Chapter 13

Micropropagation of Cannabis sativa

L.—An Update

Hemant Lata, Suman Chandra, Ikhlas A. Khan

and Mahmoud A. ElSohly

Abstract Cannabis is one of the oldest economically important plant yielding

fiber, food and medicine. It is a natural source of D9-tetrahydrocannabinol
(THC) and Cannabidiol (CBD). These two molecules have a tremendous thera-
peutic potential and commercial value in the pharmaceutical area. Cannabis is a
highly heterozygous species. Being dioceous (male and female flowers appear on
two different plants) and wind pollinated species, it is difficult to maintain the
chemical profile of biomass product, if grown from seed. Plant to plant variation is
observed even though plants are grown from seeds obtained from a single female
plant. Therefore, to maintain consistency in the end product, elite female plants are
screened and multiplied using vegetative propagation and/or tissue culture. Micro
propagation can play a vital role in the conservation of elite Cannabis clones and
rapid multiplication of novel germplasm. On the other hand, it can also be used in
genetic modification for the enhanced cannabinoid production. Research on in vitro
propagation of Cannabis has resulted in the development of protocols for callus
production, cell suspension cultures, agrobacterium mediated hairy root cultures
and regeneration of plants. This chapter provides an overview of in vitro propa-
gation of Cannabis and addresses the current applications of modern biotechnology
in propagation of elite Cannabis plants.

H. Lata
S. Chandra
I.A. Khan
M.A. ElSohly (&)

National Center for Natural Product Research, Research Institute of Pharmaceutical Sciences,
School of Pharmacy, The University of Mississippi, University, MS 38677, USA
e-mail: melsohly@olemiss.edu
I.A. Khan
Division of Pharmacognosy, Department of BioMolecular Sciences, School of Pharmacy,
The University of Mississippi, University, MS 38677, USA
M.A. ElSohly
Department of Pharmaceutics and Drug Delivery, School of Pharmacy, The University of
Mississippi, University, MS 38677, USA

© Springer International Publishing AG 2017 285

S. Chandra et al. (eds.), Cannabis sativa L. - Botany and Biotechnology,
DOI 10.1007/978-3-319-54564-6_13
286 H. Lata et al.

13.1 Introduction

Cannabis sativa L., the principle source of a group of terpenophenolic compounds,

the cannabinoids, is an open pollinated crop belonging to the family Cannabaceae.
At present this species has been cultivated widely in the world as a resource of fiber,
food and drug. Grown for fiber (Hemp) was introduced in Western Asia and Egypt,
and subsequently to the Europe between 1000 and 2000 BCE. Cultivation of hemp
in Europe became widespread after 500 CE. The crop was first brought to South
America in 1545, in Chile, and to North America in Port Royal, Acadia in 1606
(Small and Marcus 2002). Meijer and Soest (1992) have described the Netherlands
Cannabis breeding program for paper pulp production and establishment of CPRO
(Center for plant breeding and reproduction research) germplasm collection. Other
countries such as France, Russia, Poland and China have maintained Cannabis as a
fiber crop.
On the other hand, the use of C. sativa as a medicine is well known. As a plant it
is valued for both its hallucinogenic and medicinal properties and has been used to
treat a variety of ailments including pain, glaucoma, nausea, asthma, depression,
insomnia and neuralgia (Mechoulam et al. 1976; Duke and Wain 1981). The
therapeutic values of Cannabis derivatives have also been highlighted against
HIV/AIDS (Abrams et al. 2007) and multiple sclerosis (Pryce and Baker 2005). The
pharmacologic and therapeutic potency of preparations of Cannabis sativa L. and
its main active constituent D9-tetrahydrocannabinol (THC) has been extensively
reviewed (Long et al. 2005; Sirikantaramas et al. 2007).
Cannabis flowers are cross pollinated. Seed propagation is relatively straight-
forward but seed derived progeny can display considerable heterozygosity. Most
Cannabis presently used for medical purposes is grown indoors through vegetative
means, to maintain uniformity and genetic purity. However, propagation through
cuttings is very time consuming and labor intensive process and moreover, the crop
grown indoors (grow room) become susceptible to pests that reproduce rapidly such
as spider mites and aphids. The use of in vitro techniques is a promising alternative
for germplasm collections and breeders. In vitro propagation cannot only play an
important role in rapid multiplication of cultivars with desirable traits but can also
aid in the production of healthy and disease free plants (Lineberger 1983).
The objective of this article is to overview the work done in tissue and organ
culture of Cannabis and bring forth the new challenges for the refinement of
protocols as the major thrust of the future research.
13 Micropropagation of Cannabis sativa L.—An Update 287

13.2 Strategies Used for the Propagation of Cannabis

Sativa L.

