plants

Review
The Past, Present and Future of Cannabis sativa Tissue Culture
Adrian S. Monthony , Serena R. Page , Mohsen Hesami and Andrew Maxwell P. Jones *

Department of Plant Agriculture, Gosling Research Institute for Plant Preservation, University of Guelph,
Guelph, ON N1G 2W1, Canada; monthona@uoguelph.ca (A.S.M.); spage01@uoguelph.ca (S.R.P.);
mhesami@uoguelph.ca (M.H.)
* Correspondence: amjones@uoguelph.ca

Abstract: The recent legalization of Cannabis sativa L. in many regions has revealed a need for effective
propagation and biotechnologies for the species. Micropropagation affords researchers and producers
methods to rapidly propagate insect-/disease-/virus-free clonal plants and store germplasm and
forms the basis for other biotechnologies. Despite this need, research in the area is limited due to
the long history of prohibitions and restrictions. Existing literature has multiple limitations: many
publications use hemp as a proxy for drug-type Cannabis when it is well established that there is
significant genotype specificity; studies using drug-type cultivars are predominantly optimized
using a single cultivar; most protocols have not been replicated by independent groups, and some
attempts demonstrate a lack of reproducibility across genotypes. Due to culture decline and other
problems, the multiplication phase of micropropagation (Stage 2) has not been fully developed in
many reports. This review will provide a brief background on the history and botany of Cannabis as
well as a comprehensive and critical summary of Cannabis tissue culture. Special attention will be
paid to current challenges faced by researchers, the limitations of existing Cannabis micropropagation
studies, and recent developments and future directions of Cannabis tissue culture technologies.

Keywords: Cannabis; marijuana; marihuana; tissue culture; review; regeneration; floral reversion;





 micropropagation; TDZ; DKW
Citation: Monthony, A.S.; Page, S.R.;
Hesami, M.; Jones, A.M.P. The Past,
Present and Future of Cannabis sativa
Tissue Culture. Plants 2021, 10, 185. 1. Introduction
https://doi.org/10.3390/plants Cannabis sativa L. is rising to prominence as a commercial crop for industrial, food,
10010185 medical, and recreational applications. The current wave of interest has been characterized
by a growing number of countries easing restrictions around research, commercial cultiva-
Received: 29 December 2020 tion, and sale of dried Cannabis flowers, extracts, and consumable, medicinal, or industrial
Accepted: 14 January 2021
products. With interest renewed in this crop, which has been cultivated for thousands
Published: 19 January 2021
of years, research and innovation in the coming decades is expected to deepen our un-
derstanding of the growth, physiology, and biochemistry of C. sativa. Our improved
Publisher’s Note: MDPI stays neutral
understanding of this important plant will enable large-scale micropropagation, genetic
with regard to jurisdictional claims in
preservation, and the development of plant biotechnologies for advanced new plant breed-
published maps and institutional affil-
ing technologies (NPBTs). The application of plant biotechnologies and NPBTs will require
iations.
effective, high-throughput In Vitro culture systems that will allow for transformation
and subsequent scale-up of any novel cultivars developed by micropropagation. Once
developed, an effective transformation system will require regeneration and clonal propa-
gation systems that can be reliably replicated in multiple lab environments and that can
Copyright: © 2021 by the authors. effectively be scaled up to meet the commercial industry’s needs. To build these robust
Licensee MDPI, Basel, Switzerland.
regeneration and micropropagation systems, methods must be tested across multiple drug
This article is an open access article
and fiber-type genotypes, be flexible with the age and condition of plant tissues, and be
distributed under the terms and
able to accommodate small differences in culture conditions that will inevitably arise from
conditions of the Creative Commons
different lab environments and from the transfer to a large-scale commercial tissue culture
Attribution (CC BY) license (https://
operation. This review offers a critical analysis of the existing published and pre-print
creativecommons.org/licenses/by/
C. sativa micropropagation and regeneration literature and highlights the current shortfalls
4.0/).

Plants 2021, 10, 185. https://doi.org/10.3390/plants10010185 https://www.mdpi.com/journal/plants

Plants 2021, 10, 185 2 of 28

to help direct current and future research to catch up on decades of lost opportunities due
to the criminalization and overregulation of Cannabis.

2. Brief History of C. sativa in North America

The relevance of Cannabis as a versatile crop for oilseed, fiber, medicinal, and recre-
ational drug production spans millennia. Between 1000 and 2000 BCE, Cannabis was
introduced to Western Asia, Europe, and Egypt as a fiber crop for producing cloth, ship
ropes, and paper. After 500 CE, the cultivation of Cannabis was widespread across Eu-
rope [1,2]; however, it was not until 1545 and 1606 that it was introduced to South and
North America, respectively [3]. Despite its centuries-long cultivation, the beginning of the
20th century saw its recreational use outlawed and medicinal use strongly curtailed by an
addendum to the League of Nations’ 1912 Opium Convention. This act pushed countries
around the globe to restrict and criminalize Cannabis [4].
In Canada, Cannabis was made illegal following its addition to the Opium and Drug
Act in 1923 [5,6], and the United States followed suit with the 1937 Marijuana Tax Act,
severely restricting the medicinal use of Cannabis in the United States [6,7]. Cannabis had
been included in the United States Pharmacopoeia since 1850 and was removed in 1942,
a few years after passage of the Marijuana Tax Act of 1937 [8]. In the United States, Cannabis
is classified under the most restrictive drug class (Schedule I) as part of the Comprehensive
Drug Abuse Prevention and Control Act of 1970. This 1970 act overturned the 1937
Marijuana Tax Act and states that Cannabis has “no apparent medical potential and a high
likelihood of abuse” [9,10]. These restrictions, which made no distinction between fibrous
hemp and drug-type Cannabis, had the unfortunate consequence of limiting most Cannabis
research by making its acquisition for research purposes challenging [8,9]. Commercial
production of industrial hemp (C. sativa with <0.3% ∆9 -tetrahydrocannabinol (THC) by
dry weight [11]) has faced many of the same restrictions as drug-type (>0.3% THC by dry
weight) Cannabis in North America, as the distinction between the two has been largely
ignored by government and law enforcement [9].
The strict conditions that regulate Cannabis research have created challenges through-
out the research pipeline [10,12]. Early small-scale clinical trials have investigated the use
of cannabinoids to treat comorbidities of autism spectrum disorder, anxiety, chronic pain,
and seizures and have shown promising results, but research in this area has been highly
restricted and progress has been slow [13–17]. Likewise, these restrictions and the lack
of a legal industry have limited research on agronomic, horticultural, and biotechnolog-
ical aspects of the crop. As a result, relative to the economic importance, technological
development is in its infancy and many techniques that are routine for most species are not
developed in Cannabis.
In recent years, this has started to change as countries around the world have started to
lift some restrictions. In Canada, commercial production of hemp was legalized in 1998 [9];
however, regulatory barriers and a lack of market interest resulted in a very slow-growing
industry until recently [18]. In the United States, a pilot-scale production of industrial
hemp was legalized in 2014 followed by commercial-scale federal legalization in the 2018
farm bill [9,18]. Prior to this change, federally funded research in the US could only be
conducted with Cannabis obtained from the National Institute on Drug Abuse (NIDA). With
the passing of the 2018 farm bill, hemp can now be used for research, but drug-type Cannabis
is still highly restricted at the federal level. In 2013, the Marihuana for Medical Purposes
Regulations were implemented by the Government of Canada, laying the groundwork for
commercial production of medicinal Cannabis [8,19]. The legalization of the possession,
growth, and consumption of Cannabis for recreational purposes followed in October 2018.
At the international level, regulations are also beginning to change; a landmark decision
by the United Nations Commission on Narcotic Drugs (CND) voted to remove Cannabis
from Schedule IV of the 1961 Single Convention on Narcotic Drugs in December 2020,
thereby recognizing the medicinal and therapeutic uses of Cannabis [20]. While still highly
regulated, the legalization of Cannabis for medical and recreational consumption in Canada,
Plants 2021, 10, 185 3 of 29

Plants 2021, 10, 185 3 of 28

Narcotic Drugs in December 2020, thereby recognizing the medicinal and therapeutic uses
of Cannabis [20]. While still highly regulated, the legalization of Cannabis for medical and
recreational consumption
the legalization of hemp ininthe
Canada,
UnitedtheStates,
legalization of hemptrend
and a similar in the Unitedthe
around States,
world and a
has
similar trend around the world has resulted in a renaissance
resulted in a renaissance period for Cannabis research. period for Cannabis research.

