas Misra,
University of Florida, USA
Felix Stehle,
Technical University of Dortmund,
Christelle M. Andre
Specialty section:
This article was submitted to
Plant Biotechnology,
a section of the journal
Frontiers in Plant Science
Received: 27 October 2015
Accepted: 08 January 2016
Published: 04 February 2016
Andre CM, Hausman J-F
and Guerriero G (2016) Cannabis
sativa: The Plant of the Thousand
and One Molecules.
Front. Plant Sci. 7:19.
doi: 10.3389/fpls.2016.00019
Cannabis sativa: The Plant of the
Thousand and One Molecules
Christelle M. Andre*, Jean-Francois Hausman and Gea Guerriero
Environmental Research and Innovation, Luxembourg Institute of Science and Technology, Esch-sur-Alzette, Luxembourg
Cannabis sativa L. is an important herbaceous species originating from Central Asia,
which has been used in folk medicine and as a source of textile fiber since the dawn
of times. This fast-growing plant has recently seen a resurgence of interest because
of its multi-purpose applications: it is indeed a treasure trove of phytochemicals and
a rich source of both cellulosic and woody fibers. Equally highly interested in this
plant are the pharmaceutical and construction sectors, since its metabolites show
potent bioactivities on human health and its outer and inner stem tissues can be
used to make bioplastics and concrete-like material, respectively. In this review, the
rich spectrum of hemp phytochemicals is discussed by putting a special emphasis
on molecules of industrial interest, including cannabinoids, terpenes and phenolic
compounds, and their biosynthetic routes. Cannabinoids represent the most studied
group of compounds, mainly due to their wide range of pharmaceutical effects in
humans, including psychotropic activities. The therapeutic and commercial interests of
some terpenes and phenolic compounds, and in particular stilbenoids and lignans, are
also highlighted in view of the most recent literature data. Biotechnological avenues to
enhance the production and bioactivity of hemp secondary metabolites are proposed
by discussing the power of plant genetic engineering and tissue culture. In particular
two systems are reviewed, i.e., cell suspension and hairy root cultures. Additionally,
an entire section is devoted to hemp trichomes, in the light of their importance as
phytochemical factories. Ultimately, prospects on the benefits linked to the use of
the -omics technologies, such as metabolomics and transcriptomics to speed up
the identification and the large-scale production of lead agents from bioengineered
Cannabis cell culture, are presented.
Keywords: fibers, hemp, Cannabis, cellulose, lignin, cannabinoids, terpenes, lignans
The current climatic and economic scenario pushes toward the use of sustainable resources to
reduce our dependence on petrochemicals and to minimize the impact on the environment. Plants
are precious natural resources, because they can supply both phytochemicals and lignocellulosic
biomass. In this review, we focus on hemp (Cannabis sativa L.), since it is a source of fibers, oil
and molecules and as such it is an emblematic example of a multi-purpose crop. We treat the
aspects related to the use of hemp biomass and, more extensively, those linked to its wide variety
of phytochemicals.
Frontiers in Plant Science | 1February 2016 | Volume 7 | Article 19
Andre et al. Cannabis sativa Fibers and Phytochemicals
Known since the ancient times for its medicinal and textile
uses (Russo et al., 2008;Skoglund et al., 2013), hemp is
currently witnessing a revival, because of its rich repertoire of
phytochemicals, its fibers and its agricultural features, namely
quite good resistance to drought and pests, well-developed root
system preventing soil erosion, lower water requirement with
respect to other crops, e.g., cotton. This shows the great versatility
of this fiber crop and encourages future studies focused on
both Cannabis (bio)chemistry and genetic engineering. Hemp
varieties producing oil, biomass or even both are currently
cultivated and the availability of the hemp genome sequence
greatly helps molecular studies on this important crop (van
Bakel et al., 2011). In addition, the scientific community is very
much interested in harnessing Cannabis pharmacological power:
for example microorganisms are being engineered to produce
9-tetrahydrocannabinolic acid (THCA) and cannabidiolic acid
(CBDA) (Taura et al., 2007a;Zirpel et al., 2015).
The final scope of this review is to discuss the potential
of hemp for industry and to highlight its importance for the
bio-economy. More specifically, we: (i) describe the use of
hemp biomass (i.e., the fibers), (ii) discuss hemp molecules of
industrial interest (namely cannabinoids, terpenes and phenolic
compounds), (iii) describe the potential of hemp trichomes
as pharma-factories and (iv) discuss the potential of genetic
engineering, by describing the use of plant cell suspension and
hairy root cultures.
Plant lignocellulosic biomass is an abundant renewable resource,
which can provide biopolymers, fibers, chemicals and energy
(Guerriero et al., 2014, 2015, 2016). Trees are important for
the provision of wood, however, also fast-growing herbaceous
species, like textile hemp (which has a THC content <0.3%;
Weiblen et al., 2015), can provide high biomass quantities in a
short time. The stem of this fiber crop supplies both cellulosic
and woody fibers: the core is indeed lignified, while the cortex
harbors long cellulose-rich fibers, known as bast fibers (Figure 1)
(Guerriero et al., 2013).
This heterogeneous cell wall composition makes hemp stem
an interesting model to study secondary cell wall biosynthesis,
in particular the molecular events underlying the deposition of
cortical gelatinous bast fibers and core woody fibers.
Cannabis woody fibers (a.k.a “hurds” or “shivs”) are used for
animal bedding because of their high absorption capacity and for
the creation of a concrete-like material.
Hemp bast fibers are used in the biocomposite sector as a
substitute of glass fibers. The automotive industry is particularly
keen on using hemp bast fibers to produce bioplastics: this
material is stronger than polypropylene plastic and lighter in
weight (Marsh, 2003).
Beyond the applications in the construction and automotive
industries, hemp fibers are attractive also in the light of their
natural antibacterial property. Hemp bast fibers have been
indeed described as antibacterial (Hao et al., 2014;Khan et al.,
2015) and their use for the manufacture of an antibacterial
finishing agent (Bao et al., 2014), surgical devices (Gu, 2006)or
functionalized textiles (Cassano et al., 2013) has been reported.
This property is linked to the chemical composition of hemp
bast fibers: both free and esterified sterols and triterpenes
have been identified, among which β-sitosterol and β-amyrin
(Gutiérrez and del Río, 2005). These compounds possess known
antibacterial properties (Kiprono et al., 2000;Ibrahim, 2012).
Hemp bast fibers were also found to contain cannabinoids (2%
of the total metabolite extract) (Bouloc et al., 2013 and references
therein). More recently hemp hurd powder showed antibacterial
properties against Escherichia coli (Khan et al., 2015). Since
the hurd has a higher lignin content than the bast fibers, its
antibacterial property may be linked to lignin-related compounds
such phenolic compounds, as well as alkaloids and cannabinoids
(Appendino et al., 2008;Khan et al., 2015).
Numerous chemicals are produced in hemp through the
secondary metabolism. They include cannabinoids, terpenes and
phenolic compounds (Flores-Sanchez and Verpoorte, 2008)and
will be further described in the next sections. Although the
pharmacological properties of cannabinoids have extensively
been studied and are the most recognized hemp bioactives,
the other components have no reasons to envy them, as
they have also been associated with potent health-promoting
properties. Research on Cannabis phytochemicals, as well as
the widespread therapeutic use of Cannabis products, has been
limited due to various reasons, including illegality of cultivation
(due to its psychoactivity and potential for inducing dependence),
variability of active components, and low abundance of some of
them in planta. Further attentions is now drawn toward non-
THC Cannabis active components, which may act synergistically
and contribute to the pharmacological power and entourage
effects of medicinal-based Cannabis extract (Russo, 2011).
Phytocannabinoids represent a group of C21 or C22 (for the
carboxylated forms) terpenophenolic compounds predominantly
produced in Cannabis. They have also been reported in plants
from the Radula and Helichrysum genus (Appendino et al., 2008)
but our knowledge on non-Cannabis source of cannabinoids is
still in its infancy (Gertsch et al., 2010). More than 90 different
cannabinoids have been reported in the literature, although
some of these are breakdown products (ElSohly and Slade, 2005;
Brenneisen, 2007;Radwan et al., 2009;Fischedick et al., 2010)and
they are generally classified into 10 subclasses (Brenneisen, 2007).