13.2.1 Conventional Propagation

Propagation through seeds and vegetative cuttings are the most common and
popular methods of cultivating Cannabis. Seeds has been the choice of starting
material by many researchers for conducting growth and physiological studies
(Quimby et al. 1973; Lisson et al. 2000; Yoshimatsu et al. 2004), in vitro studies for
regeneration (Slusarkiewicz-Jarzina et al. 2005; Plawuszewski et al. 2006; Wieglus
et al. 2008) and production of secondary metabolites in vitro (Itokawa et al. 1977;
Feenay and Punja 2003; Flores-Sanchez et al. 2009; Wahby et al. 2013) and in vivo
(Vogelmann 1988; Meijer et al. 1992).
Different methods have been adopted for seed germination. Seeds are generally
planted in moist aerated soil and photoperiod of 18 h of cool fluorescent lights is
used for establishment of seedlings (Chandra et al. 2013). Whereas, (Plawuszewski
et al. 2006; Wieglus et al. 2008) used DARIA ind medium for planting seeds,
Wahby et al. (2013) have used moist Whatman filter paper as induction medium.
Optimum seed germination temperature is reported 21–26 °C with a photoperiod of
12 h by Feenay and Punja (2003), Slusarkiewicz-Jarzina et al. (2005) and
Plawuszewski et al. (2006). Wieglus et al. (2008) and Wahby et al. (2013) have
used dark conditions for seed germination. Different cultivars of Cannabis show
different germination response with optimal germination within 4–7 days (Weiglus
et al. 2008). Although, propagation by seeds in Cannabis is a predominant tech-
nique, however, it is impossible to maintain the elite cultivar/clone by seed and
growing from seeds result in a large portion of crop being male plants. Since,
female plants of this species contain higher levels of THC/CBD than male plants,
cultivation of female plants is preferred. Most of the researchers so far have used
seedling parts (cotyledon, epicotyl, hypocotyl and radicle), to initiate the propa-
gation studies, however, researchers at the University of Mississippi (Chandra et al.
2010), have screened and selected clones and have used, nodal segments containing
axillary buds, from the mother plant for conventional or in vitro studies since it
upholds genetic uniformities among the clones.

13.2.2 In Vitro Propagation

Tissue culture technology has emerged as a promising biotechnological tool for

multiplication and genetic enhancement of medicinally important plants. For C.
sativa, the in vitro propagation has superiority over conventional methods of
propagation not only because of high multiplication rate and production of disease
free elite plants but also overcoming the problems of heterozygosity due to its
allogamous. Although, in vitro techniques have been employed for Cannabis over
288 H. Lata et al.

the past 40 years, the regeneration of Cannabis via in vitro propagation, has been a
challenge with very few reports available so far (Table 13.1). Mostly, the propa-
gation of Cannabis has been achieved by two different routes of organogenesis i.e.
direct and indirect organogenesis. The Murashige and Skoog (MS) formulation is
the most commonly used medium for in vitro propagation of Cannabis genotypes (
Murashige and Skoog 1962). However, the use of media such as DARIA ind,
Millers medium, B5 and MB medium has also been reported (Feenay and Punja
2003; Plawuszewski et al. 2006; Wieglus et al. 2008).