3.
3. Botany
Botany andand Taxonomy
Taxonomy of C. sativa
Cannabis
Cannabis sativa
sativa L.
L. (Cannabis,
(Cannabis, hemp,
hemp, marijuana)
marijuana) is is an
an annual
annual flowering
flowering plant of the
family Cannabaceae. Although Cannabis is usually dioecious,
family Cannabaceae. Although Cannabis is usually dioecious, hermaphroditism hermaphroditism occurs
occursin
some
in some cultivars
cultivars(Figure
(Figure 1A) and
1A) andboth
bothformal
formaland andinformal
informal breeding
breeding programs
programs have
resulted
resulted inin some monoecious
monoecious cultivars,
cultivars, primarily
primarily restricted
restricted to hemp [21–23]. The family
Cannabaceae
Cannabaceae consists
consists of
of ten
ten genera, containing
containing over 100 accepted species, with Humulus
lupulus
lupulus L.L. (hops;
(hops; the
thechief
chiefingredient
ingredientininbeer)
beer)being
beinga anotable
notablemember
member[24,25].
[24,25].C. C.
sativa is
sativa
is native
native to to central
central Asia,
Asia, likelyininthe
likely thefoothills
foothillsofofthe
theHimalayan
HimalayanMountain
Mountain Range
Range [1,2].
[1,2].
Cannabis is a fast-growing
fast-growing plant,
plant,growing
growingup uptoto1010cmcma aday
dayand
andreaching heights
reaching heightsof of
6m6min
itsits
in native habitat,
native while
habitat, whilegrowth
growth in in
temperate
temperateclimates
climatesis usually lower
is usually [23,26,27].
lower [23,26,27].

Figure
Figure 1.1. InInVitro
Vitroflowering
floweringofof
Cannabis sativa.
Cannabis (A) (A)
sativa. Flowering C. sativa
Flowering malemale
C. sativa plantplant
displaying a
displaying a
hermaphroditic phenotype, showing female flowers (left) adjacent to male flowers
hermaphroditic phenotype, showing female flowers (left) adjacent to male flowers (right). Scale (right). Scale
bar—1 mm. (B) In Vitro male inflorescences of C. sativa. Scale bar—1 mm (C) A pair of female C.
bar—1 mm. (B) In Vitro male inflorescences of C. sativa. Scale bar—1 mm (C) A pair of female
sativa florets obtained from In Vitro flowering C. sativa. Scale bar—1 mm. (D) Glandular trichomes
C. sativa florets obtained from In Vitro flowering C. sativa. Scale bar—1 mm. (D) Glandular trichomes
developing on the bract surrounding the ovary of a female C. sativa inflorescence. Scale bar—2
developing
mm. (E) Matureon the bract surrounding
flowering the ovary
In Vitro explant of C.of ScaleC.bar—1
a female
sativa. sativa inflorescence. Scale bar—2 mm.
cm. (F) Four-week-old
(E) Matureexplants
vegetative flowering In Vitro
reverted explant
from of C.C.sativa.
In Vitro Scale bar—1 cm.
sativa inflorescences. (F) bar—1
Scale Four-week-old
cm. vegetative
explants reverted from In Vitro C. sativa inflorescences. Scale bar—1 cm.
When grown from a seed, the first true leaves are pairs of oppositely oriented single
When
leaflets grown
(Figure 2A).from a seed,
As the plantthe first true
matures, theleaves are pairs
phyllotaxy of oppositely
shifts oriented
from opposite single
to alternate
leaflets (Figure 2A). As the plant matures, the phyllotaxy shifts from opposite
leaf arrangement and the number of leaflets per leaf increases (Figure 2B; Clarke 1999; to alternate
leaf arrangement
Spitzer-Rimon and
et al. the Leaves
2019). numberonofa leaflets
mature per leaf
plant areincreases (Figure
digitate with 2B; Clarke
anywhere from1999;
5 to
Spitzer-Rimon et al. 2019). Leaves on a mature plant are digitate with anywhere from 5 to
11 leaflets and have a long petiole, although during flowering, they often revert to
11 leaflets and have a long petiole, although during flowering, they often revert to produc-
producing lower numbers of leaflets [23,28]. Cannabis is predominantly a short-day plant,
ing lower numbers of leaflets [23,28]. Cannabis is predominantly a short-day plant, with
with flowering induced by 12- to 14-h photoperiods [29]; however, some photoperiod-
flowering induced by 12- to 14-h photoperiods [29]; however, some photoperiod-insensitive
insensitive cultivars have been developed. Male and female plants cannot easily be
cultivars have been developed. Male and female plants cannot easily be distinguished
distinguished until flowers begin to appear [26,30]. Male flowers have five green or yellow
until flowers begin to appear [26,30]. Male flowers have five green or yellow petals and
petals and are larger than female flowers (Figure 1B). Female flowers consist of an ovule
are larger than female flowers (Figure 1B). Female flowers consist of an ovule enclosed
enclosed in a thin green bract with two yellow/whiteish stigma emerging from the closed
in a thin green bract with two yellow/whiteish stigma emerging from the closed bracts
bracts (Figure 1C) [26,30]. During the development of the flower, before the elongation of
(Figure 1C) [26,30]. During the development of the flower, before the elongation of the
stigma, glandular trichomes develop on the bract surrounding the ovary (Figure 1D) [22].
Plants 2021, 10, 185 4 of 29

Plants 2021, 10, 185 4 of 28

the stigma, glandular trichomes develop on the bract surrounding the ovary (Figure 1D)
[22]. Two main types of trichomes can be found covering Cannabis plants: glandular and
Two main typestrichomes.
non-glandular of trichomes canthe
Only be found produceCannabis
formercovering plants:inglandular
cannabinoids and non-
any considerable
glandular trichomes. Only the former produce cannabinoids in any considerable
quantity, and glandular trichomes are predominantly found on the bracts and floral quantity,
leaves
and glandular trichomes are predominantly found on the bracts and floral leaves of
of female plants (Figure 1D). Male plants produce few, if any, glandular trichomes [28,31]. female
plants
Due to(Figure 1D).levels
their low Maleofplants produce few,
cannabinoids, male if any, glandular
plants trichomes
are generally [28,31]. Due
not consumed astoa
their low levels of cannabinoids, male plants are generally not consumed as
medicinal or recreational drug and will not be extensively discussed in this review.a medicinal or
recreational drug and will not be extensively discussed in this review.

Figure 2. (A) In Vitro germinated seedling of C. sativa demonstrating opposite leaf arrangement.
Figure 2. (A) In Vitro germinated seedling of C. sativa demonstrating opposite leaf arrangement.
White arrows show oppositely oriented first true leaves. Scale bar—1 cm. (B) A Stage 2 vegetative ex-
White arrows show oppositely oriented first true leaves. Scale bar—1 cm. (B) A Stage 2 vegetative
plant (subcultured from a nodal explant) of C. sativa demonstrating alternate leaf arrangement (black
explant (subcultured from a nodal explant) of C. sativa demonstrating alternate leaf arrangement
arrows),
(black a change
arrows), in phyllotaxy
a change resulting
in phyllotaxy from explant
resulting maturation.
from explant ScaleScale
maturation. bar—1 cm.C.
cm. (C)
bar—1 (C)sativa
C.
growngrown
sativa in controlled environment
in controlled growth
environment chambers
growth underunder
chambers fluorescent lighting.
fluorescent (D) C.(D)
lighting. sativa grown
C. sativa
outdoors
grown under aunder
outdoors shadea cloth
shadeincloth
Colombia. ImageImage
in Colombia. supplied courtesy
supplied of Avicanna™.
courtesy (E) Hyperhy-
of Avicanna™. (E)
Hyperhydric C. sativa explants
dric C. sativa explants growing growing on Murashige
on Murashige and
and Skoog Skoog
(MS) (MS) medium
medium supplemented
supplemented with 0.5 µM
with 0.5 µM (TDZ).
thidiazuron thidiazuron (TDZ).

