Pharmaceutical Sciences Asia Pharm Sci Asia 2020; 47 (1), 21-29

DOI:10.29090/psa.2020.01.019.0030

Review Article

Factors affecting micropropagation of Cannabis sativa L.

: A review
Panitch Boonsnongcheep1,2,
Benyakan Pongkitwitoon3*
ABSTRACT
1
Faculty of Pharmaceutical Sciences, Khon Kaen
University, Khon Kaen, Thailand Cannabis sativa L. is a well-known herb that has been
2
Research Group for Pharmaceutical Activities used for medicines and textile fibers for thousands of years. Its
of Natural Products using Pharmaceutical
Biotechnology (PANPB), National Research
biomass and phytochemicals are precious sources for pharmaceuticals.
University Khon Kaen University, Khon Kaen, Various preparations featuring bioactive compounds from C. sativa
Thailand
including tetrahydrocannabinol and cannabidiol have been
3
Department of Pharmaceutical Botany, Faculty
of Pharmacy, Mahidol University, Bangkok, reported to link with a wide range of health benefits. Micropropaga-
Thailand tion is an alternative method for production of these resources
as the field-cultivation of C. sativa is strictly regulated in many
countries. This review highlights the conditions for micropropagation
*Corresponding author: of C. sativa. Factors affecting establishment of in vitro cultures
benyakan.pon@mahidol.ac.th
including selection of explants and disinfection protocols, media
and culture conditions, plant growth regulators used for cultivation
of C. sativa, and protocols for acclimatization are reviewed and
discussed. These successful established protocols for micro-
propagation of C. sativa are advantageous for further applied
approaches such as mutation breeding, genetic engineering,
KEYWORDS: somaclonal variation, and in vitro production of valuable
Cannabis sativa; Micropropagation; bioactive components.
Plant tissue culture
1. INTRODUCTION
Cannabis sativa L. is an annual herb of the family
Cannabaceae extensively recognized as source of precious
phytochemicals. The plant has been used for medicines and textile
fibers throughout the history. During the past decade, C. sativa
has attracted great interests in as much as attempts to legalize uses
of C. sativa in many parts of the world. To date, medical cannabis
is legal in some countries including Netherlands, Germany,
Canada, and in some states of the United States.
Various preparations of C. sativa have been used in
associated with several pharmacological disorders including
pain, nausea and vomiting, anxiety, colitis, sleep disorder, multiple
sclerosis Alzheimer’s disease and Parkinson’s disease1. The most
recognized phytochemicals produced in C. sativa are cannabinoids,
namely tetrahydrocannabinol (THC), cannabidiol (CBD), and
up to 90 different minor cannabinoids2. Other active components
that may contribute to pharmacological activities of C. sativa
https://www.pharmacy.mahidol.ac.th/journal/ are terpenes and phenolic compounds.
© Faculty of Pharmacy, Mahidol University Cannabis sativa is conventionally propagated via seeds
(Thailand) 2020
and cuttings. Limitations of field cultivated C. sativa plants are
 P. Boonsnongcheep & B. Pongkitwitoon Pharm Sci Asia 2020; 47 (1), 21-29

their heterozygosity and their susceptibility to micropropagation. Generally, meristematic tissues

diseases and pests. Furthermore, the cultivation of are used for direct regeneration. Protocols for C.
C. sativa in many countries is strictly regulated. sativa micropropagation by direct organogenesis
Therefore, micropropagation using plant tissue using cotyledons, axillary buds, and shoot tips as
culture is a promising alternative for propagation explants were established3-5 (Table 1). For callus-
of pathogen-free true-to-type C. sativa stock plants. mediated organogenesis, various explants are used
In this review, we focused on factors affecting for callus induction before shoot multiplication
micropropagation of C. sativa from the establishment and rooting. Slusarkiewicz-Jarzina et al. reported
of in vitro cultures, media and culture conditions, that, among young leaves, petioles, internodes,
plant growth regulators for shoot multiplication and and axillary buds, the highest callus frequency
rooting, as well as protocols for acclimatization. was obtained from petioles and young leaves6.
Another study reported the effect of C. sativa explant
2. ESTABLISHMENT OF IN VITRO types on callus induction and plant regeneration.
CULTURE Although there was no significant difference in
callus induction among the explants tested, cotyledon-
2.1. Selection of plant materials
derived callus had the highest regeneration efficiency
The efficient clonal multiplication of desired when compared with callus originated from stems
true-to-type plants starts with the healthy mother and roots7. In addition to explant types, different
plants. Selection of explants as starting materials cultivars also had different effects on callus
to establish an in vitro culture is a crucial step for formation and plant regenerationʻʻ6-7ʼʼ.