13.2.3 Callus Production

The early work of growing Cannabis in vitro was on callus cultures, particularly on
cell suspension cultures. Most of the studies were aimed at developing cell culture
system to obtain secondary metabolites, particularly the THC class of cannabinoids
those are specific to the genus Cannabis (Turner et al. 1980). Different explants of
Cannabis sativa, including cotyledons, hypocotyls, epicotyls, leaves, petioles have
been used for the production of callus cultures by many researchers (Itokawa et al.
1975; John et al. 1978; Francoise and Vincent 1981; Fisse et al. 1981; Heitrich and
Binder 1982; Verzar-Petri et al. 1982; Loh et al. 1983; Braut-Boucher et al. 1985;
Fisse and Andres 1985). Using seed explants many different varieties of hemp have
also been studied to obtain callus cultures. Mandolino and Ranalli (1999) used
Carmagnola, Fibranova, Uniko and Kompolti varieties, while, Feeney and Punja
(2003) used Uniko-B, Kompolti Anka and Felina-34. Slusarkiewicz-Jarzina et al.
(2005) worked on Sileia, Fibriman-24, Novosadska, Juso-15 and Fedrina-74,
whereas, Wielgus et al. (2008) have used Beniko, Silesia and Bialobrzeskie for the
callus production. Lata et al. (2009a, b, 2010, 2012) have worked on MX variety for
the propagation studies of C. sativa
In 1972, Veliky and Genest reported the first studies on Cannabis cell suspension
and investigated the accumulation of cannabinoids and phenolics in culture using
modified Gamborg’s medium (67-V) based on the research done in (1970) by
Veliky and Martin. This was followed by research done by Itokawa et al. (1975) on
the biosynthesis of Cannabis callus cultures obtained from various explants like
hypocotyls, cotyledon, roots and floral parts on MS medium supplemented with
0.1–0.01 ppm KIN and 1.0 ppm 2,4-D. Further, in 1977, Itokawa et al. studied the
biotransformation of cannabinoid precursors and alcohols using cell suspension
cultures of C. sativa. In 1983, Loh et al. induced callus and suspension cultures
from various explants of embryo, leaf and stem explants using different combina-
tions of auxins [2,4-dichlorophenoxyacetic acid (2,4-D) and
2,4,5-trichlorophenoxyacetic acid (2,4,5-T)] and reported 2, 4, 5-T (3 mg/l) as the
best medium for calli growth using MS medium. Hartsel et al. (1983) also reported
the biotransformation of CBD to CBE in cell cultures of C. sativa grown on MS
medium solidified with agar containing the vitamins of B5 medium, supplemented
Table 13.1 In vitro protocols developed for Cannabis sativa L.
13

Explant Response Medium Reference

Seedling parts Cell suspension cultures Modified Gamborg’s medium Veliky and Genest (1972)
Root, hypocotyl, leaves of seedling, male and Callus cultures MS + 0.1–0.01 ppm Itokawa et al. (1975)
female floral parts KIN + 1.0 ppm 2,4-D
Seedling parts Cell suspension cultures Itokawa et al. (1977)
Embryo, leaf, stem Callus and cell suspension cultures MS + 3 mg/l 2,4,5-T Loh et al. (1983)
Seedling parts Cell suspension cultures MS + B5 vitamins + 3 mg/l Hartsel et al. (1983)
2,4,5-T
Leaf Cell suspension cultures B5 + 0.5 mg/l KIN + 1 mg/l 2,4 - Braemer et al. (1987)
D
Anthers Cell suspension cultures: cryopreservation 10% DMSO Jekkel et al. (1989)
Leaf Cell culture MS + B5 vitamins + 1 mg/l Flores-Sanchez et al.
2,4-D + 1 mg/l KIN (2009)
Leaf Callus culture MS + 0.5 µM NAA + 1.0 µM Lata et al. (2010)
TDZ
Internodes Callus culture MS + 1 mg/l BAP + 0.5 mg/l Jiang et al. (2015)
NAA
Micropropagation of Cannabis sativa L.—An Update

Cotyledon Callus culture MS + 2 mg/l TDZ + 0.5 mg/L Movahedi et al. (2015)
IBA
Stem and leaf segment from seedling Callus culture; Agrobacterium mediated MS + B5 vitamins + 5 µM 2,4 Feeney and Punja (2003)
transformation D + 1 µM KIN
Hypocotyl Hairy root cultures A. rhizogenes and A. tumefaciens Wahby et al. (2006, 2013)
strains
Seedling Hairy root cultures from callus B5 + 4 mg/l NAA Farag and Kayser (2015)
Root development from callus Hemphill et al. (1978)
Root development from callus Fisse et al. (1981)
Stem, cotyledon, root Callus formation MS + NAA Fisse and Andres (1985)
(continued)
289
Table 13.1 (continued)
290