In this review, we will focus on the most abundant compounds
found in the drug- and fiber-type Cannabis.Thepredominant
compounds are THCA, CBDA and cannabinolic acid (CBNA),
followed by cannabigerolic acid (CBGA), cannabichromenic
acid (CBCA) and cannabinodiolic acid (CBNDA) (ElSohly and
Frontiers in Plant Science | 2February 2016 | Volume 7 | Article 19
Andre et al. Cannabis sativa Fibers and Phytochemicals
FIGURE 1 | Anatomical details of Cannabis stem. (A) Stem of an adult plant (ca 2 months); (B) The stem can be peeled off and shows a lignified core and a
cortex with bast fibers. (C) Longitudinal section of hemp stem stained with toluidine blue showing the cortex with a bundle of bast-fibers (white asterisk) and the core
with xylem vessels (black asterisk).
Slade, 2005). THCA is the major cannabinoid in the drug-
type Cannabis, while CBDA predominates in fiber-type hemps.
CBCA has been reported to dominate in the cannabinoid
fraction of young plants and to decline with maturation (Meijer
et al., 2009). The phytocannabinoid acids are non-enzymatically
decarboxylated into their corresponding neutral forms, which
occur both within the plant and, to a much larger extent, upon
heating after harvesting (Flores-Sanchez and Verpoorte, 2008).
Phytocannabinoids accumulate in the secretory cavity of the
glandular trichomes, which largely occur in female flowers and
in most aerial parts of the plants, as further described in the
next section. They have also been detected in low quantity in
other parts of the plants including the seeds (Ross et al., 2000),
roots (Stout et al., 2012) and the pollen (Ross et al., 2005), in
an extent depending on the drug- or fiber-type of Cannabis,
as described in Ta b l e 1 . More generally, the concentration of
these compounds depends on tissue type (Table 1), age, variety,
growth conditions (nutrition, humidity, light level), harvest
time and storage conditions (Khan et al., 2014). The level of
phytocannabinoids in hempseeds, and thereby of hempseed oil,
should be very low as the kernel contains only trace amount of
THC or CBD (Leizer et al., 2000;Ross et al., 2000). However,
higher THC concentrations are found on the outside surface
of the seed coat, possibly as the result of contamination with
plant leaves or flowers (Ross et al., 2000). Recently, significant
amounts of cannabinoids, and particularly of THC, were found
in five out of 11 hempseed oil samples available on the Croatian
market, suggesting that both contaminations are due to improper
processing procedures and the illegal use of drug-type hemp
(with a THC +CBN/CBD ratio >1) for nutritional purposes
c et al., 2015). Cannabinoids in the leaves have been
shown to decrease with the age and along the stem axis, with
the highest levels observed in the leaves of the uppermost nodes
(Pacifico et al., 2008). Cannabinoid contents in the stem are
scarce in the literature. An analysis performed on the dust
obtained from the top section of the stem of fiber-type hemp (low
percentage of bast fibers) revealed a low THC and CBD content
(0.04 and 1.3% on average, respectively) (Cappelletto et al., 2001).
Kortekaas et al. (1995) analyzed the cannabinoid content of hemp
black liquor. The sum of the THC and CBD fractions (without
reporting the distinct amounts of each of them) in hemp stem
wood and bark extractives was 2 and 1%, respectively, which
represented 0.003 and 0.0005% of the total fiber content.
Biosynthetic Pathway Leading to
The biosynthesis of cannabinoids from C. sativa has only been
recently elucidated. The precursors of cannabinoids actually
originate from two distinct biosynthetic pathways: the polyketide
pathway, giving rise to olivetolic acid (OLA) and the plastidal
2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, leading to
the synthesis of geranyl diphosphate (GPP) (Sirikantaramas et al.,
2007)(Figure 2). OLA is formed from hexanoyl-CoA, derived
from the short-chain fatty acid hexanoate (Stout et al., 2012),
by aldol condensation with three molecule of malonyl-CoA.
This reaction is catalyzed by a recently discovered polyketide
synthase (PKS) enzyme and an olivetolic acid cyclase (OAC)
(Gagne et al., 2012). The geranylpyrophosphate:olivetolate
geranyltransferase catalyzes the alkylation of OLA with GPP
leading to the formation of CBGA, the central precursor
of various cannabinoids (Fellermeier and Zenk, 1998). Three
oxidocyclases will then be responsible for the diversity of
cannabinoids: the THCA synthase (THCAS) converts CBGA to
THCA, while CBDA synthase (CBDAS) forms CBDA and CBCA
synthase (CBCAS) produces CBCA (Sirikantaramas et al., 2004,
2005;Taura et al., 2007b). Propyl cannabinoids (cannabinoids
with a C3 side-chain, instead of a C5 side-chain), such as
tetrahydrocannabivarinic acid (THCVA), synthetized from a
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Andre et al. Cannabis sativa Fibers and Phytochemicals
TABLE 1 | Summary of the concentrations in cannabinoids found in different parts of the hemp plants, in vitro hairy roots, and some commercial medicinal products.
Root Seed Stem Leaves Pollen Flower Bedrocan R
Molecules Fiber-
Drug-type Drug-
THC 1.04a0–12
(<0.5 in
CBD 1.67a14.3b67–
CBN 2–7d3.4–
CBG 1.63a2000f1000f1310h<600i1000–10000i11200i1700i
THCV 510h<600i(<600) – 1300i1300i<600i
CBC 3240h4 600i900–2200i2300i5400i
Data are expressed in μgg
-1 of dry weight. The most recent references have been used, when available. Abbreviations: THC, 9-tetrahydrocannabinol; CBD, cannabidiol; CBN, cannabinol; CBG, cannabigerol; THCV,
tetrahydrocannabivarin; CBC, cannabichromene. References: aFarag and Kayser, 2015;bAdapted from Stout et al., 2012;cRoss et al., 2000;dPetrovi ´
c et al., 2015 (concentration in hempseed oil); ePotter, 2004;
fPacifico et al., 2008 (growth curve experiment, the maximum concentrations are represented); gBruci et al., 2012;hRoss et al., 2005;iFischedick et al., 2010;jCappelletto et al., 2001, data from stem dust. Commercial
pharmaceutical preparations.
divarinolic acid precursor, have also been reported in Cannabis
(Flores-Sanchez and Verpoorte, 2008).
Health Benefits Linked to Cannabinoids
The pharmacology of phytocannabinoids has previously been
reviewed elsewhere (Pacher et al., 2006;Russo, 2011;Hill et al.,
2012;Giacoppo et al., 2014;Burstein, 2015) and a brief summary
and update will be presented hereafter.
Most of the biological properties related to cannabinoids
rely on their interactions with the endocannabinoid system in
humans. The endocannabinoid system includes two G protein-
coupled cannabinoid receptors, CB1 and CB2, as well as two
endogenous ligands, anandamide and 2-arachidonylglycerol.
Endocannabinoids are thought to modulate or play a regulatory
role in a variety of physiological processing including appetite,
pain-sensation, mood, memory, inflammation, insulin,
sensitivity and fat and energy metabolism (De Petrocellis
et al., 2011;Di Marzo and Piscitelli, 2015). The psychoactive
decarboxylated form of THCA, THC, is a partial agonist
of both CB1 and CB2 receptors, but has higher affinity for
the CB1 receptor, which appears to mediate its psychoactive
properties. In addition to being present in the central nervous
system and throughout the brain, CB1 receptors are also found
in the immune cells and the gastrointestinal, reproductive,
adrenal, heart, lung and bladder tissues, where cannabinoids
can therefore also exert their activities. CB2 receptors are
thought to have immunomodulatory effects and to regulate
cytokine activity. But THC has actually more molecular
targets than just CB1 and CB2 receptors, and exhibit potent
anti-inflammatory, anti-cancer, analgesic, muscle relaxant,
neuro-antioxidative (De Petrocellis et al., 2011), and anti-
spasmodic activities (Pacher et al., 2006). However, THC has
been also associated with a number of side effects, including
anxiety, cholinergic deficits, and immunosuppression (Russo,
2011). CBDA is the most prevalent phytocannabinoid in the
fiber-type hemp, and the second most important in the drug
chemotypes. CBD (decarboxylation of CBDA) presents a large
array of pharmacological properties, as recently reviewed in
Burstein (2015), which has been downplayed for many years,
as compared to THC. CBD acts yet as an important entourage
compound as it is able to reduce the side effects of THC
(Englund et al., 2012), and may thereby increase the safety of
Cannabis-based extracts. CBD itself has been shown in in vitro
and animal studies to possess, among others, anti-anxiety,
anti-nausea, anti-arthritic, anti-psychotic, anti-inflammatory,
and immunomodulatory properties (Burstein, 2015). CBD is
a very promising cannabinoid as it has also shown potential
as therapeutic agents in preclinical models of central nervous
system diseases such as epilepsy, neurodegenerative diseases,
schizophrenia, multiple sclerosis, affective disorders and the
central modulation of feeding behavior (Hill et al., 2012).