Table 1. Micropropagation protocols for C. sativa

Micropropagation Explants used Culture medium Culture Optimal

strategy Steps (Explant /PGRs conditions experimental References
disinfection) outcome
Direct regeneration Shoot Nodal segments MS medium/ 25 ± 2°C, 0.5 μM TDZ Lata
induction containing axillary 0.05 - 9.0 μM BA or 16/8 h resulted et al., 2009
buds 0.05 - 9.0 μM KN or photoperiod in 100%
(0.50% sodium 0.05 - 9.0 μM TDZ response
hypochlorite for With or without (average
20 min) 7.0 μM GA3 12.6 shoots)
Rooting ½ MS medium/ 25 ± 2°C, 2.5 µM IBA
2.5 - 5.0 μM IAA or 16/8 h resulted in
2.5 - 5.0 μM IBA or photoperiod 94% response
2.5 - 5.0 μM NAA (average
with 500 mg/L 4.8 roots)
activated charcoal
Direct regeneration Culture Seeds 1/2 MS medium 25±1°C, Wang
initiation (75% (v/v) (1%(w/v) sucrose) 16/8 h et al., 2009
ethanol for 30 s photoperiod
and 0.1% mercuric
chloride for
10-15 min)
Regeneration Shoot tips MS medium/ 25±1°C, 0.2 mg/L TDZ
1.0 - 5.0 mg/L BA or 16/8 h and 0.1 mg/L
1.0 - 5.0 mg/L KN or photoperiod NAA showed
0.1 – 0.5 mg/L TDZ the highest
with or without auxiliary bud
0.05 - 0.5 mg/L NAA multiplication
rate (3.22 buds
per shoot tip)

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 Pharmaceutical Sciences Asia

Table 1. Micropropagation protocols for C. sativa (cont.)

Micropropagation Explants used Culture medium Culture Optimal
strategy Steps (Explant /PGRs conditions experimental References
disinfection) outcome
Rooting MS medium/ 25±1°C, 0.1 mg/L IBA
0.1 - 0.5 mg/L IBA 16/8 h and 0.05 mg/
with photoperiod L NAA
0.05 - 0.25 mg/L NAA showed the
or highest
0.05 - 0.25 mg/L IAA rooting (85%)
Direct regeneration Culture Seeds (sulfuric acid MS medium 22±2◦C, Cheng
initiation for 20 s 16/8 h et al., 2016
tap water 20 min, photoperiod
75% (v/v) ethanol
for 2 min, 3% (w/v)
sodium hypochlorite
for 20 min)
Shoot In vitro MS medium/ 22±2◦C, 3-day old
induction cotyledons 0.1 - 0.6 mg/L TDZ 16/8 h cotyledon on
(2-6 day old) with or without photoperiod MS medium +
0.2 - 0.6 mg/L NAA 0.4 mg/L TDZ
+ 0.2 mg/L NAA
resulted in 51.7%
induction
frequency
(3.0 shoots
per explant)
Rooting 1/2 MS medium/ 22±2◦C, 0.5 - 2 mg/
with or without 16/8 h L IBA show
0.2 -2.0 mg/L IBA photoperiod 80% rooting
Direct regeneration Shoot Nodal segments MS medium/ 25 ± 2°C, MS medium Lata
induction/ containing 0.05 - 5.0 μM TDZ 16/8 h + 2.00 μM mT et al., 2016
rooting axillary buds or 1/2 MS medium/ photoperiod showed 100%
(0.50% sodium 0.05 - 5.0 μM IBA shoot response,
hypochlorite or MS medium/ average 13.4
+0.1%Tween 20 for 0.05 - 5.0 μM mT shoot and 96%
20 min) root response,
13.8 roots)
Indirect regeneration Culture Seeds MS medium 22°C, Slusarkiewicz-
via callus initiation (5% calcium 12/12 h Jarzina
hypochlorite for 6, photoperiod et al., 2005
8, 15 min)
Callus In vitro young MS medium/ 24°C, Petiole explants
induction leaves, petioles, 1.0 - 4.0 mg/L KN or Darkness with 2.0-3.0 mg/
and indirect internodes, and 0.5 - 2.0 mg/L NAA or for 2-3 weeks L DIC showed
regeneration axillary buds 2.0 - 4.0 mg/L 2,4-D or callus highest frequency
2.0 - 3.0 mg/L DIC induction, then, of callus
16/8 h formation (82.7%
photoperiod of explants)
for indirect and plant
regeneration regeneration
(6.0% of callus
from all cultivars)

