fpls-09-01969 January 8, 2019 Time: 15:54 # 1
published: 09 January 2019
Council for Agricultural and
Economics Research, Bologna, Italy
CREA-CIN Rovigo, Italy
Council for Agricultural and
Economics Research, Rome, Italy
Ethan B. Russo
This article was submitted to
a section of the journal
Frontiers in Plant Science
Received: 31 October 2018
Accepted: 19 December 2018
Published: 09 January 2019
Russo EB (2019) The Case
for the Entourage Effect
and Conventional Breeding of Clinical
Cannabis: No “Strain,” No Gain.
Front. Plant Sci. 9:1969.
The Case for the Entourage Effect
and Conventional Breeding of
Clinical Cannabis: No “Strain,” No
Ethan B. Russo*
International Cannabis and Cannabinoids Institute, Prague, Czechia
The topic of Cannabis curries controversy in every sphere of inﬂuence, whether politics,
pharmacology, applied therapeutics or even botanical taxonomy. Debate as to the
speciation of Cannabis, or a lack thereof, has swirled for more than 250 years. Because
all Cannabis types are eminently capable of cross-breeding to produce fertile progeny,
it is unlikely that any clear winner will emerge between the “lumpers” vs. “splitters” in
this taxonomical debate. This is compounded by the profusion of Cannabis varieties
available through the black market and even the developing legal market. While labeled
“strains” in common parlance, this term is acceptable with respect to bacteria and
viruses, but not among Plantae. Given that such factors as plant height and leaﬂet
width do not distinguish one Cannabis plant from another and similar difﬁculties in
deﬁning terms in Cannabis, the only reasonable solution is to characterize them by their
biochemical/pharmacological characteristics. Thus, it is best to refer to Cannabis types
as chemical varieties, or “chemovars.” The current wave of excitement in Cannabis
commerce has translated into a ﬂurry of research on alternative sources, particularly
yeasts, and complex systems for laboratory production have emerged, but these
presuppose that single compounds are a desirable goal. Rather, the case for Cannabis
synergy via the “entourage effect” is currently sufﬁciently strong as to suggest that one
molecule is unlikely to match the therapeutic and even industrial potential of Cannabis
itself as a phytochemical factory. The astounding plasticity of the Cannabis genome
additionally obviates the need for genetic modiﬁcation techniques.
Keywords: cannabis, cannabinoid, marijuana, hemp, genomics, genetically modiﬁed organism,
INTRODUCTION: DEFINING TERMS
Earlier data on taxonomy of Cannabis was previously reviewed (Russo, 2007), which will be
herein summarized and supplemented. Cannabis is a dioecious annual of the Cannabaceae
family which traditionally includes hops, Humulus spp. Alternatively, Cannabis has also been
assigned to Moraceae, Urticaceae, or even in the Celtidaceae families on the basis of chloroplast
restriction site maps (Weigreﬀe et al., 1998), and chloroplast mat K gene sequences (Song
et al., 2001). More recently, the Cannabaceae have subsumed eight genera: Celetis, Pteroceltis,
Aphananthe, Chaetachme, Gironniera, Lozanella, Trema, and Parasponia, comprising 170 odd
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species (McPartland, 2018), a ﬁnding supported by genetic
analysis of four plastid loci (Yang et al., 2013). Current
research on fossil pollen samples associated with the ecological
associations of Cannabis with steppe companion species
(Poaceae, Artemisia, Chenopodiaceae), and Humulus (hops) with
forest genera (Alnus, Salix, Populus), have established that
although Cannabis seems to have originated in the Tibetan
Plateau at least 19.6 million years ago, it has also been indigenous
to Europe for at least a million years (McPartland et al., 2018),
and refuted the conventional wisdom that this “camp follower”
was brought there by man.
The species assignation of Cannabis itself is fraught with
great debate. Cannabis sativa, meaning “cultivated Cannabis,”
was so named by Fuchs, among others, in 1542 (Fuchs,
1999), an assignation 211 years before the systematization of
botanical binomials Linnaeus in his Species Plantarum (Linnaeus,
1753). Lamarck subsequently suggested Cannabis indica, a more
diminutive intoxicating Indian plant from India, as a separate
species (Lamarck, 1783). The issue has remained unresolved
in the subsequent centuries with two opposing philosophies.