Interestingly, CBD presents also strong anti-fungal and anti-
bacterial properties, and more interestingly powerful activity
against methicillin-resistant Staphylococcus aureus (MRSA)
(Appendino et al., 2008). After THC and CBD, CBC is the
third most prevalent phytocannabinoid. CBC presents notably
anti-inflammatory (Delong et al., 2010), sedative, analgesic
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Andre et al. Cannabis sativa Fibers and Phytochemicals
FIGURE 2 | Schematic view of the biosynthetic pathways leading to the Cannabis secondary metabolites discussed in this review. Tr a nsp o r t of
precursors is represented by dashed arrows, while direct catalytic reactions are depicted by bold arrows. See text for detailed pathways. Abbreviations used: IPP,
isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate; MVA, mevalonate; MEP, methylerythritol
(Davis and Hatoum, 1983), anti-bacterial and antifungal
properties (Eisohly et al., 1982). CBC is also a potent inhibitor
of anandamide uptake, an endogenous ligand of CB receptors
(De Petrocellis et al., 2011). CBN is a degradation product of
THC and is mostly found in aged Cannabis. CBN has a twofold
lower affinity for CB1 receptors and a threefold higher affinity
for the CB2 receptors, as compared to THC. It thus affects
cells of the immune system more than the central nervous
system, as reviewed in (McPartland and Russo, 2001). Current
cannabinoid-based therapeutic treatments is limited to special
cases, i.e., spasticity associated to multiple sclerosis in adult
patients, to treat nausea/vomiting linked to cancer therapies,
to stimulate appetite in HIV-positive patients (Giacoppo et al.,
2014;Lynch and Ware, 2015). Borrelli et al. (2013),after
highlighting the beneficial effects of CBG on murine colitis,
suggest that this cannabinoid should also be considered for
clinical experimentation in patients affected by inflammatory
bowel disease.
Adverse Health Effects of Cannabinoids
As mentioned earlier, the recreational and medical use of
Cannabis as well as of THC and other synthetic cannabinoids
have also been associated with numerous side effects. Two recent
reviews (Volkow et al., 2014;van Amsterdam et al., 2015)
notably reported the adverse health effects linked to the use
of natural Cannabis and synthetic cannabinoids, respectively.
When adjusted for confounders such as cigarette smoking,
the impact of short- and long-term use appear to be similar
for both types of consumption and are directly linked to the
level of THC or its synthetic analog. The THC content of
recreational Cannabis has indeed drastically increased in the last
30 years (from 3% in 1980s to almost 20% now, as reported
in Tab l e 1), with very low level of the other cannabinoids
such as CBD. Effects of short-term use include memory and
cognitive deficits, impaired motor coordination, and psychosis.
Long-term use of THC has been associated to an increased risk
of addiction, cognitive impairment, altered brain development
when initial use was done early in adolescence, and an increased
risk of chronic psychosis disorder including schizophrenia. The
protective role that CBD could play to alleviate these negative
effects is now well established and documented (Iseger and
Bossong, 2015).
Terpenes form the largest group of phytochemicals, with
more than 100 molecules identified in Cannabis (Rothschild
et al., 2005;Brenneisen, 2007). Terpenes are responsible for
the odor and flavor of the different Cannabis strains. They
have therefore likely contributed to the selection of Cannabis
narcotic strains under human domestication (Small, 2015).
Terpenes are classified in diverse families according to the
number of repeating units of 5-carbon building blocks (isoprene
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Andre et al. Cannabis sativa Fibers and Phytochemicals
units), such as monoterpenes with 10 carbons, sesquiterpenes
with 15 carbons, and triterpenes derived from a 30-carbon
skeleton. Terpene yield and distribution in the plant vary
according to numerous parameters, such as processes for
obtaining essential oil, environmental conditions, or maturity
of the plant (Meier and Mediavilla, 1998;Brenneisen, 2007).
Mono- and sesquiterpenes have been detected in flowers, roots,
and leaves of Cannabis, with the secretory glandular hairs
as main production site. Monoterpenes dominate generally
the volatile terpene profile (from 3.1 to 28.3 mg g1of
flower dry weight, Fischedick et al., 2010) and include mainly
D-limonene, β-myrcene, α-andβ-pinene, terpinolene and
linalool. Sesquiterpenes, and β-caryophyllene and α- humulene
in particular, occur also to a large extent in Cannabis extracts
(from 0.5 to 10.1 mg g1of flower dry weight, Fischedick et al.,
2010). Triterpenes have also been detected in hemp roots, as
friedelin and epifriedelanol (Slatkin et al., 1971), in hemp fibers
as β-amyrin (Gutiérrez and del Río, 2005) and in hempseed
oil as cycloartenol, β-amyrin, and dammaradienol (Paz et al.,
Terpenes, along with cannabinoids, have successfully been
used as chemotaxonomic markers in Cannabis, as they are
both considered as the main physiologically active secondary
metabolites (Fischedick et al., 2010; Elzinga et al., 2015).
When grown in standardized conditions, a significant and
positive correlation was found between the level of terpenes
and cannabinoids (Fischedick et al., 2010). This may be
explained by the fact that mono- and sesquiterpenes are
synthesized in the same glandular trichomes in which the
cannabinoids are produced (Meier and Mediavilla, 1998). This
association was, however, not confirmed on a larger panel
of samples coming from different origins (Elzinga et al.,
Biosynthetic Pathways Leading to the
Different Classes of Terpenes
Two different biosynthetic pathways contribute, in their early
steps, to the synthesis of plant-derived terpenes (Figure 2).
Whereas the cytosolic mevalonic acid (MVA) pathway is
involved in the biosynthesis of sesqui-, and tri-terpenes, the
plastid-localized MEP pathway contributes to the synthesis of
mono-, di-, and tetraterpenes (Bouvier et al., 2005). MVA and
MEP are produced through various and distinct steps, from
two molecules of acetyl-coenzyme A and from pyruvate and
D-glyceraldehyde-3-phosphate, respectively. They are further
converted to isopentenyl diphosphate (IPP) and isomerised to
dimethylallyl diphosphate (DMAPP), the end point of the MVA
and MEP pathways. In the cytosol, two molecules of IPP (C5)
and one molecule of DMAPP (C5) are condensed to produce
farnesyl diphosphate (FPP, C15) by farnesyl diphosphate synthase
(FPS). FPP serves as a precursor for sesquiterpenes (C15),
which are formed by terpene synthases and can be decorated
by other various enzymes. Two FPP molecules are condensed
by squalene synthase (SQS) at the endoplasmic reticulum to
produce squalene (C30), the precursor for triterpenes and
sterols, which are generated by oxidosqualene cyclases (OSC)
and are modified by various tailoring enzymes. In the plastid,
one molecule of IPP and one molecule of DMAPP are
condensed to form GPP (C10) by GPP synthase (GPS). GPP is
the immediate precursor for monoterpenes (Kempinski et al.,
Health Benefits Associated with
Terpenes are lipophilic compounds that easily cross membranes
and the blood-brain barrier in particular (Fukumoto et al.,
2006). They present a wide-array of pharmacological properties,
which have recently been described in several reviews (Russo,
2011;Singh and Sharma, 2015). The biological activities of
D-limonene, also commonly found in Citrus essential oils, have
been well described in the literature. It notably exhibits potent
anti-cancer, anxiolytic and immunostimulating properties in
humans (Komori et al., 1995). β-myrcene, a terpene commonly
found in hop, is recognized as a potent anti-inflammatory,
analgesic, and anxiolytic component (Cleemput et al., 2009).