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 P. Boonsnongcheep & B. Pongkitwitoon Pharm Sci Asia 2020; 47 (1), 21-29

Table 1. Micropropagation protocols for C. sativa (cont.)

Micropropagation Explants used Culture medium Culture Optimal
strategy Steps (Explant /PGRs conditions experimental References
disinfection) outcome
Rooting MS medium / 24°C, MS medium/
1.0 mg/L IAA and 16/8 h 1.0 mg/L IAA
1.0 mg/L NAA photoperiod and
1.0 mg/L NAA
Indirect regeneration Culture Seeds (10 s 70% DARIA medium 24°C, Wielgus
via callus initiation ethanol and 1% darkness for et al., 2008
sodium hypochlorite 4-7 days
for 20 min)
Callus Cotyledons, stems, DARIA medium/ 24-26°C, Cotyledon
induction and roots 1 mg/L KN and 16/8 h explants
0.05 mg/L NAA photoperiod showed the
highest callus
induction
efficiency and
stem explants
showed the
highest
regeneration
percentage
Indirect DARIA medium/ 24–26°C, DARIA medium/
regeneration 0.2 mg/L BA and 16/8 h 0.2 mg/L BA and
0.03 mg/L NAA photoperiod 0.03 mg/L NAA
Rooting DARIA medium/ 24–26°C, DARIA
2.0 mg/L IAA 16/8 h medium/
photoperiod 2.0 mg/L IAA
Indirect regeneration Callus Young leaves (0.50% MS medium/ 25 ± 2°C, 0.5 μM NAA Lata
via callus induction sodium hypochlorite 0.5 - 2.0 μM IAA or 16/8 h + 1.0 μM TDZ et al., 2010
+0.1% Tween 20 for 0.5 - 2.0 μM IBA or photoperiod resulted in 93%
20 min) 0.5 - 2.0 μM NAA or response and
0.5 - 2.0 μM 2,4-D or the highest
1.0 μM TDZ amount of callus
Shoot MS medium/ 25 ± 2°C, 0.5 μM TDZ
induction 0.5 - 10.0 μM BA or 16/8 h resulted in 93.3%
0.5 - 10.0 μM KN or photoperiod response
0.5 - 10.0 μM TDZ (12.3 shoots)
Rooting MS medium/ 25 ± 2°C, 2.5 μM IBA
0.5 - 10.0 μM IAA or 16/8 h resulted in 96.6%
0.5 - 10.0 μM IBA or photoperiod response
0.5 - 10.0 μM NAA (10.0 roots)
Indirect regeneration Culture Seeds (30 s MS medium 25°C, Movahedi
via callus initiation 70% alcohol, 2% 16/8 h et al., 2015
sodium hypochlorite photoperiod
+ 1 drop of tween
20 for 20 min,
5 min 0.05%
mercuric chloride

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Table 1. Micropropagation protocols for C. sativa (cont.)

Micropropagation Explants used Culture medium Culture Optimal
strategy Steps (Explant /PGRs conditions experimental References
disinfection) outcome
Callus In vitro cotyledon MS medium/ 25°C, Cotyledon
induction and epicotyl 0.1 - 3 mg/L BA or 16/8 h explants
0.1 - 3 mg/L TDZ photoperiod treated with
with or without 2 mg/L TDZ
0.5 mg/L IBA + 0.5 mg/L IBA
showed the
highest callus
fresh weight
Shoot Same media as 25°C, Callus from
induction callus induction 16/8 h epicotyl explant
photoperiod treated with
2 mg/L BA
+ 0.5 mg/L IBA
showed the
highest shoot
regeneration rate
Rooting MS medium/ 25°C, 0.1 mg/L IBA
0.1 - 1 mg/L NAA or 16/8 h showed the
0.1 - 1 mg/L IBA photoperiod highest
rooting rate