Cannabis is a matter
The taxonomy of the genus Cannabis matter of
of spirited
spirited debate
debate and
and nono consensus
consensus
has emerged
has emerged on on whether
whether itit is
is aa monospecific
monospecific or or polyspecific
polyspecific genus
genus [32–34].
[32–34]. The
The ability
ability to
to
distinguish between
distinguish between hemp
hempand drug-typeCannabis
anddrug-type Cannabishas
hasbeen
been thethe
subject of of
subject much
muchinterest by
interest
lawlaw
by enforcement, whichwhich
enforcement, relies on THConcontent
relies THC for distinction
content [35]. From a[35].
for distinction law From
enforcement
a law
and regulatory standpoint, the two main categories of Cannabis
enforcement and regulatory standpoint, the two main categories of Cannabis have have been described
been
as “drug-type”
described (medicinal(medicinal
as “drug-type” or recreational) and “fiber-type”
or recreational) (industrial(industrial
and “fiber-type” hemp), thehemp),
drug-
typedrug-type
the generally generally
being dioecious, with a short,
being dioecious, withwide, bush-like
a short, wide, growth
bush-like pattern,
growthwhile the
pattern,
fiber-type can be either dioecious or monoecious with a tall and thin growth
while the fiber-type can be either dioecious or monoecious with a tall and thin growth pattern [36].
However,
pattern this
[36]. distinction
However, this is further complicated
distinction by hemp by
is further complicated developed for seed for
hemp developed or seed
non-
psychoactive cannabinoids, which often morphologically resemble
or non-psychoactive cannabinoids, which often morphologically resemble drug-type drug-type Cannabis.
Two distinct
Cannabis. Two Cannabis
distinctchemotypes have been identified,
Cannabis chemotypes have beenwhich also fall
identified, in line
which with
also fallthe
in two
line
aforementioned morphological groups and are largely defined by their
with the two aforementioned morphological groups and are largely defined by their THC THC content. The
fiber-type Cannabis, or “hemp”, has a THC dry weight in the flowering heads of <0.3% or
content. The fiber-type Cannabis, or “hemp”, has a THC dry weight in the flowering heads
<0.2% depending on the jurisdiction [9,11,18]. Hemp can often be accompanied by a higher
of <0.3% or <0.2% depending on the jurisdiction [9,11,18]. Hemp can often be accompanied
cannabidiol (CBD) content (THC:CBD < 1), while the elite drug-type cultivars typically
by a higher cannabidiol (CBD) content (THC:CBD < 1), while the elite drug-type cultivars
report a THC:CBD ratio >1, or >0.3% THC in the flower heads [9,37].
typically report a THC:CBD ratio >1, or >0.3% THC in the flower heads [9,37].
However, a taxonomic system based on THC:CBD ratios has faced scrutiny [38] and
However, a taxonomic system based on THC:CBD ratios has faced scrutiny [38] and
other classification systems that further divide the species based on chemotype have been
other classification systems that further divide the species based on chemotype have been
suggested. These include classifications based on other secondary metabolites produced
by the Cannabis rather than solely the THC and CBD levels [39,40]. Early genetic studies
Plants 2021, 10, 185 5 of 28

attempting to distinguish between the genetic fingerprints of hemp and Cannabis have
suggested that the chief differentiation factor between the two plants was a single locus that
determined the production of THC or CBD synthases [41]. These findings have been echoed
by whole genomic and transcriptomic assemblies of hemp and drug-type Cannabis, which
have shown that hemp plants have high levels of cannabidiolic acid synthase (CBDAS)
genes and transcripts, while the THCAS gene encoding the oxidocyclase enzyme, which
forms tetrahydrocannabinolic acid (THCA), is dominant in drug-type cultivars [2,42].
However, recent work using single-nucleotide polymorphisms (SNPs) has shown that
the genetic differences between hemp extend beyond the loci responsible for cannabinoid
production and are instead found throughout the entire genome [35].
Drug-type Cannabis has been historically described by enthusiasts as consisting of three
species: C. sativa, C. indica, and C. ruderalis. The diversity of chemical and morphological
traits within Cannabis has led some taxonomists to agree with this and propose that Cannabis
should be considered a polyspecific genus containing multiple individual species: sativa,
indica, and ruderalis [43–45]. Further sub-speciation has even been suggested within these
groups [34,46]; however, this nomenclature has yet to be widely used. The taxonomy of
drug-type Cannabis is complicated by years of prohibition, which resulted in informal,
clandestine breeding programs that caused decades of interbreeding and hybridization
without records of parentage [40,46]. The ability to consistently and reliably distinguish
between sativa and indica types of Cannabis has been scrutinized [35], and as a result of
these underground breeding programs, establishing the pedigree of Cannabis is incredibly
challenging and has resulted in unpredictability for consumers of C. sativa products [33].
Concerns have also been raised that this ever-increasing introgression is leading to a decline
in biodiversity in the species and a loss of native indigenous C. sativa varieties [46]. The
ease of interbreeding within Cannabis has resulted in a highly polymorphic genome, which
has led many researchers to classify Cannabis sativa as a monospecific, highly polymorphic
species [32,47–49]. This debate is ongoing and has been reviewed extensively [32,34,45].
However, it is not the focus of this review and we will refer to species as presented by the
authors when possible.

4. Current Production Practices

Cannabis is a highly adaptable species that can be grown in a variety of conditions,
including outdoors in tropical or temperate climates or in controlled environments ranging
from rudimentary greenhouse structures to sophisticated controlled environment facilities
(Figure 2C,D) [36]. The production system of choice is determined based on the end-use
of the plant. Plants grown to produce low-value commodities such as oilseed or fiber
are typically cultivated exclusively outdoors, where production costs are low. In contrast,
plants cultivated for dried flowers for recreational or medicinal use can be cultivated
outdoors, in greenhouses, or in indoor production facilities. While production costs for
recreational/medicinal products are also lower outdoors, there is a general belief that
indoor production facilities produce higher-quality products, which justifies the extra
costs for premium flowers [50]. However, with the growing trend toward extracts and
purified cannabinoids, it is likely that much of the medicinal/recreational production (CBD
from hemp, THC from drug-type Cannabis) will be done outdoors to capitalize on these
lower production costs. The higher level of oversight offered in controlled environments
also allows for easier management of insects and diseases, which is important in order
to meet strict government health and safety regulations surrounding the use of chemical
control agents, microbial load, and other quality assurance (QA) requirements [51,52].
These regulations have driven most of the commercial drug-type Cannabis production into
greenhouses and indoor facilities for now [53].
As with production systems, the approach to plant propagation is influenced by the
end-use of the plants. Traditionally, hemp has been cultivated by seed using large-scale,
highly mechanized, production practices similar to other grain crops [54]. In contrast,
drug/recreational Cannabis is generally propagated using clonal methods and treated as a
Plants 2021, 10, 185 6 of 28

horticultural crop [51,52,55]. This is done to mitigate the high level of phenotypic diversity
displayed within seedling populations and to consistently produce high quality, uniform
crops that meet consumer preferences and comply with government regulations [56].
While this variability also exists in hemp seed, the benefits of clonal propagation and
manual planting do not justify the costs for oilseed or fiber [57]. However, new regulations
surrounding the use of hemp to produce CBD and other non-psychoactive cannabinoids
have led some hemp producers to use clonal propagation [18,54,57].
Clonal propagation can take many forms, but traditionally, Cannabis has been prop-
agated through stem cuttings. In general, Cannabis is relatively easy to root, and large
numbers of plants can be produced from a single mother plant [55]. While more expensive
than seed, this approach can be efficiently used to mass-produce genetically and phenotyp-
ically uniform plants at a commercial scale to produce a more uniform crop. However, this
approach requires the maintenance of mother plants in a vegetative state and can occupy
10–15% of the floor space in a commercial operation. The maintenance of mother plants
also requires them to remain in a vegetative state. While this is easily accomplished for
most genotypes, it presents challenges for day-neutral genotypes as they do not respond
to photoperiod [29]. Perhaps of greatest importance, though, is that mother plants are
susceptible to insects, pathogens, and viruses and can transmit these biotic factors to their
cuttings and lead to problems during production. This is of importance in Cannabis as
there are currently very few control options registered for the crop and there is a strong
consumer preference for no pesticide use [18,51,52].