α-Pinene is an acetylcholinesteral inhibitor, and may thereby aid
memory abilities (Kennedy et al., 2011), which could counteract
the memory deficits induced by THC. Linalool, commonly
found in Lavandula angustifolia, possesses similar properties
to the ones described for its monoterpene counterparts, i.e.,
analgesic, anti-anxiety, anti-inflammatory, and anticonvulsant
(Russo, 2011). β-caryophyllene, a well-known active principle
of black pepper and Copaiba balsam, possesses potent anti-
inflammatory and gastric cytoprotector activities (Singh and
Sharma, 2015). Interestingly, it selectively binds to the CB2
receptor and could therefore technically be considered as a
phytocannabinoid (Gertsch et al., 2008). Pentacyclic triterpenes
such as β-amyrin and cycloartenol have been shown to possess
numerous biological activities including anti-bacterial, anti-
fungal, anti-inflammatory and anti-cancer properties ( z quez
et al., 2012;Moses et al., 2013). These triterpenes are key
contributors to the pharmacological properties of numerous
medicinal herbs (Kirby et al., 2008;Yadav et al., 2010;Sawai and
Saito, 2011).
Phenolic Compounds
Phenolic compounds, also known as phenylpropanoids,
constitute one of the most widely distributed group of secondary
metabolites in the plant kingdom. They present more than
10,000 different structures, including phenolic acids, such
benzoic and hydroxycinnamic acids, flavonoids such as flavonols
and flavones, stilbenes and lignans (Andre et al., 2010). In
Cannabis, about 20 flavonoids have been identified, mainly
belonging to the flavone and flavonol subclasses (Flores-Sanchez
and Verpoorte, 2008). These include the O-glycoside versions
of the aglycones apigenin, luteolin, kaempferol and quercetin,
as well as cannflavin A and cannflavin B, which are methylated
isoprenoid flavones that are unique to Cannabis (Figure 2)
(Ross et al., 2005). Phenolic amides and lignanamides have also
been described in Cannabis fruits and roots (Sakakibara et al.,
1992;Lesma et al., 2014). The lignanamides belong to the lignan
class of compounds and include cannabisin-like compounds
(of the types A-, B-, C-, D-, E-, F-, and G) and grossamide
(Flores-Sanchez and Verpoorte, 2008). Similar compounds
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Andre et al. Cannabis sativa Fibers and Phytochemicals
such as cannabisin D, have been described in Cannabis leaves,
where it was strongly induced upon the UV-C treatment (Marti
et al., 2014). Interesting amounts of lignans were recently
found in the hydrophilic extract of hemp seeds. The hemp seed
lignan profile was shown to be dominated by syringaresinol
and medioresinol, followed by secoisolariciresinol, lariciresinol,
and pinoresinol (Smeds et al., 2012). Hemp seeds contain,
however, about 20-times less total lignans (32 mg of total lignans
per 100 g of dry weight) than flax seeds, a well-known source
of lignans. Interestingly, the lignan content of hulled hemp
seeds represents only 1% of the content in whole seed (Smeds
et al., 2012). Nineteen stilbenes have been isolated in Cannabis
with characteristic structural backbones such as spirans,
phenanthrenes and bibenzyls (Flores-Sanchez and Verpoorte,
2008). They include molecules such as cannabistilbene I, IIa
and IIb, as well as dihydroresveratrol. Interestingly, bibenzyl
stilbenes, including the putative 3-O-methylbatatasin, were
strongly induced in Cannabis leaves by UV radiations (Marti
et al., 2014).
Biosynthetic Pathway Leading to the
Different Classes of Phenolic
Phenolic compounds are produced through the phenylpropanoid
pathway in the cytoplasm and are subsequently transported
in the vacuole or deposited in the cell wall (Figure 2).
Routes to the major classes of phenolic compounds involve
(i) the core phenylpropanoid pathway from phenylalanine to
an activated (hydroxy) cinnamic acid derivative (p-coumaroyl
CoA), via the actions of the phenylalanine-ammonia-lyase
(PAL), cinnamate 4-hydroxylase (C4H, a cytochrome P450)
and 4-coumarate-CoA ligase (4CL), as well as specific branch
pathways for the formation of (ii) simple esters, lignins and
lignans, (iii) flavonoids, (iv) coumarins, and (v) stilbenes
(Andre et al., 2009;Naoumika et al., 2010;Docimo et al.,
2013)(Figure 2). Although the flavonoid pathway has been
extensively studied in several plants, there is no specific data
on the biosynthesis of flavonoids in Cannabis. Generally,
lignans such as secoisolariciresinol are produced in planta by
stereoselective coupling of coniferyl alcohol moieties, via two
distinct dirigent proteins, giving rise to (+)or() pinoresinol.
Each pinoresinol can then be further enantiospecifically reduced
to lariciresinol and secoisolariciresinol (Dalisay et al., 2015).
The key molecular events associated with the biosynthesis
of lignanamides are still unknown. The structure of these
molecules suggests, however, a condensation of the precursors
tyramine and CoA-esters of coumaric, caffeic, and coniferic
acid (Flores-Sanchez and Verpoorte, 2008), followed by an
oxidative coupling reaction catalyzed by a dirigent protein,
as described for lignans. The flavonoid pathway is initiated
by condensation of p-coumaroyl CoA with three molecules
of malonyl-CoA (Figure 2). Naringenin chalcone is rapidly
isomerized by the enzyme chalcone isomerase (CHI) to form
naringenin, the branch point of flavonols on one hand and
flavones on the other one. Flavanone 3-hydroxylase (F3H)
may subsequently hydroxylate naringenin to produce the
dihydroflavonol, dihydrokaempferol, which can be further
hydroxylated by flavonoid 3hydroxylase (F3H) to form
dihydroquercetin. Dihydrokaempferol and dihydroquercetin
are substrates of flavonol synthase (FLS), which catalyzes
the production of the flavonols kaempferol and quercetin,
respectively. Naringenin may alternatively be converted to
apigenin, by a reaction catalised by a flavone synthase (FNS).
Apigenin can be further hydroxylated by a flavonoid 3
hydroxylase (F3H) to form luteolin which is likely the precursor
of the diverse cannflavins (Flores-Sanchez and Verpoorte, 2008).
Health Benefits Associated with Phenolic
In plants, phenolic compounds may act as antioxidants
under certain physiological conditions and, thereby, protect
plants against oxidative stress. In humans, it was shown that
there is a correlation between dietary phenolic compound
intake and a reduced incidence of chronic diseases such as
cancers, cardiovascular and neurodegenerative diseases (Arts
and Hollman, 2005), but these positive health effects may not
be entirely explained by the phenolic antioxidant properties,
as they are poorly bioavailable. Phenolic compounds may
induce the up-regulation of endogenous antioxidant enzymes
in vivo, due to their ability to act as pro-oxidants and
generate Reactive Oxygen Species (ROS) (Halliwell et al.,
2005). They may also exert their action through non-specific
protein binding interactions (Gertsch et al., 2010). The flavones
and flavonols found in Cannabis exert a wide range of
biological effects, including properties shared by terpenes and
cannabinoids. They present anti-inflammatory, anti-cancer and
neuro-protective properties as reviewed in (Andre et al., 2010).
In addition, apigenin has been shown to possess anxiolytic
(Murti et al., 2012) and oestrogenic properties (Wang and
Kurzer, 1998). The specific cannflavin A et B are potent
anti-inflammatory compounds, via inhibition of prostaglandin
E2 and 5-lipoxygenase (Werz et al., 2014). Health-related
studies concerning lignanamides are scarce and showed in vitro
anti-inflammatory (Sun et al., 2014) and cytotoxic activities
(Cui-Ying et al., 2002). Lignans in general show a wide
array of health-promoting properties including antioxidant,
antiviral, antidiabetic, antitumorigenic and anti-obesity activities.