Explant Response Medium Reference

Apical and axillary buds Shoot, root MS + 0.45 mg/l BAP + 20 mg/l Richez-Dumanois et al.
IBA (1986)
Leaf callus Shoot Mandolino and Ranalli
(1999)
Callus Root Mackinnon et al. (2000)
Internodes, axillary buds, petioles Callus, shoot regeneration MS + 2.0 mg/l and 3.0 mg/l Slusarkiewicz-Jarzina
dicamba et al. (2005)
Roots, leaves, stem shoot regeneration DARIA medium Plawuszewski et al.
(2006)
Cotyledon, stem root Callus, shoot regeneration DARIA medium Weiglus et al. (2008)
Shoot regeneration Formula b based medium Casano and Grassi (2009)
Lateral buds from seedling Shoot and root regeneration MS + TDZ + NAA Bing et al. (2007)
MS + IBA
Nodal segments with axillary buds Direct organogenesis; Shoot and root regeneration, MS + 0.5 µM TDZ Lata et al. (2009a, b,
synthetic seed MS + 2.5 µM IBA 2012)
Nodal segments with axillary buds Direct organogenesis; Shoot and root regeneration MS + 2 µM m-topolin Lata et al. (2016)
Shoot tips Shoot, root MS + 0.2 mg/l TDZ + 0.1 mg/l Wang et al. (2009)
NAA
MS+ 0.1 mg/l IBA + 0.05 mg/l
NAA
Epicotyl Shoot and root regeneration MS + 2 mg/l BAP + 0.5 mg/l IBA Movahedi et al. (2015)
Cotyledons Shoot and root regeneration MS + 0.4 mg/l TDZ + 0.5 mg/l Chaohua et al (2016)
IBA
MS Murashige & Skoog medium; B5 Gamborg medium; BAP 6-Benzylaminopurine; DMSO Di methyl sulphoxide; 2,4-D 2, 4-di chloro phenoxy acetic acid; 2,4,5–T: 2, 4,
5-tri chloro phenoxy acetic acid; IBA Indole 3 butyric acid; KIN Kinetin; NAA Napthalein acetic acid; TDZ Thidiazuron
H. Lata et al.
13 Micropropagation of Cannabis sativa L.—An Update 291

with 3 ppm 2,4,5-T. More biosynthesis studies in 1987 were conducted by Braemer
and Paris (1987) for investigating the conversion of flavonoids to glucosides using
suspension cultures of C. sativa. The cells were grown in B5 medium supplemented
with 0.5 mg/l KIN and 1 mg/l 2,4-D. After a gap of almost a decade,
Flores-Sanchez et al. (2009) employed elicitation using biotic and abiotic elicitors
on cannabinoid production in C. sativa cultures. The cell cultures initiated from leaf
explants were maintained in MS medium supplemented with B5 vitamins, 1 mg/l 2,
4-D and 1 mg/l KIN. However, no cannabinoids were found in elicited or con-
trolled cultures. Lata et al. (2010) used young leaf tissues as explant for obtaining
callus on MS medium supplemented with different concentrations (0.5, 1.0, 1.5, and
2.0 µM) of IAA, IBA, NAA, and 2,4-dichlorophenoxyacetic acid (2,4-D) in
combination with 1.0 µM TDZ for the production of callus. The optimum callus
growth and maintenance was in 0.5 µM NAA plus 1.0 µM TDZ. On the other
hand, Jiang et al. (2015) have used internodes of the new cultivar Long-ma of C.
sativa as explants for tissue culture. Best combination for callus induction has been
reported on MS medium supplemented with 1 mg/l BAP and 0.5 mg/l NAA. In
another recent study conducted by Movahedi et al. (2015), the best callus were
obtained using cotyledon explant treated with 2 mg/1 TDZ and 0.5 mg/1 IBA.