5. Micropropagation of C. sativa
An alternative approach to clonal propagation that addresses many of the challenges
of conventional C. sativa propagation is the use of micropropagation, which uses plant
tissue culture to mass-propagate plants in a highly controlled environment using aseptic
techniques. In micropropagation, plants are cultivated in culture vessels, typically in a
multi-tier culture room or even in stackable vessels outfitted with light emitting diode (LED)
lighting [58] (Figure 3). This allows large numbers of plants to be maintained in a very
small space, thereby reducing the amount of floor space required to maintain mother plants.
This is particularly attractive for producers that want to maintain a large genetic library
but do not want to dedicate the amount of floor space that would be required otherwise.
Tissue culture techniques also offer a variety of approaches that may help in maintaining
Plants 2021, 10, 185 day-neutral genotypes and for long-term genetic preservation. Most importantly, due to7 the of 29
sterile nature of plant tissue culture, it can be used to produce insect-/pathogen-/virus-free
propagules to reduce biotic pressures.

Figure3.3.Healthy
Figure Healthy C.
C. sativa explants growing
growing inin We-V
We-V boxes
boxes (A).
(A).Callus
Calluscultures
culturesgrowing
growingininglass
glass
culturevessels
culture vesselsunder
underLED
LEDlighting
lightinginin a controlled
a controlled environment
environment growth
growth chamber
chamber (B) (B)
andand high-
high-density
density stackable
stackable culture(We-V)
culture vessels vesselswith
(We-V) with individually
individually programmableprogrammable LED(C)
LED lighting lighting (C)
demonstrate the
demonstrate the variety and density with which C. sativa can be cultured under
variety and density with which C. sativa can be cultured under In Vitro conditions. In Vitro
conditions.

The use of plant tissue culture for the propagation of disease-free plants has provided
the foundation for clean plant programs in various crops since the late 1900s [59–61]. In
some cases, certified disease-free plants produced through tissue culture are planted
Plants 2021, 10, 185 7 of 28

The use of plant tissue culture for the propagation of disease-free plants has provided
the foundation for clean plant programs in various crops since the late 1900s [59–61].
In some cases, certified disease-free plants produced through tissue culture are planted
directly in the field for production, while in other cases, they are used as clean material
that is further propagated through other means in highly sanitary conditions and tested
for important diseases before being used for commercial production [62]. The latter model
provides most of the benefits of micropropagation while reducing costs. This approach has
been successful in the seed potato industry for developing a disease eradication system [59].
In the case of Cannabis, either approach could be taken, and the decision would need to be
based on a careful analysis of the costs and benefits by the producer, which will include
many factors such as the efficiency of micropropagation, labor costs, the value of additional
floor space, risk assessment, and other factors.
The principal challenge in developing effective micropropagation methods is species
and genotype specificity, resulting in many variations at each stage of micropropagation.
Micropropagation is often broken down into five stages, where each stage needs to be opti-
mized to establish a fully developed micropropagation method (Figure 4) [63,64]. These
include Stage 0: Selection/maintenance of parent plant material; Stage 1: Initiation of
cultures; Stage 2: Multiplication of shoots/embryos; Stage 3: Shoot elongation and rooting;
Stage 4: Acclimatization (Figure 4). While the selection and maintenance of ex vitro stock
plants are often ignored, the importance of stock plant health for the subsequent success
of the cultures can have a significant impact on further results. Provided that the stock
plants from Stage 0 are in good condition, the explants generally respond well to surface
disinfection and produce an initial flush of growth during Stage 1. This initial flush of
growth is often followed by a more sporadic growth pattern until the explants acclimatize
to In Vitro conditions. It is in Stage 2, after plants acclimatize to In Vitro growth, where the
largest benefit of micropropagation becomes apparent: the exponential multiplication of
plants. Many horticultural crops are maintained for extended periods of time in Stage 2 and
continuously sub-cultured for commercial-scale plant production. To illustrate the capabil-
ity for rapid plant production, an In Vitro protocol using Stage 2 plants with a reasonable
multiplication rate of 10 would produce one million plants after only six subcultures (106 ).
When a sufficient quantity of plants has been produced in Stage 2, they are then transferred
to Stage 3 to elongate and develop roots, or alternatively, they are transferred directly
from their In Vitro environment to an indoor growth facility/greenhouse to acclimatize,
thereby combining Stages 3 and 4 (Figure 4). Combining these stages is often preferred for
commercial applications as it reduces the number of steps In Vitro, thereby saving time
and labor costs [65].
The earliest In Vitro studies of Cannabis were conducted in hemp and focused on
determining its suitability for In Vitro culture and whether tissue culture would affect the
agronomic and chemical characteristics of the plant (Table 1) [66–69]. Richez-Dumanois
et al. [66] showed that hemp could be micropropagated using nodal cuttings and the
inclusion of IBA and BAP promoted the growth of shoots from existing meristematic
tissues. They also demonstrated the successful acclimatization of In Vitro grown hemp to
greenhouse conditions [66]. Importantly, their work showed that the In Vitro grown plants’
chemical and physical profiles were similar to their greenhouse-grown counterparts. This
finding has been reasserted by contemporary studies on medicinal Cannabis that found
micropropagation from nodal cuttings had no significant effect on the cannabinoid contents
of the mature flowering plant [69].
021, 10, 185 8 of 29

Plants 2021, 10, 185 8 of 28

(Figure 4). Combining these stages is often preferred for commercial applications as it
reduces the number of steps In Vitro, thereby saving time and labor costs [65].