Interestingly, secoisolariciresinol, lariciresinol and pinoresinol
are converted into enterolignans by the anaerobic intestinal
microflora, which are mammalian oestrogen precursors (phyto-
oestrogens) (Wang et al., 2010). Due to the structural similarity
of enterolignans with mammalian oestrogens, these compounds
are potentially interesting for combating some hormone-
dependent cancers. The mechanisms of action of the lignans are,
however, complex, with multiple targets involved (Sainvitu et al.,
It is now well accepted that the health benefits of fruits, vegetables
and other plant foods are due to the synergy or interactions
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Andre et al. Cannabis sativa Fibers and Phytochemicals
between the different bioactive compounds or other nutrients
present in the whole foods, and not to the action of a sole
compound (Liu, 2013). Similarly, Cannabis-based therapeutics
exert their pharmacological effects in humans via synergistic
or antagonistic interactions between the various phytochemicals
described above. These interactions may occur through various
mechanisms including: (i) bioavailability, (ii) interference with
cellular transport processes, (iii) activation of pro-drugs or
deactivation of active compounds to inactive metabolites, (iv)
action of synergistic partners at different points of the same
signaling cascade (multi-target effects) or (v) inhibition of
binding to target proteins (Efferth and Koch, 2011). A good
example is the stronger muscle-antispastic effect of a Cannabis
extract compared to pure THC, which represents an important
finding for the treatment of multiple sclerosis (Wagner and
Ulrich-Merzenich, 2009). Non-THC cannabinoids have shown
positive influence on the side effects induced by THC such
as anti-anxiety activities. CBD may also reduce the induced
cognitive and memory deficits in subjects smoking Cannabis
(Wright et al., 2013). CBD affects the pharmacokinetics of THC
through different mechanisms: (i) by fluidizing the membranes
and therefore increasing the penetration of THC in muscle
cells, and (ii) by inhibiting the P450-mediated hepatic drug
metabolism, which is involved in the degradation and elimination
of the molecule (Klein et al., 2011). Terpenes may also alter
the pharmacokinetics of THC by increasing the blood-brain
barrier permeability. This characteristic has notably been used to
patent a transdermal patch, which delivers cannabinoids into the
bloodstream by using a terpene as a permeation agent (Smith,
2015). Terpenes may also modulate the affinity of THC for
the CB1 receptor and interact with neurotransmitter receptors,
which may support contributions of terpenes on cannabinoid-
mediated analgesic and psychotic effects (McPartland and Russo,
2001;Russo, 2011). In view of the potential of phytocannabinoid-
terpene synergy, it has been suggested to tailor novel therapeutic
treatments such as CBD-terpene extracts to be used against acne,
MRSA, depression, anxiety, insomnia, dementia and addiction
(Russo, 2011).
Flavonoids may also modulate the pharmacokinetic of THC,
via inhibition of the hepatic P450 enzymes (3A11 and 3A4)
(McPartland and Russo, 2001;Russo, 2011).
Finally, there is an example of predator-targeted synergy
between terpenes and phytocannabinoids in the Cannabis plant
itself: on one side, the specific mixture of monoterpenes and
sesquiterpenes determines viscosity and thereby the stickiness of
Cannabis exudations necessary to trap the insects, and on the
other one, the phytocannabinoid acid acts as potent insecticidal
molecules (Sirikantaramas et al., 2005;Russo, 2011).
Trichomes are epidermal protuberances covering the leaves,
bracts and stems of plants and some of them, like the
glandular trichomes, are capable of secreting (or storing)
secondary metabolites as a defense mechanism. Several papers
have focused on the characterization of these specialized
structures using -omics (Wang et al., 2009a;Schilmiller et al.,
2010;McDowell et al., 2011;Jin et al., 2014), because their
integrated study can favor the development of technologies
harnessing their rich biochemical potential (Schilmiller et al.,
2008). An -omics database (TrichOME; available at: http:// enabling comparative analyses in plant
trichomes has also been created with the purpose of providing
the researchers with the possibility to mine data relative
to metabolites, genes, expression profiles (Dai et al., 2010).
Additionally, several procedures (in some instances supported
by a video demonstration; e.g., Nayidu et al., 2014)for
the isolation of trichomes from the leaves of different plant
species are available (e.g., Marks et al., 2008;Balcke et al.,
Hemp has different types of trichomes (Figures 3A–F)which
belong to two categories, i.e., glandular and non-glandular
(Happyana et al., 2013). Capitate sessile, capitate stalked and
bulbous hemp trichomes are secretory structures (Figures 3C–F).
In Cannabis THCA is accumulated in the heads (glands)
of both capitate-stalked and capitate sessile trichomes, but
in the former the content is higher (Mahlberg and Kim,
2004). Notably, in the textile variety, the cannabinoids CBDA
and CBCA occur at high concentrations instead of THCA,
while the reverse is true for drug strains (Mahlberg and Kim,
Studies on hemp have demonstrated that THCA is synthesized
in the storage cavity and that the enzyme responsible for THCA
production, i.e., THCAS, follows a sorting pathway from the
secretory cells to the storage cavity (Sirikantaramas et al., 2005).
The accumulation in the storage cavity is due to the cytotoxicity
of cannabinoids: they induce indeed death via apoptosis, when
supplied for 24 h to both hemp and tobacco cell suspension
cultures (Sirikantaramas et al., 2005). Heterologous expression
of THCAS fused to GFP in tobacco leads to fluorescence
of the trichome heads, thereby confirming the localization
of the enzyme in the storage cavity (Sirikantaramas et al.,
Depending on their color, hemp glandular trichomes show
different secretory phases (Mahlberg and Kim, 2004): the mature
secreting gland appears translucent (at this stage the cannabinoid
content is the highest), while aging glands are yellow and
senescing brown.
According to the current model cannabinoids are produced
via terpenes secreted by plastids present in the disk cells
and phenols stored in their vacuole (Mahlberg and Kim,
2004): analyses using the electron microscope have shown
that oily secretions (most likely terpenes) round in shape
are secreted from the plastids (which have the appearance
of reticulate bodies). Subsequently vesicles are released into
the cavity together with fibrillar matrix originating from
the cell walls of the disk cells. The fibrillar matrix is
transported to the subcuticular cell wall and contributes to its
thickening via yet unidentified mechanisms (Mahlberg and Kim,
Besides cannabinoids, Cannabis trichomes produce other
secondary metabolites, namely terpenes (see previous paragraph
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Andre et al. Cannabis sativa Fibers and Phytochemicals
FIGURE 3 | Hemp trichome types. (A) Unicellular non-glandular trichome; (B) cystolythic trichomes; (C) capitate sessile trichome; (D) capitate-stalked trichome;
(E) simple bulbous trichome; (F) complex bulbous trichome. Images kindly provided by Dr. David J. Potter.
FIGURE 4 | Workflow showing the achievements (in green) and potential future approaches (in light blue) to produce cannabinoids in cultures of
Cannabis, as well as other plant hosts.
on Cannabis phytochemicals), which are responsible for the
typical plant aroma (Russo, 2011). Among the Cannabis terpenes
of low abundance, is nerolidol (0.09% of the total terpene
content, Ross and ElSohly, 1996), which, interestingly, has anti-
malarial and anti-leishmanial effects (reviewed by Russo, 2011).
Given the pharmacological importance of these compounds, it
would be interesting to devise engineering strategies aiming
at either boosting the secondary metabolism, or increasing
the density of trichomes in Cannabis. Among the possible
genetic engineering approaches, it is here worth mentioning two
examples recently reported in Artemisia annua.Wewillhere
discuss only these two examples, as further discussion on how to
scale up the production of cannabinoids is presented later in this
It has been recently shown that the transformation of A. annua
with the rolB and rolC genes of Agrobacterium rhizogenes led to
plants with an increased content of artemisinin (Dilshad et al.,
2015). The rol genes are known for their stimulatory action
on plant secondary metabolism (Bulgakov, 2008). The study on
A. annua showed that rolB and rolC trigger different effects,
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Andre et al. Cannabis sativa Fibers and Phytochemicals
with rolB showing enhanced production with respect to rolC.
An additional study on A. annua has shown that the expression
of a β-glucosidase from Trichoderma reesei increases glandular
trichome density and artemisinin production (Singh et al., 2015).