Abbreviations : PGRs plant growth regulators, MS Murashige and Skoog medium, BA benzyladenine, KN kinetin, TDZ thidiazuron,
GA3 gibberellic acid, IAA indole-3-acetic acid, IBA indole-3-butyric acid, NAA 1-naphthaleneacetic acid, mT meta-
topolin, 2,4-D 2,4-dichlorophenoxyacetic acid, DIC 3,6-dichloroanisic acid

2.2. Disinfection 3. MEDIA AND CULTURE CONDITIONS

To initiate contamination-free in vitro 3.1. Culture media
culture, plant materials from field cultivation need
Artificial media for in vitro cultures are
to be surface-sterilized. Several studies germinated
generally composed of macronutrients, micro-
seeds of C. sativa on culture medium and used seed-
nutrients, carbon sources, and vitamins, with
derived in vitro explants for further experiments.
optional gelling agent and some additives or plant
Generally, seeds were washed thoroughly with
running tap water and optional 70-75% ethanol growth regulators. The most frequently used basal
followed by a disinfection reagent. The prepared medium for cultivation of C. sativa is Murashige
seeds were then rinsed with distilled water before and Skoog (MS) medium10 with 3% (w/v) sucrose
transferred to the culture medium for germination. as carbon source. Modified MS media were also
Commonly used disinfectants for seeds sterilization reported. Wang et al. used half-strength MS medium
are 1-3% sodium hypochlorite5,7. In addition, 5% with 1% (w/v) sucrose for seed germination and
calcium hypochlorite6 and 0.1% mercuric chloride4 transferred the in vitro explants to full-strength MS
were also used. In some studies, cultivated nodal medium with 3% (w/v) sucrose supplemented with
segments and young leaves were directly surface various plant growth regulators for shoot and root
sterilized to establish the in vitro cultures. The induction4. Only one study used Daria ind medium,
concentration of disinfectant was lower, i.e. 0.5% which the authors explained as a modification of
sodium hypochlorite with 0.1% Tween 20 as Knopp’s solution medium, for seed germination
surfactant3,ʻʻ8,9ʼʼ, since these tissues were more delicate and Daria medium supplemented plant growth
than seeds. regulators for regeneration via callus of C. sativa7.

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P. Boonsnongcheep & B. Pongkitwitoon Pharm Sci Asia 2020; 47 (1), 21-29

Figure 1. Schematic diagram of steps in micropropagation

3.2. Culture conditions 4.1. Shoot multiplication by direct organogenesis

In most studies, in vitro C. sativa cultures To establish a protocol for propagation of
were cultivated at 22-26 ºC with a 16-h photo period. elite C. sativa clones, Lata et al. studied the effects
However, seed germination and callus induction of different concentrations (0.05 - 9.0 µM) of
were carried out in darkness in some studiesʻʻ6-7ʼʼ. cytokinins- benzyladenine (BA), kinetin (KN),
Details on effects of culture conditions and light and thidiazuron (TDZ)- on multiplication and
sources on C. sativa plant regeneration were limited. proliferation of nodal explants containing axillary
However, a study in field-cultivated C. sativa buds3. Among the three cytokinins, TDZ showed
clonally propagated via cuttings suggested that the highest response of shoot induction. Shoots
bud biomass and phytochemical profiles were were proliferated on all explants within 14 d when
improved when treated with supplemental red-blue cultured on MS medium supplemented with 0.5
and red-green-blue subcanopy lights11. Therefore, µM TDZ, with average 13 shoots per explant and
future studies could pay attention on effects of average 7.1 cm of shoot length. Slight increase,
different light spectra and intensities on in vitro although statistically insignificant, in shoot growth
development of C. sativa. was observed when transferring cultures to MS
medium supplemented with 0.5 µM TDZ in
4. PLANT GROWTH REGULATORS combination with 7.0 µM gibberellic acid (GA3)3.
Although cytokinin alone is sufficient for shoot
Plant growth regulators, especially auxins multiplication, studies showed that adding a low
and cytokinins, play a very important role on in vitro concentration of auxin is beneficial in some cases12.
development, morphogenesis, and growth of plant In this matter, Wang et al. studied effects of BA,
tissue cultures12. Shoots can be induced directly from KN, and TDZ, either alone or in combination with
explants (direct organogenesis) or via callus (indirect auxin 1-naphthaleneacetic acid (NAA), on bud
organogenesis), which generally involves appropriate formation of shoot tip explants4. The best response
combination of auxins and cytokinins. After shoot was obtained from explants cultured on MS medium
elongation and multiplication, adventitious root containing 0.2 mg/L TDZ and 0.1 mg/L NAA,
formation is stimulated. Plant growth regulators with the highest auxiliary bud multiplication rate
used in micropropagation of C. sativa are discussed of 3.22 per shoot tip. Similarly, Cheng et al. found
according to stages of culture development. that MS medium supplemented with 0.4 mg/L