Ernest Small has championed the single species concept (Small
and Cronquist, 1976). Polytypic treatments of Cannabis also
gained adherents (Schultes et al., 1974;Anderson, 1980)
on morphological criteria suggesting separation of Cannabis
sativa L. Cannabis indica Lam. and Cannabis ruderalis Jan.,
a scheme supported by systematic chemotaxonomy. Principal
component analysis (PCA) of 157 Cannabis accessions from
around the world assessed allozyme frequencies at 17 gene
loci suggested a split (Hillig, 2005b). “Sativa” gene pools from
eastern European ruderal samples were linked to narrow-
leaﬂet European and Central Asian ﬁber and seed plants,
while an “indica” grouping encompassed Far Eastern seed
and ﬁber plants and drug plants with broad-leaﬂets from
most of the rest of the world, along with wild accessions
from the Indian subcontinent. Central Asian roadside samples
(Cannabis ruderalis) were thought to represent a third group.
Gas chromatography (GC) and starch-gel electrophoresis studies
also suggested species separation of sativa and indica (Hillig and
Agronomic factors in 69 samples suggested inclusion of
eastern hemp and drug plants in Cannabis indica (Hillig,
2005a), a division supported by fragment length polymorphisms
(Datwyler and Weiblen, 2006).
More recently, PCA seemed to point to terpenoid content
as the most convincing distinguishing chemotaxonomic markers
between putative sativa and indica species (Elzinga et al., 2015).
Similarly, PCA was felt to separate drug Cannabis from hemp
(Sawler et al., 2015). A recent study demonstrated demarcation
of Cannabis drug from hemp accessions via genotyping of
13 microsatellite loci across the genome, not merely genes
aﬀecting cannabinoid or ﬁber production (Dufresnes et al.,
2017). Professor Giovanni Appendino has reported the presence
of the cis-19-THC stereo-isomer only in the hemp accessions
(Giovanni Appendino, personal communication). However,
these distinctions may well pass by the wayside given the current
trend to crossbreed hemp with drug cultivars to avoid legislative
restrictions on THC content.
The Cannabis species controversy, Cannabis sativa vs. indica
vs. afghanica, has continued unabated to the current day
with impassioned arguments advanced by the protagonists
(Clarke and Merlin, 2013, 2016;Small, 2015;McPartland
and Guy, 2017;Small, 2017). This author, having been
on every side of the issue at one time or another, has
chosen to eschew the irreconcilable taxonomic debate as
an unnecessary distraction (Piomelli and Russo, 2016), and
rather emphasize that only biochemical and pharmacological
distinctions between Cannabis accessions are relevant. In
his recent seminal review, McPartland agreed, “Categorizing
Cannabis as either ‘Sativa’ and ‘Indica’ has become an exercise in
futility. Ubiquitous interbreeding and hybridization renders their
distinction meaningless.” (McPartland, 2018) (p. 210).
An additional non-sensical nomenclature controversy
pertains in common parlance to Cannabis “strains,” an
appellation that is appropriate to bacteria and viruses, but
not plants (Bailey and Bailey, 1976;Usher, 1996;Brickell et al.,
2009), especially so with Cannabis where the chemical variety,
abbreviated “chemovar” is the most appropriate appellation
(Lewis et al., 2018).
THE CANNABIS GENOME AND
ALTERNATIVE HOST BIOCHEMICAL
2011 was a landmark year for Cannabis genomics, as Medical
Genomics and Nimbus Informatics issued an online report on
the complete 400 million base-pair genomic sequence, which was
shortly joined by a draft genome and transcriptome (van Bakel
et al., 2011).
This development sparked prominent publicity and
controversy as to what it might portend. Whereas, the human
genome was analyzed some 20 years earlier, the implications for
Cannabis were subject to great speculation.
The news catalyzed a ﬂurry of new research, but considerable
progress had already been achieved in applied Cannabis genetics.
The identiﬁcation and synthesis of 19-tetrahydrocannabinol
(THC) was accomplished in Israel 1964 (Gaoni and Mechoulam,
1964), but it was not until much later before successful cloning
of its biosynthetic enzyme, tetrahydrocannabinolic acid synthase
(THCA synthase) (Sirikantaramas et al., 2004;Figure 1). Enzyme
crystallization followed (Shoyama et al., 2005). Cannabidiolic
acid synthase, which catalyzes cannabidiolic acid (CBDA), the
precursor of cannabidiol (CBD), had been previously identiﬁed
and produced in pure form (Taura et al., 1996;Figure 1).
These developments stimulated additional ﬁndings, including the
archeological phytochemical discovery of THCA synthase in a
2700 year old Cannabis cache from a tomb in Central Asia along
with two previously unreported single nucleotide polymorphisms
(SNPs) in the enzyme’s gene sequence (Russo et al., 2008).