The hydrolytic enzyme favors the release of active plant growth
regulators from the conjugates stored in the plastids, thereby
favoring trichome formation, as well as biomass production and
leaf area (Singh et al., 2015). It would be interesting to devise an
engineering strategy aimed at increasing the density of trichomes
in Cannabis, by adopting a similar strategy. –Omics studies on
Cannabis trichomes will help identify important genes, among
which transcription factors (involved in trichome formation),
which can be likewise used for engineering approaches.
Cannabis is a precious plant with multiple applications, hence
the possibility of engineering it genetically to produce useful
compounds/raw products is highly valuable. In this section of
the review we will: (i) discuss the progress made in Cannabis
in vitro propagation together with the biotechnological prospects
of Cannabis genetic engineering, by highlighting the challenges
and benefits, (ii) describe the hairy root culture system as
a tool for the scalable production of cannabinoids and (iii)
discuss the advantages of the Cannabis cell suspension culture
Cannabis In Vitro Propagation and
The cultivation of Cannabis is severely regulated in many
countries; therefore alternative in vitro growth techniques
are receiving a lot of attention. The in vitro cultivation
of Cannabis is also an advantageous way to preserve
cultivars/clones (Lata et al., 2009a) with specific metabolite
Methods to multiply C. sativa plants in vitro via stimulation
of axillary buds on nodal segments, or induction of adventitious
buds in the shoot tips have been described (Lata et al., 2009a;
Wang et al., 2009b). It was shown that micro-propagated plants
are genetically stable; therefore the method is appropriate and
useful for the clonal multiplication of this important crop (Lata
et al., 2010).
A protocol has also been developed for the propagation
of hemp via the synthetic seed technology. According to this
procedure, axillary buds or nodal segments are encapsulated in
calcium alginate beads (Lata et al., 2009b, 2011), which can then
be stored and subsequently used for clonal propagation of the
plant. This system was shown to allow the successful growth of
homogeneous and genetically stable Cannabis plants even after
6 months of storage (Lata et al., 2011).
To set up a successful Cannabis transformation protocol,
the mastery of in vitro culture techniques is necessary: whether
the strategy adopts plant explants or undifferentiated calli
as starting material, the regeneration of the whole plant is a
mandatory step. Organ regeneration, in particular shoots, can
be quite cumbersome and therefore the screening of different
plant growth regulator concentrations and combinations
has to be carried out to find the right culture medium
Cannabis sativa is a notorious recalcitrant plant to
transformation, because the regeneration efficiencies are
quite low and dependent upon the cultivar, tissue, plant age
and growth regulator combination (Slusarkiewicz-Jarzina et al.,
2005). As an example, although successful transformation of
hemp calli via Agrobacterium tumefaciens was reported by Feeney
and Punja (2003), the undifferentiated cells failed to regenerate
the shoots. The cells were transformed with phosphomannose
isomerase and colorimetric assays showed successful expression
of the transgene.
Nevertheless some success in hemp regeneration was reported
and shown to be linked to the choice of specific plant growth
regulators. For example the addition of thidiazuron (TDZ),
which has cytokinin-like activity, was shown to increase the
development of shoots in hemp explants (Lata et al., 2009a)and
in leaf-derived calli of a high yielding THCA clone (Lata et al.,
2010). The herbicide DICAMBA was also reported to favor the
regeneration of hemp shoots from calli (Slusarkiewicz-Jarzina
et al., 2005).
Cannabis transformation protocols using plant explants
(thereby avoiding the passage to undifferentiated cells) have been
described for several important crops (e.g., cotton, Zapata et al.,
1999;jute,Saha et al., 2014). Notably, successful transformation
of hemp plants was reported by MacKinnon et al. (2001)
using shoot tips: the protocol uses shoot tip explants and
the regeneration potential of the shoot apical meristem after
infection with A. tumefaciens. Additionally a patent application
was filed describing Cannabis transformation using 1–2 cm
hypocotyl explants, the plant growth regulators zeatin and 6-
benzylaminopurine (BAP) for shoot regeneration (Sirkowski,
Hairy Root Cultures for the Production of
An additional system offering interesting applications for the
industrial production of compounds showing pharmaceutical
effects in humans is the hairy root system, a type of
Agrobacterium-transformed plant tissue culture used to
study plant metabolic processes. Transformation of hemp
and subsequent establishment of hairy root culture has been
described by Wahby et al. (2013) using both A. rhizogenes
and A. tumefaciens. In this study hypocotyls were found to
be the most responsive tissue for infection. The hairy root
system is very interesting for the production of secondary
metabolites in medicinal plants (Jiao et al., 2014;Patra and
Srivastava, 2014;Wawrosch et al., 2014;Gai et al., 2015;Tian,
valuable metabolites. For example, in tobacco transgenic hairy
roots the production of THCA was successfully obtained by
expressing hemp THCAS (Sirikantaramas et al., 2007). The
hairy root system is characterized by hormone-independent
high growth rate and by the same metabolic potential as
the original organ (Pistelli et al., 2010). A protocol for the
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Andre et al. Cannabis sativa Fibers and Phytochemicals
establishment of hairy roots from Cannabis callus cultures has
also been described (Farag and Kayser, 2015). In this study
calli were grown on full-strength B5 medium supplemented
with 4 mg/L 1-Naphthaleneacetic acid (NAA) and their
potential of cannabinoid production was evaluated. The authors
found that after 28 days of cultivation in the dark, a peak
could be observed in the accumulation of cannabinoids in
culture media supplemented with different concentrations
of indole-3-acetic acid (IAA). However, the yield remained
below 2 μg/g of dry weight, thereby showing that further
optimizations are still required in this field. The induction of
rhizogenesis in undifferentiated Cannabis cells is important,
because it can be performed on calli overexpressing key
transcription factors and/or genes involved in the cannabinoid
The production of cannabinoids in hemp hairy root cultures
can be then further implemented with adsorbents to avoid
toxicity issues (a more detailed discussion concerning possible
ways to avoid toxicity is present in the section dedicated to
heterologous plant hosts). In alternative, inducible promoters
can be used, like for instance the glucocorticoid-inducible
promoter, which was already shown to be effective in inducing
a controlled, reversible and dosage-dependent expression of
GFP in Catharanthus roseus hairy roots (Hughes et al.,
Cannabis Cell Suspension Cultures for
the Production of Cannabinoids
Plant cell suspension cultures offer important advantages, as they
can be transformed and then cultivated in bioreactors for the
production of useful metabolites (Weathers et al., 2010;Bortesi
et al., 2012;Liu et al., 2012;Han et al., 2014). Cannabis callus
cultures are not able to produce any cannabinoids, irrespective
of the chemotypes (drug-, hybrid-, or fiber-type) used as mother
plants or growth regulators used in the culture medium (Pacifico
et al., 2008). The transformation of hemp cell suspension cultures
with genes involved in specific metabolic pathways can offer the
possibility of enhancing the production of important classes of
metabolites such as cannabinoids but also of others with potential
pharmacological use. In this paragraph we will discuss about
potential biotechnological approaches to boost the production of
cannabinoids in Cannabis cell suspension culture.
The increased production of cannabinoids in Cannabis
cell suspension cultures can be achieved via the expression of
transcription factors involved in Cannabis gland biochemistry
(Figure 4). Transcription factors represent a powerful tool
in plant metabolic engineering, because of their “cascade”
mechanism of action: if master regulators involved in
cannabinoid biosynthesis are identified in C. sativa trichomes,
they could be expressed constitutively or inducibly in Cannabis
cell suspension cultures. It is important to mention here that two
transcription factors belonging to the MYB family were already
shown to be preferentially expressed in Cannabis glands (Marks
et al., 2009) and therefore represent ideal candidates to express.
These genes show homology with Arabidopsis thaliana MYB112
and MYB12, which are known to be involved in the tolerance to
oxidative stress and flavonol biosynthesis, respectively (Marks
et al., 2009 and references therein). The expression of these
transcription factors in an inducible manner is a strategy worth
being tested for the production of cannabinoids. The inducible
expression will limit the negative effects caused by the toxicity
of the accumulating cannabinoids during the growth of the
transformed plant cells (as more thoroughly described in the
next section).