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TDZ and 0.2 mg/L NAA yielded the best response induction is generally achieved by supplementation
of shoot regeneration from cotyledon explants, of auxin alone whereas exogenous cytokinins often
with 51.7 ± 7.2 % of explant forming shoots and inhibit root induction12. For micropropagation of
average 3.0 ± 0.4 shoots per explants. Recently, C. sativa, the most frequently used auxins are
efficient micropropagation of C. sativa was achieved IBAʻʻ3-5ʼʼ, 8 and IAAʻʻ6-7ʼʼ. In some studies, highest
using novel aromatic cytokinin, meta-topolin (mT). rooting frequencies were obtained by using
The best response of shoot formation was obtained combination of natural auxin and synthetic auxin
from nodal segments cultured on MS medium such as NAAʻʻ4,6ʼʼ. Interestingly, recent study
supplemented with 2 µM mT, with 100% of explant reported that a separate rooting step by medium
producing shoots, average 13.44 ± 1.38 shoots supplemented with auxin was not required for
per explant, and average shoot length of 11.44 ± C. sativa regenerated on MS medium containing
0.80 cm13. mT, a novel aromatic cytokinin, as the healthy
roots were able to regenerate from the shoots
4.2. Indirect organogenesis multiplied within 4-6 weeks13.
Indirect organogenesis requires shoot
regeneration from morphogenic callus. Slusarkiewicz- 5. ACCLIMATIZATION
Jarzina et al. studied influence of plant growth
Acclimatization is a crucial step when
regulators–KN, NAA, 2,4-dichlorophenoxyacetic
transferring plants from in vitro cultures into filed
acid (2,4-D), and 3,6-dichloroanisic acid (DIC)– on
or greenhouse. In the in vitro cultures, plants are
callus induction and plant regeneration of C. sativa.
cultivated under special conditions including
The results showed that, of all media used, MS
availability of nutrient and growth regulators,
medium supplemented with 2.0 – 3.0 mg/L DIC
yielded the highest frequency of callus formation aseptic condition, and high humidity15. Efficient
(82.7% of explants). However, plant regeneration acclimatization process provides environmental
rate from all media tested were very low (1.4% of adaptation for minimal damage of plants before
plated callus)6. Successful callus-mediated C. sativa being transferred to soil. To increase survivability of
regeneration was reported by using combination C. sativa in vitro plantlets, protocols for acclima-
of auxin and cytokininʻʻ7-8ʼʼ, 14. In order to initiate callus tization had been established and reported. Wang
cultures, leaf explants were cultured on MS medium et al. deflasked C. sativa plantlets when the intro
supplemented with different concentrations of roots reached 0.5-1.0 cm length and cultured in
various auxins, indole-3-acetic acid (IAA), indole- the bottle poured with sterile water. After a week,
3-butyric acid (IBA), NAA and 2,4-D, in combi- the plantlets were washed in running tap water to
nation with cytokinin TDZ. High callus amount remove any remained medium followed by 0.2%
with 93% callus response were obtained from (w/v) Bavistin1, a fungicide solution. The plantlets
explants cultured on MS medium containing 0.5 were transferred to be cultured on perlite and
µM NAA and 1.0 µM TDZ. Then the shoots were hardened by an extra 3-4 weeks in controlled
induced by MS medium containing 0.5 µM TDZ8. conditions of culture room, followed by 2 weeks
Similarly, Movahedi et al. obtained high callus cultured in semi controlled conditions in the shade
formation from cotyledon explants cultured on house, and finally transplanted in field, after which
MS medium supplemented with 0.5 mg/L IBA in allowed 95% of in vitro plantlets to survive for 3
combination with 3 mg/L TDZ14. Another group of months4. Another acclimatization protocol using
researchers also obtained successful C. sativa plant coco natural growth medium, a commercially
regeneration using Daria medium supplemented available medium for hydroponic plants made
with combination of auxin and cytokinin – 0.05 from coconut fibers, was reported. In this protocol,
mg/L NAA and 1 mg/L KN for callus induction, C. sativa rooted shoots were taken out of the in
and 0.03 mg/L NAA and 0.2 mg/L BA for shoot vitro medium and washed thoroughly in running
regeneration7. tap water. The plantlets were preincubated coco
natural growth medium in a thermocol cup covered
4.3. Rooting with polythene bags to maintain humidity for 10
Unlike shoot and callus induction, root day. After that, they were acclimatized in ferti-