By 2011, the enzymes for the production of the major
phytocannabinoids had been identiﬁed. Similarly, selective
advanced Mendelian breeding yielded Cannabis varieties rich
in speciﬁc single components. Thus, high-THC and high-
CBD plants were produced for pharmaceutical development
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FIGURE 1 | Biosynthetic pathways and enzymes (red) of Cannabis sativa, indication the natural species Helichrysum umbraculigerum, and alternative species
(in color) that have been genetically modiﬁed to produce subsequent products [redrawn and updated from (Russo, 2011) using ACD/ChemSketch 2017.2.1].
(de Meijer et al., 2003;de Meijer, 2004), with analogous breeding
of high-cannabigerol (CBG) (de Meijer and Hammond, 2005)
and cannabichromene (CBC) lines (de Meijer et al., 2009a). The
selective breeding also extended to propyl phytocannabinoid
analogs, tetrahydrocannabivarin (THCV), cannabidivarin
(CBDV), cannabigerivarin (CBGV), and cannabichromivarin
(CBCV) (de Meijer, 2004). The availability of plants with high
titers of these “minor cannabinoids” portend interesting new
pharmaceutical applications (Russo, 2011;Russo and Marcu,
Access to the Cannabis genome might simplify production
of THC-knockout plants via CRISPR technology (clustered
regularly-interspaced short palindromic repeats). While this
could be attractive for industrial hemp breeding, a prior
generation of plant husbandry has already yielded hemp cultivars
that easily fulﬁll international restrictions that require 0.1% or
less THC content (Wirtshafter, 1997;McPartland et al., 2000;
Small and Marcus, 2003). In fact, cannabinoid-free Cannabis
with no functional cannabigerolic acid synthase (Figure 1) has
also been produced conventionally (de Meijer et al., 2009b).
Thus, it remains unclear that genetic engineering of Cannabis
is even necessary for this plant whose incredible plasticity
already displays bountiful biochemical diversity. Introduction
of genetically modiﬁed organism (GMO) Cannabis would
incite considerable controversy among certain segments of the
population, and likely provoke a ﬂurry of legal entanglements
over patent and breeding rights.
One may easily imagine a variety of additional science ﬁction
scenarios. In the 1990s an Internet hoax spread the rumor that
an apocryphal Professor Nanofsky had introduced genes for
THC production into oranges (Citrus x. sinensis (L.) Osbeck).
Although this could be technologically achievable, such an eﬀort
would be no more than a laboratory carnival act in light of
the prodigious cannabinoid production from Cannabis itself.
A stealthy peppermint chemovar (Mentha xpiperita Lamiaceae)
sporting illicit phytocannabinoids in the glandular trichomes of
its leaves might be more logical choice for such underground
subversive daydreams and send rhizomes and runners along
Prior claims of production of cannabidiol from hops
(Humulus lupulus L. Cannabaceae) and ﬂax (Linum
usitatissimum L. Linaceae) are unsubstantiated, but
cannabigerolic acid and cannabigerol were detected in
South African Helichrysum umbraculigerum Less. Asteraceae
(Bohlmann and Hoﬀmann, 1979;Appendino et al., 2015;Russo,
2016;Figure 1), but without reference to its concentration.
This claim was conﬁrmed recently with trace amounts observed
from dried samples of aerial parts (Mark Lewis, personal
Because the complexity of purely de novo biochemical
synthesis of cannabinoids has been deemed non-cost eﬀective
(Carvalho et al., 2017), alternative microbial hosts have been
suggested (Zirpel et al., 2017). In 2004, cDNA cloning of THCA
synthase was achieved, allowing conversion of cannabigerolic
acid (CBGA) to THC (Sirikantaramas et al., 2004), and an 8%
THCA production in tobacco hairy roots (Nicotiana tabacum
cv.Xanthi Solanaceae) was demonstrated on CBGA feeding
(Figure 1). The enzyme was also expressed in the insect,
Spodoptera frugiperda (J.E. Smith) Noctuidae (fall armyworm)
via a recombinant baculovirus. Subsequently, this research group
turned to yeasts, Pichia pastoris (now Komagataella phaﬃi Phaﬀ
Saccharomycetaceae) (Taura et al., 2007;Figure 1), and achieved
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a CBGA to THCA conversion of 98% over 24 h, with yield of
32.6 mg/L of medium. A recombinant form of THCA synthase
proved 4.5X more eﬃcient than in Cannabis and 12X that in
S. frugiperda. This process was subsequently optimized with a
64.5-fold improvement in activity (Zirpel et al., 2018), with a
reported production in K. phaﬃi of 3.05 g/L of THCA after 8 h of
incubation at 37◦C. A simple calculation provides that this yield
could also be achieved from extraction of just 15 g of 20% THCA
Cannabis terpenoid production is similarly possible in
alternative hosts. Saccharomyces cerevisiae Meyen ex E.C. Hansen
Saccharomycetaceae mutants deﬁcient in farnesyl diphosphate
synthase enzyme accumulate geranyl pyrophosphate instead,
which is shunted into the production of medically useful
terpenoid, linalool (Oswald et al., 2007;Figure 1). Similarly,
other researchers have harnessed the biosynthetic capabilities of
mitochondria in S. cerevisiae to increase farnesyl diphosphate
production of sesquiterpenoids (Farhi et al., 2011), although not
ones common to Cannabis.