In addition to the genetic engineering approach, plant cell
suspension cultures can be elicited to boost the production
of secondary metabolites. The literature is rich in examples
concerning the increased expression of secondary metabolites
in plant cells elicited with different factors (reviewed recently
by Ncube and Van Staden, 2015). Both biotic and abiotic stress
factors can indeed be used to re-direct the plant metabolism:
nutrients, light, temperature, fungal elicitors are among the most
common factors manipulated.
In hemp suspension cells, elicitation with biotic and abiotic
elicitors did not induce an increase in cannabinoids (Flores-
Sanchez et al., 2009); however, jasmonic acid was shown to elicit
the production of the antioxidant tyrosol (Pec et al., 2010).
It is here worth mentioning the effect of a so far neglected
element, silicon (Si). Despite being a non-essential element
for plant growth, Si is known to increase plant vigor and to
alleviate the effects of exogenous stresses (Epstein, 2009). Very
recently Si was shown to alleviate the effects of salt stress and to
induce the production of chlorogenic acid in Lonicera japonica
(Gengmao et al., 2015). Given the stimulatory effects that Si has
on plant metabolism, it is interesting to further investigate, from
a molecular perspective, the effects of Si supplementation on
Cannabis secondary metabolite production. Cyclodextrins have
also been used in plant cell suspension cultures to enhance the
production of various non-polar metabolites such as stilbenes
(Yang et al., 2015), phytosterols (Sabater-Jara and Pedreño, 2013)
or triterpenes (Goossens et al., 2015). Cyclodextrins are cyclic
oligosaccharides consisting of five or more α-D-glucopyranose
residues. They are known to form inclusion complexes with
lipophilic compounds, including cannabinoids (Hazekamp and
Verpoorte, 2006), in their hydrophobic cavity, thereby improving
metabolite solubility in an aqueous environment. In addition,
cyclodextrins, thanks to their chemical structure similar to that
of the alkyl-derived oligosaccharides released from plant cell wall
when a fungal infection occurs, act as elicitors of secondary
metabolite production (Sabater-Jara and Pedreño, 2013).
It would therefore be worth investigating the effect of
cyclodextrins on the production of the non-polar cannabinoids
in hemp suspension cell cultures.
The expression of genes involved in the cannabinoid biosynthetic
pathway in cell suspension cultures of plants other than
Cannabis represents an interesting alternative for the scalable
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Andre et al. Cannabis sativa Fibers and Phytochemicals
production of cannabinoids (Figure 4). For example synthetic
biology could be used to recreate the cannabinoid biosynthetic
pathway in heterologous plant cells via the expression of
THCAS, together with the upstream enzymes involved in
the synthesis of CBG, i.e., the tetraketide synthase (the
type III PKS), the aromatic prenyltransferase and the OAC
(Gagne et al., 2012). In this respect tobacco bright yellow
2 (BY-2) cells are very interesting expression hosts, given
their wide use in plant biotechnology as “workhorse” for
the production of recombinant proteins (e.g., Reuter et al.,
The biomimetic production of cannabinoids in heterologous
plant hosts is challenging, however, one strategy that is worth
taking into account concerns the use of synthetic “metabolons”
(Singleton et al., 2014). A “metabolon” is the association of
enzymes which carry out a series of sequential reactions in
a given pathway. Examples for the occurrence of metabolons
exist in plants for pathways involving, e.g., the synthesis of
phenylpropanoids (Chen et al., 2014) and the cyanogenic
glycoside dhurrin (Nielsen et al., 2008). Entire metabolic
pathways can be engineered via the use of synthetic metabolons
enabling the association of enzymes in close proximity: this
allows a more efficient shunting of intermediates at the active
siteofenzymesactinginchain(Singleton et al., 2014). One
possible way to assemble a synthetic metabolon is via the
use of a scaffolding protein enabling the association of the
enzymes (Singleton et al., 2014;Pröschel et al., 2015). In the
specific case of cannabinoid production, the creation of a
synthetic metabolon comprising for instance the type III PKS
and OAC (Gagne et al., 2012), together with the aromatic
prenyltransferase and the THCAS, can be achieved via (i)
the use of dockerin-cohesin modules, or (ii) the metazoan
signaling proteins SH3-, PDZ-, GBD binding domains, or (iii)
the SpyTag/SpyCatcher domains (recently reviewed by Pröschel
et al., 2015).
The assembly of multimodular constructs for expression in
plants is no longer an insurmountable challenge, thanks to the
development of methods like the Gateway-mediated cloning
(reviewed by Dafny-Yelin and Tzfira, 2007), Golden Gate (Binder
et al., 2014), GoldenBraid (Sarrion-Perdigones et al., 2011), to
name a few.
When cannabinoids are produced in heterologous plant
hosts, toxicity effects have to be taken into account, as it
was shown that THCA and CBGA cause cell death via
apoptosis in cells of Cannabis and tobacco BY-2 (Sirikantaramas
et al., 2005). For plant cell suspension cultures cultivated
in bioreactors, the in situ product removal via a two-phase
culture system might be useful to favor the accumulation of
the toxic metabolites produced in sites which are separated
from the cells (Cai et al., 2012)(Figure 4). The use of
adsorbents in the culture medium can not only sequester
the toxic compounds, but also stimulate the secondary
metabolite biosynthesis (Cai et al., 2012 and references
One additional approach that can be used to avoid
product toxicity in plant cell suspension cultures is
artificial compartmentalization (Figure 4). This approach
has been recently proposed in A. annua cell cultures
for the production of artemisinin (Di Sansebastiano
et al., 2015). The authors induced the formation of
an artificial compartment (generated by membranes
deriving from endocytosis and the endoplasmic
reticulum-vacuole trafficking) via the expression of a
truncated SNARE protein, AtSYP51. The creation of an
artificial compartment can be used for the production
of cannabinoids, because it can trap and stabilize
the toxic secondary metabolites until extraction is
performed, in a manner analogous to what discussed for
Hemp is a unique versatile plant, which can provide high biomass
quantities in a short time. Hemp stem is used as a source of woody
and bast fibers for the construction and automotive industries,
while hemp seeds are used as a source of dietary oil and hemp
leaves and flowers as a source of bioactive components.
To date, more than 540 phytochemicals have been described
in hemp (Gould, 2015), and their pharmacological properties
appear to go much beyond psychotic effects, with the capacity to
address needs like the relief of chemotherapy-derived nausea and
anorexia, and symptomatic mitigation of multiple sclerosis.
Continuously discovering new prototypes of drugs is of
tremendous importance to meet tomorrow’s challenges in terms
of public health (Atanasov et al., 2015). Nature has already
provided a large source of new molecules and new skeletons.
A recent review reporting the new drugs available on the market
during the last 30 years showed that more than 35% of these
new drugs have a direct natural origin. This percentage rises
to over 60% if we take into account all the drugs whose
structure is inspired by a natural pharmacophore (Newman
and Cragg, 2012). Cannabis presents a colossal potential for
enlarging the library of bioactive metabolites. Compounds can
be obtained from hemp trichomes, cell suspension cultures,
hairy root systems, or via the biotransformation of THCA or
CBDA using fungal, bacterial, or plant cells (Akhtar et al.,
Our increasing knowledge on the key molecular components
triggering the diverse phytochemical pathways in planta
(Figure 2), may also allow, through a genetic engineering
approach, to further increase the production of specific
cannabinoids, terpenes, or phenolic compounds, or to
reconstruct the pathway in heterologous systems using a
synthetic biology approach. Apart from the importance
of studies focused on improving Cannabis genetic
transformation, it is necessary to know more about the
regulatory mechanisms involved in secondary metabolite
production in C. sativa. For example enzymological and
structural studies will help devise protein engineering
approaches to improve the catalytic functions of key
enzymes (Taura et al., 2007a). However, further studies
would still be needed to elucidate other key genes
Frontiers in Plant Science | 12 February 2016 | Volume 7 | Article 19
Andre et al. Cannabis sativa Fibers and Phytochemicals
involved in biosynthetic pathways of, for instance, less-
abundant cannabinoid derivatives. For that purpose,
the combination of metabolomics with genome-based
functional characterizations of gene products would
provide an accelerated path to discovering novel
biosynthetic pathways to specialized metabolites. Indeed,
the functions of numerous genes have been identified and
characterized through the correlation of gene expression
and metabolite accumulation (Sumner et al., 2015).