13.2.4 Agrobacterium Mediated Transformation

The use of hairy root cultures technology has revolutionized the role of plant cell
culture technology in fine chemical synthesis (Toivonen 1993). In addition, the
hairy root technology offers an alternative and a promising in vitro source for the
production of valuable secondary metabolites as compared to plant suspension
cultures due to more biochemical and genetic stability (Liu et al. 1998; Farag and
Kayser 2015).
The first reports of Cannabis transformation was reported by Feeney and Punja
(2003). The agrobacterium transformation approach resulted in well developed calli
on MS medium with B5 vitamins supplemented with 5 lM 2, 4 D and 1 lM KIN.
However, the cultures were unresponsive to plant regeneration. One of the early
attempts of working with Cannabis root infection using A. rhizogenes in 2006,
Wahby et al. identified secondary metabolites (choline and atropine) in Cannabis
roots. Wahby et al. later extended the work in 2013, on transformation of Cannabis
roots with A. rhizogenes and transforming Cannabis calli with A. tumefaciens.
Hypocotyl of intact seedlings was reported as the most responsive material for the
establishment of C. sativa hairy root cultures, however, no regenerated shoots were
observed. Most recently, Farag and Kayser (2015) have reported hairy root cultures
of C. sativa from callus induced using B5 medium supplemented with 4 mg/l NAA
under dark conditions, for the production of cannabinoids. However, very low
amount of cannabinoids have been detected.
292 H. Lata et al.

13.2.5 Regeneration

Efficient plant regeneration protocol is essential for mass production of pharma-

ceutically superior elite clones of C. sativa. The induction of direct shoot regen-
eration depends on the nature of the plant organ from which the explants were
derived and the interaction between endogenous growth substances and the syn-
thetic growth regulators added to the media (George and Eapen 1994; Jones et al.
2007). There are only few reports on induction of organogenesis of Cannabis
sativa. Early reports dates back to 1970s, Hemphill et al. (1978), obtained root
development but no shoot formation from callus. Similar results were reported by
Fisse et al (1981). In 1985, Fisse and Andres used different explants (stem, leaf,
cotyledon, root and callus cultures) for Cannabis micropropagation. NAA stimu-
lated rhizogenesis and gibberellic acid was reported to promote stem elongation.
Richez-Dumanois et al. (1986) induced direct shoot multiplication of explants from
apical and axillary buds using BAP. Whereas, Mandolino and Ranalli in (1999),
demonstrated occasional shoot regeneration of hemp (C. sativa L.) from leaf callus.
Mackinnon et al. (2000) obtained root development but no shoot formation from
callus. Slusarkiewicz-Jarzina et al. (2005), too reported shoot regeneration of C.
sativa, from calli regenerated from different explants (internodes, axillary buds and
petioles) on MS media supplemented with various combinations of KN, NAA,
2,4-D and dicamba. However, only 2% of calli were able to regenerate into whole
plants of which highest regeneration frequency was obtained from petiole explants
on medium supplemented with 2.0 and 3.0 mg/l dicamba. Plawuszewski et al.
(2006), worked on three different polish cultivars of hemp to regenerate in vitro
growth from explants, roots, leaves and stem grown on DARIA medium. But only
were able to obtain partial regeneration. Shoot regeneration from calli was extended
to 14% by Weiglus et al. (2008) using different explants from cotyledons, stem and
root on DARIA medium. It was observed that interaction between tested explant
and cultivar (cv.) had significant effect on the efficiency of plant regeneration, with
highest regeneration observed for cotyledon explants (cv. Beniko) and the lowest
for stem explants (cv. Silesia). Casano and Grassi (2009), reported a higher
micropropagation rate of meristem of selected clones of Cannabis in Formula b
based medium as compared to the MS based medium.
Further studies on organogenesis in Cannabis show the predominance of use of
TDZ in inducing shoot morphogenesis. Thidiazuron, is a substituted phenylurea
(N-phenyl-1,2,3-thidiazol-5-ylurea) with intrinsic cytokinin like activity (Huetteman
and Preece 1993). Compared to most other compounds, with cytokinins activity, TDZ
can stimulate better shoot proliferation and regeneration (Lata et al. 2009a; Parveen and
Shahzad 2010). The use of lateral buds obtained from germinating seeds were inves-
tigated by Bing et al. (2007), using a combination of TDZ and NAA for shoot
regeneration and IBA for rooting on MS medium. Lata et al. (2009a) have successfully
established a direct organogenesis protocol using nodal segments containing axillary
buds as explants. The quality and quantity of regenerants were better with thidiazuron
(0.5 lM thidiazuron) than with benzyladenine or kinetin. Adding 7.0 lM of gibberellic
13 Micropropagation of Cannabis sativa L.—An Update 293