Figure 4. The five-stage

Figure 4. Themicropropagation process for tissue
five-stage micropropagation culture.
process The red
for tissue arrowThe
culture. is indicative
red arrowofisthe 1-3-4 approach
indicative of (where
the 1-3-4Stage
Stage 2 is skipped). approach (where Stage
2 is commonly 2 is skipped).
skipped Stage
in C. sativa 2 is commonly skipped
micropropagation methodsin C. to
due sativa
the plant’s recalcitrance to
micropropagation
long-term culture characterized methods due
by a slow to the in
decline plant’s recalcitrance
fitness. Inclusion oftoStage
long-term culture
2 allows characterized
for repeated by a of In Vitro
subcultures
slow decline
plants (indicated in fitness.
by the circular Inclusion
arrows), of Stage
therefore 2 allows for
facilitating repeatedmultiplication
large-scale subcultures oforInlong-term
Vitro plants
germplasm storage.
(indicated by the circular arrows), therefore facilitating large-scale multiplication or long-term
germplasm storage.
Table 1. Summary of the published micropropagation studies on C. sativa. The table summarizes studies that rely on shoot
multiplication (SM) to increase explant number. SM refers to the proliferation of multiple shoots from an existing meristem,
The earliest In Vitro studies of Cannabis were conducted in hemp and focused on
such as axillary or apical nodes and floral meristems. Cannabis type is defined in this table as either psychoactive “drug-type”
determining its suitability for In Vitro culture and whether tissue culture would affect the
(Cannabis; tetrahydrocannabinol (THC) > 0.3% in flowering head) or known industrial hemp genotypes “fiber-type” (Hemp;
agronomic and chemical characteristics of the plant (Table 1) [66–69]. Richez-Dumanois et
THC < 0.3% in flowering head). A breakdown of the cultivars (CVs) used in the study and the number which responded to
al. [66] showed that hemp could be micropropagated using nodal cuttings and the
the treatment are included for each study. N.S. Not specified is assigned to data that were not specified, instances of “data
inclusion of IBA and BAP promoted the growth of shoots from existing meristematic
not shown”, or when data are omitted in the original research article.
tissues. They also demonstrated the successful acclimatization of In Vitro grown hemp to
greenhouse conditions C. [66]. Importantly,
sativa Type their work showed that the In Vitro grown
Explant Stages
Source plants’(Response)
chemical and physical(#CVs profiles wereBest similar
Media Best Results
to their greenhouse-grown Reported
Responded/Used)
counterparts. This finding has been reasserted by contemporary studies on medicinal
Richez- Apical and axillary Fiber-type SM: SM: Stage 0: Y
Dumanois et al.,
Cannabisnodes
that found micropropagation from nodal cuttings had no significant effect on the Stage 1: Y
(2/2) MS + 0.5 µM BAP + 0.1 µM 2 shoots/explant (apical
1986 cannabinoid(SM) contents of the mature flowering plant IBA[69]. meristem), % response N.S. Stage 2: N
[66] Rooting: Rooting: Stage 3: Y
MS + 0.2% activated charcoal 47.7% response Stage 4: Y
+ 10 µM IBA
Lata et al., 2009a Axillary nodes (SM SM: SM: Stage 0: Y
Drug-type (1/1) Stage 1: Y
[70] and rooting) MS + 0.5 µM TDZ 12.6 shoots/explant
100% response Stage 2: N
Rooting: Rooting: Stage 3: Y
1 Stage 4: Y
2 MS + 2.5 µM IBA + 0.05% 4.8 roots/explant
activated charcoal 95% response
Lata et al., 2009b Alginate Drug-type Shoot induction: Shoot induction: Stage 0: Y
[71] encapsulated axillary (1/1) MS + 0.5 µM TDZ + 0.075% 11.8 shoots/explant (90 days; Stage 1: Y
nodes (Shoot PPM avg. 30 explants) Stage 2: Y
induction and Rooting: Rooting: Stage 3: Y
rooting) (1:1) sterile fertilome: coco 100% conversion from Stage 4: Y
natural growth medium + MS encapsulation (90 days)
+ 0.5% PPM
Lata et al., 2016 Axillary nodes (SM Drug-type SM: SM: Stage 0: Y
[72] and rooting) (1/1) MS + 2 µM mT 13.4 shoots/explant Stage 1: Y
100% response Stage 2: N
Rooting: Rooting: Stage 3: Y
MS + 2 µM mT 13.8 roots/explant Stage 4: Y
96% response
Plants 2021, 10, 185 9 of 28

Table 1. Cont.

C. sativa Type
Explant Stages
Source (#CVs Best Media Best Results
(Response) Reported
Responded/Used)
Grulichova Shoot tips Fiber-type SM: SM: Stage 0: Y
et al., 2017 (SM) (2/2) MS + 0.54 µM NAA + Shoots/explant N.S. Stage 1: Y
[73] 1.78 µM BAP a % response N.S. Stage 2: N
Stage 3: N
Stage 4: N
Piunno et al., Immature and Drug-type Shoot induction: Shoot induction: Stage 0: Y
2019 mature inflorescences (2/3) MS + 10 µM TDZ 4 shoots/floral cluster Stage 1: Y
[74] (shoot induction and % response N.S. Stage 2: N
rooting) Rooting: Rooting: Stage 3: Y
MS + 1.86 µM kinetin + Describes ‘most’ cultures as Stage 4: Y
0.54 µM NAA a rooting.
Smýkalová Shoot apex, isolated SM: SM: Stage 0: Y
apical meristem, and Fiber-type Stage 1: Y
et al., 2019 IMB4 + 6.97 µM KIN + 4.4 shoots/explant (isolated
cotyledonary nodes (1/1) Stage 2: N
[75] 0.81 µM BAP9THP + 0.11 mM meristems)
from seedlings adenine hemisulphate a ~96% response Stage 3: Y
(SM, shoot Shoot development: Shoot development: Stage 4: N
development, and 1
2 MS no PGRs N.S.
rooting) Rooting: Rooting:
1 a
2 MS + 0.20 µM NAA 50% response
Monthony et al., Single and pairs of Drug-type Floral reversion: Floral reversion: Stage 0: N
2020a florets (2/2) DKW w/vitamins + 1 µM mT Estimated 18.2 explants Stage 1: N
[76] (floral reversion and derived from one In Vitro Stage 2: Y
rooting) flowering plant Stage 3: Y
81% response Stage 4: Y
Rooting: Rooting:
DKW w/vitamins 44% rooted
Page et al., 2020 Axillary nodes Drug-type SM: SM: Stage 0: N
[77] (SM) (4/5) DKW + 0.5 µM TDZ 2.23 shoots/explant Stage 1: N
80% response Stage 2: Y
Stage 3: N
Stage 4: N
Wróbel et al., Shoot tips and nodes Fiber-type SM: SM: Stage 0: Y
1
2020 from axillary (1/1) 2 MS + 2.85 µM IAA a 2.5 shoots/explant Stage 1: Y
[78] branches 70% response Stage 2: Y
(SM and rooting) Rooting: Rooting: Stage 3: Y
1
2 MS + 2.85 µM IAA a 74.6% rooted Stage 4: Y
Codesido et al., Axillary nodes Drug-type SM: SM: Stage 0: Y
2020 (SM) (6/6) Formula βH media Shoots/explant N.S. Stage 1: Y
[79] 58% response Stage 2: N
Stage 3: N
Stage 4: N
Mestinšek Mubi Axillary nodes Drug-type b SM: SM: Stage 0: Y
et al., 2020 (SM) (2/2) MS+ 2.07 µM mT a 1.78 shoots/explant Stage 1: Y
[80] 97.8% response Stage 2: N
Rooting: Rooting: Stage 3: Y
MS + no PGRs % response N.S. Stage 4: Y
a Molarity values converted from mg/L. b Authors reported using a high-cannabidiol (CBD) drug-type C. sativa, % THC not specified.

As it has been well established that Cannabis can be cultured In Vitro without af-
fecting its biochemical outcomes, contemporary studies have shifted to determining the
optimal growth and multiplication conditions for each stage of micropropagation, a task
complicated by the numerous factors which must be considered when growing a plant
In Vitro (Figure 5). Existing Cannabis micropropagation studies have primarily taken to
optimizing freshly initiated tissues for shoot proliferation, opting to focus on plant growth
regulator (PGR) combinations that result in rapid shoot proliferation (Table 1) [69,72,75].
Once developed, the shoots are rooted on an auxin-rich medium and then transferred
back into growth facilities (Stages 3 and 4; Figure 4). These rapid and high-throughput
approaches to C. sativa micropropagation are useful but neglect to study the long-term
Plants 2021, 10, 185 10 of 28

health and maintenance of the explants in culture, evidenced by the relatively few studies
reporting results from Stage 2 (Tables 1 and 2). The result of this is that explants are not fully
acclimatized to In Vitro conditions, and consequentially, the protocols are only optimized
for Stages 1, 3, and 4 (a 1-3-4 approach) rather than for the long-term conservation and
multiplication of germplasm in Stage 2 (Figure 4 and Table 1).