At present, the existing Cannabis genomic sequences are
not fully annotated. Consequently, applied foreknowledge and
detective work will be necessary to acquire practical data on
genetic function in Cannabis. The greatest potential in such
investigation will lie in the realm of epigenetics, underlying
hereditable changes in gene expression or phenotype of the
plant. The most salient deﬁciency is a lack of knowledge
regarding regulation of cannabinoid production. Understanding
the biosynthetic pathways and regulation of terpene synthases
producing the Cannabis terpenoids has barely been initiated
(Booth et al., 2017) and remain ripe targets of additional research
An additional problem in Cannabis husbandry remains a
dearth of voucher specimens (which are prohibited by the US
Drug Enforcement Administration without Schedule I license)
and formal deposits of chemovar accessions in seed and
tissue repositories. The latter has been accomplished by GW
Pharmaceuticals, and independently by NaPro Research (Lewis
et al., 2018) in the National Collection of Industrial, Food and
Marine Bacteria (NCIMB) in Scotland. Many private companies
have eschewed sharing germplasm due to legal restrictions and
fear of loss of intellectual property.
In 1998, Professors Raphael Mechoulam and Shimon Ben-Shabat
posited that the endocannabinoid system demonstrated an
“entourage eﬀect” in which a variety of “inactive” metabolites
and closely related molecules markedly increased the activity
of the primary endogenous cannabinoids, anandamide and
2-arachidonoylglycerol (Ben-Shabat et al., 1998). They also
postulated that this helped to explain how botanical drugs
were often more eﬃcacious than their isolated components
(Mechoulam and Ben-Shabat, 1999). Although the single
molecule synthesis remains the dominant model for
pharmaceutical development (Bonn-Miller et al., 2018), the
concept of botanical synergy has been amply demonstrated
contemporaneously, invoking the pharmacological contributions
of “minor cannabinoids” and Cannabis terpenoids to the plant’s
overall pharmacological eﬀect (McPartland and Pruitt, 1999;
McPartland and Mediavilla, 2001;McPartland and Russo, 2001,
2014;Russo and McPartland, 2003;Wilkinson et al., 2003;Russo,
2011). Several pertinent examples of the entourage eﬀect in
Cannabis are illustrative:
In a randomized controlled trial of oromucosal Cannabis-
based extracts in patients with intractable pain despite optimized
opioid treatment, a THC-predominant extract failed to
demarcate favorably from placebo, whereas a whole plant extract
(nabiximols, vide infra) with both THC and cannabidiol (CBD)
proved statistically signiﬁcantly better than both (Johnson et al.,
2010), the only salient diﬀerence being the presence of CBD in
In animal studies of analgesia, pure CBD produces a
biphasic dose-response curve such that smaller doses reduce
pain responses until a peak is reached, after which further
increases in dose are ineﬀective. Interestingly, the application of
a full spectrum Cannabis extract with equivalent doses of CBD
eliminates the biphasic response in favor of a linear dose-response
curve such that the botanical extract is analgesic at any dose with
no observed ceiling eﬀect (Gallily et al., 2014).
A recent study of several human breast cancer cell lines in
culture and implanted tumors demonstrated superiority of a
Cannabis extract treatment to pure THC, seemingly attributable
in the former to the presence of small concentrations of
cannabigerol (CBG) and tetrahydrocannabinolic acid (THCA)
(Blasco-Benito et al., 2018).
Anticonvulsant eﬀects of cannabidiol were noted in animals
in the 1970s with the ﬁrst human trials in 1980 (Cunha et al.,
1980). A recent experiment in mice with seizures induced by
pentylenetetrazole employed ﬁve diﬀerent Cannabis extracts with
equal CBD concentrations (Berman et al., 2018). Although all
the extracts showed beneﬁts compared to untreated controls,
salient diﬀerences were observed in biochemical proﬁles of
non-CBD cannabinoids, which, in turn, led to signiﬁcant
diﬀerences in numbers of mice developing tonic-clonic seizures
(21.5–66.7%) and survival rates (85–100%), highlighting the
relevance of these “minor” components. This study highlights
the necessity of standardization in pharmaceutical development,
and although it could be construed to support the single
molecule therapeutic model (Bonn-Miller et al., 2018), it requires
emphasis that complex botanicals can meet American FDA
standards (Food and Drug Administration, 2015). Speciﬁcally,
two Cannabis-based drugs have attained regulatory approval,
(nabiximols, US Adopted Name) in 30 countries, and
in the United States.