acid into a medium containing 0.5 lM thidiazuron slightly increased shoot growth.
Elongated shoots when transferred to half-strength MS medium supplemented with
500 mg/L activated charcoal and 2.5 lM indole-3-butyric acid resulted in 95% rooting.
Concurrently, Wang et al. (2009) used shoot tips as explants for obtaining axillary bud
induction using MS medium supplemented with different cytokinins (BA, KN, TDZ).
Among the cytokinins tested by them, TDZ (0.2 mg/l) was found to provide the best
bud induction. For root induction different media, full strength MS, half strength MS,
B5 and NN were tested. The best rooting and elongation was obtained on 0.1 mg/l IBA
and 0.05 mg/l NAA on MS media with 85% rate of success in root development.
Movahedi et al. (2015) used cotyledon and epicotyl explant on MS medium supple-
mented with various combinations of BA, TDZ or alone, to investigate micropropa-
gation in C. sativa L. The callus formation was dominant over direct regeneration with
cotyledon giving higher callus frequency and volume in TDZ (3.0 mg/l) in combina-
tion with IBA (0.5 mg/l), whereas, epicotyl showed better regeneration than cotyledon.
Both BAP and TDZ were individually effective in shoot formation and no significant
differences were observed. Roots were obtained on 0.1 mg/l IBA. The highest shoot
regeneration rate was achieved in calli produced from epicotyl treated with 2 mg/l BAP
and 0.5 mg/l IBA. More recently, Chaohua et al. (2016) used cotyledons as explants.
TDZ in MS medium was more efficient in inducing in vitro shoots than BAP or ZT.
Based on their results 80% of shoots were able to develop roots on MS medium
supplemented with 0.4 mg/l TDZ and 0.5 mg/l IBA. Further, Lata et al. (2016), have
reported an effective one step regeneration system based on adventitious shoot
induction as well as of an effective rooting procedure for C. sativa using novel aromatic
cytokinin; meta-topolin. Nodal segments containing axillary buds from a selected
vegetatively propagated plant (mother plant) were used as explants for initiation of
shoot cultures. The highest number of shoots was obtained in the treatment with
2.0 µM mT with maximum shoot length. All the explants were capable of producing
shoots. Most of the shoots were rooted in various concentrations of mT, however, the
optimal concentration for rooting was obtained on MS medium supplemented with
2.0 µM mT, on which 100% of the regenerated shoots developed roots with an average
of 18.7 roots per shoots within 4 weeks of transfer to fresh medium.

13.2.6 Germplasm Conservation

Plant tissue culture, has been used for clonal propagation of desired clones and high
yielding elite strains through conservation. Not many studies are available on
germplasm conservation of C. sativa. In 1989, cryopreservation of hemp suspen-
sion cultures was developed as a means to preserve germplasm collections (Jekkel
et al. 1989). Of the different cryoprotectants (DMSO, glycerol, proline) used,
highest viability (58%) was obtained using 10% DMSO and −10 °C temperature
transfer. Later in 2009, Lata et al. have successfully used, synthetic seed technology
for economical large-scale clonal propagation and germplasm conservation of the
screened and selected elite germplasm. Axillary buds isolated from aseptic multiple
294 H. Lata et al.

shoot cultures were encapsulated in calcium alginate beads. The best gel com-
plexation was achieved using 5% sodium alginate with 50 mM CaCl2.2H2O.
Encapsulated explants exhibited the best regrowth and conversion frequency on
Murashige and Skoog medium supplemented with thidiazuron (TDZ 0.5 lM) and
PPM (0.075%) under in vitro conditions (Lata et al. 2009b). This system further
allowed development of an efficient conservation protocol for C. sativa that has led
to the successful growth of homogeneous and genetically stable Cannabis plants
even after 6 months of storage at 15 °C (Lata et al. 2012).

13.3 Conclusions

To the best of our knowledge, this is the first comprehensive update on Cannabis
sativa micropropagation. Establishment of an efficient in vitro regeneration system
for Cannabis is of high significance for mass production of pharmaceutically
superior elite clones. In vitro culture techniques will not only provide improved
methods of clonal propagation but also a valuable means for establishing ex situ
collection of Cannabis germplasm with minimum space, free of diseases and low
maintenance requirement. For the genetic transformation studies, in vitro propa-
gation can lay a foundation for cultivating the new varieties of Cannabis.

Acknowledgements This work was supported in part by the National Institute on Drug Abuse
(NIDA), National Institute of Health (NIH), Department of Health and Human Services, USA,
Contract No. N01DA-15-7793.

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