Figure 5. Tissue culture of healthy explants relies on the careful optimization of multiple factors. Center image: Freshly
subcultured vegetative explant of C. sativa.
Figure 5. Tissue culture of healthy explants relies on the careful optimization of multiple factors.
Table 2. Summary of the published regeneration
Center studies
image: Freshly on C. sativa.
subcultured The table
vegetative summarizes
explant studies that rely on shoot
of C. sativa.
regeneration for the production of C. sativa explants. Shoot regeneration refers to the formation of de novo shoots from
non-meristematic tissues such
Table 2. Summary of theaspublished
leaves, stems, or cotyledons.
regeneration This
studies onincludes direct
C. sativa. The and
tableindirect organogenesis
summarizes and rely
studies that somatic
on shoot
regeneration
embryogenesis fromfor the production
callus or suspensionof C. sativa explants.
cultures. Shootisregeneration
Cannabis type refers
defined in this tabletoasthe formation
either of de novo
psychoactive shoots from
“drug-type”
non-meristematic
(Cannabis; THC > 0.3% tissues such ashead)
in flowering leaves,
orstems,
known or industrial
cotyledons.hemp
This includes
genotypes direct and indirect
“fiber-type” organogenesis
(Hemp; and in
THC < 0.3% somatic
flowering head). A breakdown of the cultivars (CVs) used in the study and the number which responded to the treatment“drug-
embryogenesis from callus or suspension cultures. Cannabis type is defined in this table as either psychoactive
type” (Cannabis; THC > 0.3% in flowering head) or known industrial hemp genotypes “fiber-type” (Hemp; THC < 0.3% in
are included for each study. N.S. Not specified is assigned to data that were not specified, instances of “data not shown”,
flowering head). A breakdown of the cultivars (CVs) used in the study and the number which responded to the treatment
or when data are omitted in the original research article.
are included for each study. N.S. Not specified is assigned to data that were not specified, instances of “data not shown”,
or when data are omitted in the C. original research article.
sativa Type
Explant Stages
Source (#CVs Responded/ Optimal Media Optimal Results
(Response)
Explant C. sativa Reported
Stages
Source Used) Optimal Media Optimal Results
(Response) Type Reported
Mandolino and Leaf, hypocotyl, Fiber-type Callogenesis/ Callogenesis/shoot Stage 0: N
Ranalli, 1999 cotyledon, and root (1/12) shoot regeneration: regeneration: Stage 1: Y
[81] (Callogenesis and MS + B5 vitamins + One tested cultivar Stage 2: N
shoot regeneration) 13.57–45.24 µM 2,4-D + occasionally gave rise to Stage 3: Y
0.04–0.44 µM BAPa organogenic callus from Stage 4: N
hypocotyl tissue.
% regeneration N.S.
Ślusarkiewicz- Juvenile leaves, Callus induction: Callus induction: Stage 0: Y
petioles, internodes, Fiber-type Stage 1: Y
Jarzina et al., MS + dicamba (9.05 and 52.3% (5 CV Average; petioles)
(5/5)
2005 and axillary nodes 13.57 µM a ) Stage 2: N
[82] (Callus induction, Shoot induction: Shoot induction: Stage 3: Y
shoot induction, and MS + dicamba (9.05 and 2.5% (cv. Silesia; petioles) Stage 4: Y
rooting) 13.57 µM a )
Rooting: Rooting:
MS + 0.57 µM IAA + 0.54 µM 69.9% plantlets formed roots
NAA a
Plants 2021, 10, 185 11 of 28

Table 2. Cont.

C. sativa Type
Explant Stages
Source (#CVs Responded/ Optimal Media Optimal Results
(Response) Reported
Used)
Axillary nodes Callus induction: Callus induction: Stage 0: Y
Plawuszewski (Direct Fiber-type DARIAind+ + NAA + BAP % callusing N.S. Stage 1: Y
et al., 2006 organogenesis) Stage 2: N
(3/3) (concentrations N.S.)
[67] Stems and roots Stage 3: Y
Shoot proliferation: Shoot proliferation:
(Indirect somatic DARIApro + NAA + BAP Adventitious shoot formation Stage 4: N
embryogenesis) (concentrations N.S.) from axillary nodes and
somatic embryo formation
from stem tissue reported
% N.S.
Somatic embryogenesis: Somatic embryogenesis:
DARIApro+ + NAA + BAP N.S.
(concentrations N.S.)
Rooting: Rooting:
DARIAroot + IAA N.S.
(concentrations N.S.)
Raharjo et al., Leaves, flowers, and Drug-type Callogenesis: Callogenesis: Stage 0: Y
2006 seedling roots, stems, (0/1) MS + 0.56 mM mesoinositol + Statistical analysis N.S. Stage 1: Y
[83] and shoots 29.65 µM thiamine diHCl + Callusing was greatest using Stage 2: Y
(Callogenesis, callus 4.86 pyridoxine HCl + flowers and seedling shoots Stage 3: N
suspension cultures) 8.12 µM nicotinic acid + Stage 4: N
4.52 µM 2,4-D a
Suspension culture Suspension culture:
(2 steps): Continued callus growth, no
Step 1: MS (as above, regeneration
aqueous; 2 weeks)
Step 2: B5 media + 9.05 µM
2,4-D + 2.85 µM IAA +
2.69 µM NAA + 5.12 µM
potassium a
Wielgus et al., Cotyledons, axillary Callus induction: Callus induction: Stage 0: Y
nodes, and roots Fiber-type Stage 1: Y
2008 DARIA (ind+) + 4.65 µM Best morphogenic callus
(3/3)
[68] (Callus induction, kinetin + 0.27 µM NAA a induction: stem explants (all Stage 2: N
shoot induction, and cultivars) Stage 3: Y
rooting) Statistical analysis N.S. Stage 4: N
Shoot induction: Shoot induction:
DARIA (pro+) + 0.89 µM BAP 15.56% with cotyledon
+ 0.16 µM NAA a explants (cv. Beniko)
Rooting: Rooting:
DARIA (root+) + 11.42 µM Statistical analysis N.S.
IAA a
Flores-Sanchez Leaves Drug-type Suspension culture: Suspension culture: Stage 0: Y
et al., 2009 (Callus suspension (1/1) MS + B5 vitamins + 4.52 µM Growth rate N.S. Stage 1: Y
[84] cultures and somatic 2,4-D + 4.65 µM kinetin Stage 2: N
embryogenesis) Somatic embryogenesis: Somatic embryogenesis: Stage 3: N
Media composition N.S. Number of embryos N.S Stage 4: N
Juvenile leaves Callogenesis: Callogenesis: Stage 0: Y
Lata et al., 2010 (Callogenesis, shoot Drug-type Stage 1: Y
MS + 0.5 µM NAA + 1 µM 93.3% response
[85] induction, and (1/1) Stage 2: N
TDZ
rooting) Shoot induction: Shoot induction: Stage 3: Y
MS + 0.5 µM TDZ 12.3 shoots/explant Stage 4: Y
96.6% response
Rooting: Rooting:
1
2 MS + 2.5 µM IBA 10 roots/explant
96.6% response
Juvenile leaves Callogenesis: Callogenesis: Stage 0: Y
Farag, 2014 Drug-type Stage 1: Y
(Callogenesis and B5 + 2.69 µM NAA + 50% callusing response
[28] (1/1) Stage 2: Y
shoot regeneration) 22.20 µM BAP + 0.11 mM
adenine hemisulfate a Stage 3: Y
Shoot regeneration: Shoot regeneration: Stage 4: Y
B5 + 1.44 µM GA3 a 8.5 shoots/callus
% regeneration N.S.
Rooting: Rooting:
B5 + 8.56 µM IAA a 2.75 roots/explant
100% response
Plants 2021, 10, 185 12 of 28

Table 2. Cont.

C. sativa Type
Explant Stages
Source (#CVs Responded/ Optimal Media Optimal Results
(Response) Reported
Used)
Movahedi et al., Cotyledons and Drug-type Callogenesis/shoot Callogenesis/shoot Stage 0: Y
2015 epicotyls (1/1) regeneration: regeneration: Stage 1: Y
[86] (callogenesis+ shoot MS + 8.88 µM BAP + 2.46 µM ~2 shoots/epicotyl Stage 2: Y
regeneration, rooting) IBA % response N.S. Stage 3: Y
Rooting: Rooting: Stage 4: Y
MS + 0.49 µM IBA a % response N.S.
Chaohua et al., Cotyledon Fiber-type Callogenesis/shoot Callogenesis/shoot Stage 0: Y
2016 (callogenesis + shoot (8/8) regeneration: regeneration: Stage 1: Y
[87] regeneration, rooting) MS + 1.82 µM TDZ + 1.07 µM 3 shoots/explant (3-day-old Stage 2: N
NAA a cotyledons) Stage 3: Y
51.7% regeneration Stage 4: Y
Rooting: Rooting:
1
2 MS + 2.46–9.84 µM IBA a 80% response
Galán-Ávila Hypocotyl, cotyledon Fiber-type Organogenesis: Organogenesis: Stage 0: Y
et al., 2020 and first two true (5/5) MS + 1.82 µM TDZ + 1.07 µM 1.49 shoots/hypocotyl Stage 1: Y
[88] leaves NAA a 54.17% response Stage 2: N
(direct organogenesis Rooting: Rooting: Stage 3: Y
and rooting) MS + 1.82 µM TDZ + 1.07 µM ~18% rooted Stage 4: Y
NAA a
Monthony et al., Young leaves Drug-type Callogenesis: Callogenesis: Stage 0: N
2020b (callus induction and (10/10) MS + 0.5 µM NAA + 1 µM 100% response across all 10 Stage 1: N
[89] shoot regeneration) TDZ cultivars Stage 2: Y
Shoot regeneration: Shoot regeneration: Stage 3: N
MS + 0.5 µM TDZ Not achieved Stage 4: N
a Molarity values converted from mg/L.