Classical approaches used focused on the spatial and
temporal distribution of the targeted phytochemicals
and on the plant transcriptome, as influenced by the
developmental stage and environmental stresses. With respect
to the resurgence of interest in Cannabis phytochemicals
nowadays, the results of such studies will be soon
CA was involved in the review writing, J-FH was involved in
manuscript refinement, and GG initiated the idea of the review
and was involved in the manuscript writing.
The authors wish to thank the support by the Fonds National de
la Recherche, Luxembourg (Project CANCANC13/SR/5774202).
Laurent Solinhac is gratefully acknowledged for providing the
longitudinal cross section image of hemp stem appearing in
Figure 1. The authors are grateful to Dr David J. Potter (GW
Pharmaceuticals Ltd, Salisbury, Wiltshire, UK) for providing the
trichome pictures appearing in Figure 3.
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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
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Frontiers in Plant Science | 17 February 2016 | Volume 7 | Article 19
... In addition, a growing perianth was observed during the early stages of flower development, which later withered, lost its individuality, and resembled a thin membrane (Spitzer-Rimon et al., 2019). Commercially, the plants are grown for their psychotropic substances, which are synthesized in the trichomes that appear on flower bracts in female inflorescences (Andre et al., 2016). Trichomes are the most significant morphological part of the flower as they have chemotaxonomic and pharmaceutical significance. ...
... C. sativa is distinguishable from other cannabis species by the complexity of its chemical composition. This composition includes terpenes, carbohydrates, amides, amines, phytosterols, phenolic compounds, fatty acids and their esters, and more (Andre et al., 2016). Cannabinoids, which are classified as terpenophenolics, make up the most common category of bioactive compounds found in cannabis. ...
... The plant has more or less 483 distinct components (Rajput & Kumar, 2018). Terpenoids are very potent metabolites, capable of influencing the behaviour of animals and even humans when breathed in very little levels; moreover, some studies have postulated that terpenoids and cannabinoids work synergistically with one another (Russo et al., 2008;Andre et al., 2016;Singh & Sharma, 2015;Tahir et al., 2021). Herbal cannabis includes at least 144 cannabinoids, the most well-known of which are tetrahydrocan-nabinol (THC), cannabidiol (CBD), cannabinol (CBN), -caryophyllene, Cannabigerol, etc., (Figure 1). ...
Cannabis sativa L. is a flowering plant in the family Cannabaceae, and has been cultivated since ancient times for its fibres, oils, resins, dried inflorescences, and leaves. It can be used for a variety of industrial purposes. Over the years, the therapeutic and pharmacological efficacy of its phytoconstituents is shown in a variety of human diseases and health. The use and exploitation of the plant have sparked controversy; however, there are recent legalizations of its use for medical and other purposes in many countries within the corresponding legislative framework. In addition to this legalization, C. sativa is encouraging the very rapid growth of the cannabis oriented pharmaceutical industry. This chapter summarized recent developments in the science of C. sativa and its products about their industrial application, while also addressing gaps in the existing knowledge and future research directions for this high-value multi-use, and potential industrial plant with universal benefits.
... Herbal medicine and plants were used as remedy for many unique forms of sicknesses such as liver, kidney, coronary heart sicknesses, therefore, scientists around the world have studied unique sorts of herbal plant in phrases of chemical and physiological properties, indications, and their side effects. Cannabis species is among the herb plants that has been studied by many scientists in vitro round the world [1]. Cannabis sativa typically called hemp, its one in every of the oldest cultivated plants, and determinative its specific origin of its long history of cultivation. ...
... Cannabis sativa L. is a popular herbaceous plant from central Asia that has been utilized in traditional medicine, it can be used as a drug (marijuana) or a non-drug (marijuana) (hemp). The former is often used for different purposes such as recreational and medical purposes, whilst the latter is critical to the fiber and food industry [1]. Different preparations of Cannabis sativa have been used as a traditional medicine in Asia to cure illnesses, including diarrhea, inflammation, headache, nausea, hematochezia and alopecia. ...
... Different preparations of Cannabis sativa have been used as a traditional medicine in Asia to cure illnesses, including diarrhea, inflammation, headache, nausea, hematochezia and alopecia. Cannabis sativa has anti-inflammatory, analgesic, antipyretic, and antidiarrhea properties [1,3,4]. Due to a variety of factors, includes unlawful cultivation, diversity of active components, and low quantity of certain of them in plant [5]. ...
Full-text available
Cannabis, sometimes known as hemp, is a plant that originated in Central Asia. Cannabis seeds nutritional profile is astounding. It is most digestible, balanced, natural, and complete supply of protein or amino acids. Cannabis seeds can be used in a variety of medical fields. Each 100 gm of the seeds used in the study contains 20, 26 and 37 gm of carbohydrate, protein and fat respectively. The present study used two groups of male rats (control and treatment groups). Rats were 11 weeks of average age. 10 mg/kg of body weight of powdered cannabis seeds were administrated orally to treatment group daily for 15 days. Alkaline Phosphatase, glutamic oxaloacetic transaminase and glutamic pyruvic transaminase (ALP, GOT, and GPT), creatinine and uric acid were performed for estimating the effect of cannabis seeds on renal and liver function. Enzymes Alkaline phosphatase, glutamic oxaloacetic transaminase and glutamic pyruvic transaminase (ALP, GPT, and GOT) as well as creatinine, uric acid and very low-density lipoprotein (VLDL) were not significantly (p<0.05) different in both groups. Level of Cholesterol, triglyceride and c-reactive protein were significantly (p<0.05) reduced. Serum total protein, albumin and globulin were significantly (p<0.05) elevated in treatment group in comparison to control group. In conclusion, this study found the effect of cannabis seeds on decreasing cardiovascular risk. It was found that Keywords: cannabis seeds, renal function, liver function, anti-inflammatory effect. Kurdistan Journal of Applied Research | Volume 7-Issue 1-June 2022 | 134 cannabis have good roles in protein metabolism, as well as albumin. The study has showed the role of cannabis in strengthen the immune system via elevation of globulin level. Moreover, cannabis seed has anti-inflammatory property through decreasing the level of CRP.
... It is widely regarded as a crop with various uses and a wide range of current and future applications, including those for nutrition, energy, textiles, healthcare, and a wide range of industrial goods [3]. The cannabis plant (Cannabis sativa L.) is one of the world's oldest crops, which has been widely cultivated due to its industrial [4], nutritional [5], medicinal, and psychotropic [6] products. Regulatory perspectives and terminology vary in different countries based on the concentration of ∆ 9 -tetrahydrocannabinol (THC). ...
Full-text available
Currently, there are increased interests in growing grain and fiber hemp (Cannabis sativa L.) as well as in large-scale hemp products. Cannabis has been grown/utilized for thousands of years as a fiber, grain, and drug/medicinal plant. However, the strict control of cannabis cultivation to combat illegal use, the spread of new yarns and oilseeds, and the advent of cheap synthetic fibers caused a decreased/eliminated hemp production. Hemp has been banned in most of the world for more than seven decades; it missed out on the Green Revolution and the adoption of new technologies and varieties, creating a knowledge gap. After the 2014 and 2018 Farm Bill in the USA, hemp became legal and the land grand universities launched research programs. The ability to utilize the entire plant for multiple purposes creates opportunity for the market to value hemp products. Hemp production technology varies depending on the type of hemp cultivated (grain, fiber, or cannabinoids), soil characteristics, and environmental factors. Hemp has the potential to be a very sustainable and ecologically benign crop. Hemp roots have a significant potential for absorbing and storing heavy metals such as lead, nickel, cadmium, and other harmful substances. In addition, hemp has been proven to be an excellent carbon trap and biofuel crop. Hemp has the ability to successfully suppress weeds, and it is generally regarded a pesticide-free crop. The purpose of this paper is to examine historic and recent industrial hemp (grain and fiber) literature, with a focus on hemp agronomy and utilization.