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P. Boonsnongcheep & B. Pongkitwitoon Pharm Sci Asia 2020; 47 (1), 21-29

lome, a sterile potting mix, in large pots. The plants shoot organogenesis of Cannabis sativa L. In
reached 14-to 16-cm height within 6 week and Vitro Cell Dev Biol-Plant 2009;45:12–9.
exhibited 95% survival rate at 8 wk after transfer3. 4. Wang R, He LS, Xia B, Tong JF, Li N, Peng F.
A micropropagation system for cloning of
6. CONCLUSIONS hemp (Cannabis sativa L.) by shoot tip culture.
Pak J Bot. 2009;41: 603-8.
As C. sativa conventional field propagation
5. Cheng C, Zang G, Zhao L, Gao C, Tang Q,
proved difficult by its susceptibility to plant
Chen J, et al. A rapid shoot regeneration
pathogens, variation in bioactive components,
protocol from the cotyledons of hemp (Cannabis
and a strict regulatory in many countries, in vitro
sativa L.). Ind Crops Prod. 2016;83:61–5.
cultures can be a potent alternative for production
6. Slusarkiewicz-Jarzina A, Ponitka A, Kaczmarek
of elite C. sativa plant materials in the controlled
Z. Influence of cultivar, explant source and
conditions. Fortunately both direct and indirect
plant growth regulator on callus induction
plantlet regeneration were reported to be highly
and plant regeneration of Cannabis sativa L.
successful using various explant types. Most study
Acta Biol Cracov Ser Bot. 2005;47:145-51.
used MS medium as the basal medium for in vitro
7. Wielgus K, Luwanska A, Lassocinski W,
cultures. Combination of both auxins and cytokinins
Kaczmarek Z. Estimation of Cannabis sativa L.
as plant growth regulators were suitable for callus
tissue culture conditions essential for callus
induction and shoot multiplication in most studies
induction and plant regeneration. J Nat Fibers.
while rooting step generally required only auxin
2008;5:199-207.
alone. Despite the successful of C. sativa in vitro
8. Lata H, Chandra S, Khan I, ElSohly MA.
culture establishment, the further challenging
High frequency plant regeneration from leaf
step lies in the improvement of active compound
derived callus of high Δ9-tetrahydrocannabinol
accumulated in C. sativa in vitro cultures.
yielding Cannabis sativa L. Planta Med. 2010;
76:1629-33.
Conflict of interest
9. Chandra S, Lata H, Mehmedic Z, Khan I,
None to declare
ElSohly MA. Assessment of cannabinoids
Funding content in micropropagated plants of Cannabis
None to declare sativa and their comparison with conventionally
propagated plants and mother plant during
Ethical approval developmental stages of growth. Planta Med.
None to declare 2010;76:743-50.
10. Murashige T, Skoog F. A revised medium for
Article info:
rapid growth and bioassays with tobacco tissue
Received April 21, 2019
cultures. Physiol Plant 1962;15:473-97.
Received in revised form May 29, 2019
11. Hawley D, Graham T, Stasiak M, Dixon M.
Accepted July 2, 2019
Improving Cannabis bud quality and yield
with subcanopy lighting. Hortscience. 2018;
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