It has been hypothesized that during Stage 1, explants have residual energy and
endogenous plant growth regulators from the mother plants, resulting in an initial flush
of growth during the initiation phase, followed by the sporadic growth response that has
been observed until the cultures stabilize and acclimatize to In Vitro conditions [63,90].
Long-term culture decline of Stage 2 explants has been noted by Wróbel et al. [78], who
reported a drop of 74–82% in the number of regenerated explants taken from Stage 1
plants. This initial flush of growth followed by a culture decline has also been observed by
Page et al. [77], where previously published media compositions worked well for culture
initiation (Stage 1), producing an initial flush of shoot proliferation, but failed to support
long-term culture proliferation (Stage 2). Culture decline was manifested through high
rates of hyperhydricity, callusing, and, in many cases, death of the cultures (Figure 2D) [77].
In these same studies, feminization of male plants cultured In Vitro on media opti-
mized for Stage 1 growth was observed (Figure 1A). Feminization of Cannabis plants has
previously been reported in male plants treated with ethylene, a plant growth regulator
associated with the stress response in plants [91–93]. Reports of hermaphroditism in male
Cannabis plants suggests the accumulation of ethylene in the culture vessels over time,
likely as a response to less-than-optimal media to support Stage 2 growth. These findings
highlight the current challenge of maintaining plants In Vitro long-term (Stage 2) using
media from studies that have taken a 1-3-4 approach (Figure 4). There is a pressing need for
further optimization of Stage 2 to develop a reliable five-stage micropropagation system in
Cannabis and take full advantage of micropropagation.
Optimizing macro- and micronutrients for the culture of In Vitro plants represents
one of the keystones to developing a successful micropropagation system (Figure 5) [94].
MS-based media are the current standard for C. sativa micropropagation studies (Table 1);
however, few studies have conducted extensive comparisons between MS and other basal
salts. Recently, a comparison of several basal salt mixtures (MS, Driver and Kuniyuki
Walnut (DKW), B5, and BDS as modified at Arkansas Bioscience Institute (BABI)) by Page
et al. [77] demonstrated better performance of Stage 2 explant growth using a DKW-based
Plants 2021, 10, 185 13 of 28

medium. On this medium, explants were healthier and had higher multiplication rates
than their MS counterparts. DKW and MS are both relatively rich basal salts, suggesting
that Cannabis requires high nutrient levels. The most notable difference between these two
basal salts is that DKW contains higher levels of sulphur (~7×), calcium (~3×), and copper
(10×) [77]. Interestingly, DKW was initially developed to address similar long-term declines
in walnut cultures, comparable to what has been observed with Cannabis [95]. While Page
et al. [77] demonstrated that DKW was more suitable for Stage 2 micropropagation of
Cannabis, their findings likely represent an underestimate of the benefits, as the study was
conducted for only one subculture of Stage 2 plants, while the issues of culture decline
generally increase over multiple subcultures. Using DKW-based media, several cultivars of
Cannabis have now been maintained for multiple years with no obvious signs of decline [89].
Despite the improvement over MS-based media, some signs of nutrient deficiency were
still observed, and further improvements are possible through optimization of basal media.
Most protocols for micropropagation in Cannabis rely on shoot multiplication from
existing meristems found in the apical and axillary nodes (Table 1). These meristems are
regions of high cellular plasticity with cells early in their developmental state. Nodal
cuttings can be grown In Vitro, much like vegetative greenhouse propagation. The most
successful and frequently reported methods for In Vitro multiplication of Cannabis are those
that rely on PGRs to cause shoot multiplication (SM) from a single nodal explant, resulting
in the proliferation of shoots, often described as multiple shoot cultures (MSCs; Table 1).
The highest yielding SM methods report between 9 and 13 explants per node [70,72]
but used freshly initiated tissues from the greenhouse (Stage 1) and did not include an
evaluation of Stage 2 performance. A review of the literature reveals a notable lack of
C. sativa micropropagation studies using long-term In Vitro grown germplasms, or Stage 2,
highlighting the need for future study in this area (Table 1).
In contrast to these reports of prolific MSCs, some authors have noted that Cannabis
does not readily produce MSCs and instead tends to produce a single shoot with a high
degree of apical dominance and low levels of branching, resulting in a much lower multi-
plication rate (Table 1) [75,78,80]. A recent study of two high-CBD Cannabis cultivars by
Mestinšek Mubi et al. [80] found shoot multiplication between 0.59 and 1.78 on TDZ or
meta-Topolin (mT) media recipes, which had previously been reported to yield between 11
and 13 shoots by other research groups [72]. Furthermore, the authors found that these
media compositions did not significantly improve shoot proliferation over the PGR-free
control (MS) in their two tested cultivars [80]. Similarly, Wróbel et al. [78] report that
existing nodal propagation protocols result in multiplication rates of 0.9, resulting in a loss
of plant material. Another alternative to traditional nodal shoot multiplication is the use
of two node explants used by Page et al. [77]. Unfortunately, this alternative reduces the
number of explants that can be obtained from a single plant, thereby limiting the overall
multiplication rate.
To address this issue, a recent study proposed an alternative approach to Cannabis
micropropagation in which single shoots are grown for a period of time and the apical
meristem is removed in culture [78]. Removal of the apical shoot breaks apical dominance,
allowing the axillary buds to develop into branches. Shoot tips from the developed axillary
branches are then used as secondary explants for Stage 2 growth and multiplication, with
the authors reporting a higher survival rate from shoot tips than nodal explants [78]. Using
this approach, Wróbel et al. [78] increased the multiplication rate of Stage 2 explants from
0.9 when micropropagated on MS + 0.25 mg/L TDZ to 3.0 using this modified shoot tip
micropropagation method on MS + 0.5 mg/L indole-3-acetic acid (IAA) medium. While
this approach was effective and represents one of the few methods reporting multiplication
of Stage 2 cultures (Table 1), the added step of removing the apical shoot requires more
labor and increases the risk of contamination. It should also be noted that the explants
were initiated into culture from seeds, and juvenile tissues generally respond better In
Vitro than mature explants. However, the author reports that well-established subcultures
were used in their experiments and that a minimum of ten culture cycles were performed
Plants 2021, 10, 185 14 of 28

before proceeding to Stage 3 (rooting). Another group reported that the addition of
an auxin antagonist α-(2-oxo-2-phenylethyl)-1H-indole-3-acetic acid (PEO-IAA) could
also contribute to breaking apical dominance and increase branching in seedling tissues,
resulting in a multiplication coefficient of up to 1:10 [75]. This is a promising approach that
merits further investigation.