The question then arises: Can a Cannabis preparation or single
molecule be too pure, thus reducing synergistic potential? Recent
data support this as a distinct possibility. Anecdotal information
from clinicians utilizing high-CBD Cannabis extracts to treat
severe epilepsy, such as Dravet and Lennox-Gastaut syndromes,
showed that their patients demonstrated notable improvement
in seizure frequency (Goldstein, 2016;Russo, 2017;Sulak et al.,
2017) with doses far lower than those reported in formal
clinical trials of Epidiolex, a 97% pure CBD preparation with
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FIGURE 2 | PhytoFactsTM depiction of cannabinoid and terpenoid content of CaryodiolTM, aka “Kashmir Blue,” a Type III, cannabidiol-, and
caryophyllene-predominant chemovar. See (Lewis et al., 2018) for details of PhytoFacts and conventional breeding methodology. Copyright© 2016 BHC Group,
LLC. All rights reserved. Any unauthorized use of this document or the images or marks above may violate copyright, trademark, and other applicable laws.
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THC removed (Devinsky et al., 2016, 2017, 2018;Thiele et al.,
2018). This observation was recently subjected to meta-analysis
of 11 studies with 670 patients in aggregate (Pamplona et al.,
2018). Those results showed that 71% of patients improved
with CBD-predominant Cannabis extracts vs. 36% on puriﬁed
CBD (p<0.0001). The response rate at 50% improvement
in seizure frequency was not statistically diﬀerent in the two
groups and both groups achieved seizure-free status in about
10% of patients. However, the mean daily doses were markedly
divergent in the groups: 27.1 mg/kg/d for puriﬁed CBD vs.
only 6.1 mg/kg/d. for CBD-rich Cannabis extracts, a dose only
22.5% of that for CBD alone. Furthermore, the incidence of
mild and severe adverse events was demonstrably higher in
puriﬁed CBD vs. high-CBD extract patients (p<0.0001), a result
that the authors attributed to the lower dose utilized, which
was achieved in their opinion by the synergistic contributions
of other entourage compounds. Such observations support the
hypothesis of greater eﬃcacy for Cannabis extracts combining
multiple anticonvulsant components, such as CBD, THC, THCA,
THCV, CBDV, linalool, and even caryophyllene (Lewis et al.,
These studies and others provide a ﬁrm foundation for
Cannabis synergy, and support for botanical drug development
vs. that of single components (Bonn-Miller et al., 2018),
or production via fermentation methods in yeast or other
micro-organisms. An example of the power of conventional
selective breeding is illustrated (Figure 2), in the form of
a Cannabis chemovar named CaryodiolTM for its enhanced
caryophyllene content (0.83%) as a CB2agonist, along with
highly favorable Type III THC:CBD ratio of 1:39.4. Such
a preparation portends to be applicable to treatment of
numerous clinical conditions including: pain, inﬂammation,
ﬁbrotic disorders, addiction, anxiety, depression, autoimmune
diseases, dermatological conditions and cancer (Pacher and
Mechoulam, 2011;Russo, 2011;Xi et al., 2011;Russo and Marcu,
2017;Lewis et al., 2018). Producing such a combination from
microbial sources might require combinations of cannabinoids
from multiple yeast species and, as a result, it would represent
a combination product subject to a diﬃcult regulatory path
compared to Cannabis preparations from extracts of a single
species (e.g., nabiximols) that has been accepted as a unitary
formulation in 30 countries across the globe (Food and Drug
This article has brieﬂy outlined recently technological
attempts to “reinvent the phytocannabinoid wheel.” Cogent
arguments would support that it can be done, but should it be
done? The data supporting the existence of Cannabis synergy
and the astounding plasticity of the Cannabis genome suggests a
reality that obviates the need for alternative hosts, or even genetic
engineering of Cannabis sativa, thus proving that, “The plant
does it better.”
The author conﬁrms being the sole contributor of this work and
has approved it for publication.
The author appreciated the assistance of the staﬀ of the University
of Montana Inter-Library Loan Oﬃce of Mansﬁeld Library for
provision of research materials.
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Conﬂict of Interest Statement: I am Director of Research for the International
Cannabis and Cannabinoids Institute. We serve clients engaged in cannabis
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