Floral Reversion: An Alternate Micropropagation Approach

As previously highlighted, some of the frequent challenges in Cannabis micropropa-
gation include the lack of multiple shoot formation, a strong degree of apical dominance
leading to low levels of branching, and poor survival of single-node explants. As a
result, many studies using nodal tissues report low multiplication rates ranging from
less than one (resulting in loss of stock plants) to about four explants per nodal culture
(Table 1) [66,75,77,78,80]. From a practical standpoint, these multiplication rates are not
suitable for many applications. In order to increase the multiplication rate through shoot
proliferation, alternative approaches to increase the number of meristems per explant
are needed. One potential approach is to induce flowering and use floral reversion. The
inflorescence of the Cannabis plant is a highly branched compound racemose inflorescence
that contains a large number of meristematic regions [22]. Initial observations found that
some Cannabis plants initiate flower development In Vitro (Figure 1C,E), and recently,
Moher et al. [29] demonstrated that flowering could be reliably induced using a short-day
photoperiod, similar to what is observed in the field. As such, In Vitro flowering plants rep-
resent an alternative approach to increase the number of meristems per plant to potentially
increase the multiplication rate.
The use of inflorescence tissues from C. sativa appears to be a promising alternative
mode of micropropagation to nodal cultures [74,76], having been well studied in many
species [62,96–98]. Inflorescences tissues that demonstrate the ability to return from a
flowering phase of growth to a vegetative stage of growth are broadly described as under-
going floral or inflorescence reversion [62,96–99]. In Vitro PGR-induced floral reversion has
been shown in a variety of dicots and monocots. In monocots, it is widely used in many
commercially important crops such as grasses, palms, bananas, and grains [62,98,100–102].
In dicots, floral reversion has been employed less frequently; however, it has been shown
in the Brassicaceae family and has been employed in conservation efforts of recalcitrant
dicots [96,97,103,104].
In C. sativa, floral reversion has been studied in a very limited capacity. A recent publi-
cation from our lab provided the first known report of regeneration from floral explants of
Cannabis [74]. Piunno et al. were able to show that In Vitro floral reversion was possible
from two of the three commercially produced cultivars tested when using floral explants
collected from greenhouse/indoor plants. This study was important as it demonstrated the
ability of floral explants to produce phenotypically normal shoots but did not determine
whether they were produced from existing meristems or through regeneration from non-
meristematic tissues, or whether In Vitro plants could be used as a source of explants [74].
Subsequent work by Monthony et al. [76] shed light on the mechanism of floral reversion
using flowering In Vitro C. sativa. Based on histological observations, it appears that the
vegetative explants that reverted from floral tissues originated from existing meristems
subtending the florets, similar to what has been reported in nodal cultures (Figure 1F).
Survival was greater in explants that contained floret pairs rather than individual florets,
and the estimated multiplication rate of 18.2 (Table 1) matched or exceeded protocols using
nodal micropropagation [76]. While in-vitro-grown vegetative explants generally have
5–6 nodes, each flowering In Vitro plant produces approximately 24 florets, highlighting
the potential to dramatically increase Stage 2 multiplication rates (Table 1) [76,77]. Further-
more, vegetative explants derived from florets under a long-day photoperiod could then
be returned to short-day conditions to induce more In Vitro flowering. The re-flowering
of reverted tissues provides a continuous micropropagation cycle consisting of flowering,
reversion, vegetative growth, and re-flowering, which is ideally suited for Stage 2 growth.
Plants 2021, 10, 185 16 of 29

Plants 2021, 10, 185 15 of 28

6. Regeneration in C. sativa
While proliferation of explants from pre-existing meristems, such as the nodal
propagation methods outlined in Table 1, typically results in low rates of mutation and
This alternative method may also provide a viable approach for the clonal propagation
good genetic fidelity, de novo regeneration systems (Table 2) can offer increased
of day-neutral genotypes, which cannot be maintained in a continuous vegetative state
multiplication rates and are required for many other biotechnologies [105–108].
of growth.
Vegetative nodes used for shoot proliferation represent a small fraction of the entire tissue
composition
6. Regeneration of the
in plant and are ultimately limited. Regeneration from non-meristematic
C. sativa
somatic tissues offers a larger pool of starting materials for micropropagation. As a result,
While proliferation of explants from pre-existing meristems, such as the nodal prop-
de novo regeneration from somatic tissues through embryogenesis and organogenesis can
agation methods outlined in Table 1, typically results in low rates of mutation and good
greatly increase the number of explants produced in the same time-period. As the
genetic fidelity, de novo regeneration systems (Table 2) can offer increased multiplication
regulatory landscape evolves to facilitate research, interest in de novo regeneration of
rates and are required for many other biotechnologies [105–108]. Vegetative nodes used
Cannabis has been increasing, yet the body of literature investigating regeneration systems
for shoot proliferation represent a small fraction of the entire tissue composition of the
in Cannabis
plant and are remains limited
ultimately (FigureRegeneration
limited. 6; Table 2). Afrom
further complicating factor
non-meristematic in the
somatic body
tissues
of literature is the inconsistent use of the term regeneration, as some publications
offers a larger pool of starting materials for micropropagation. As a result, de novo re- use it
when referring
generation fromtosomatic
SM/MSCs from
tissues tissuesembryogenesis
through containing existing meristems with
and organogenesis canno clear
greatly
evidence of regeneration [67,70,74,78,82].
increase the number of explants produced in the same time-period. As the regulatory
Earlierevolves
landscape reviewstoon the state
facilitate of C. sativa
research, micropropagation
interest and regeneration
in de novo regeneration largely
of Cannabis has
failed to underscore the many challenges in the existing body of research.
been increasing, yet the body of literature investigating regeneration systems in Cannabis These
challenges
remains limitedinclude incomplete
(Figure 6; Tableand
2). Aambiguously reportedfactor
further complicating results
in [28,67,74,81,82,84,86],
the body of literature
recalcitrance to regeneration
is the inconsistent use of the [28,68,81,82,86,88];
term regeneration, genotype- and tissue-specific
as some publications responses
use it when refer-
to regeneration
ring to SM/MSCs [68,81,82,87,88];
from tissues and a lack ofexisting
containing reproducibility of successful
meristems protocols
with no clear in the
evidence of
literature [77,89].
regeneration [67,70,74,78,82].

Figure 6. Yearly publications and the number of citations for articles matching the search TOPIC:
Figure 6. Yearly publications and the number of citations for articles matching the search TOPIC:
(Cannabis sativa
(Cannabis sativa OR
OR hemp)
hemp) AND
AND TOPIC:
TOPIC:(regeneration)
(regeneration)AND
ANDTOPIC:
TOPIC:(micropropagation
(micropropagationOR ORInInVitro
OR tissue culture) on the Web of Science database. Data obtained from Web of Science®on
Vitro OR tissue culture) on the Web of Science database. Data obtained from Web of Science® 17 Novem-
on
berNovember
17 2020 using2020
Microsoft
using Edge®. Data
Microsoft presented
Edge®. may not bemay
Data presented a comprehensive representation of the
not be a comprehensive
representation of the available
available publications publications
and are limited and are limited
to publications to publications
indexed indexed by Web of
by Web of Science®.
Science®.
Earlier reviews on the state of C. sativa micropropagation and regeneration largely
failed
6.1. to underscore
Incomplete the many challenges
and Ambiguously in the existing body of research. These challenges
Reported Results
include incomplete and ambiguously reported results [28,67,74,81,82,84,86], recalcitrance to
Existing micropropagation protocols that rely on regeneration from non-
regeneration [28,68,81,82,86,88]; genotype- and tissue-specific responses to regeneration [68,
meristematic tissues often report low levels of regeneration, and in some studies, the rate
81,82,87,88]; and a lack of reproducibility of successful protocols in the literature [77,89].
or frequency of germination is not reported, and ambiguous or no visual evidence is
included (Table
6.1. Incomplete and2).Ambiguously
Flores-Sanchez et al.
Reported [84], for example, reported the induction of
Results
somatic embryogenesis from C. sativa suspension cultures but they did not provide any
Existing micropropagation protocols that rely on regeneration from non-meristematic
data or visual evidence in support of their claims. Mandolino et al. [81] report “occasional”
tissues often report low levels of regeneration, and in some studies, the rate or frequency
of germination is not reported, and ambiguous or no visual evidence is included (Table 2).
Flores-Sanchez et al. [84], for example, reported the induction of somatic embryogenesis
Plants 2021, 10, 185 16 of 28

from C. sativa suspension cultures but they did not provide any data or visual evidence
in support of their claims. Mandolino et al. [81] report “occasional” regeneration from
hypocotyl tissues; however, they do not report the frequency of shoot production or the
percentage of tissues that responded. Studies on callus cultures by Ślusarkiewicz-Jarzina
et al. [82] state that across all treatments, they were able to achieve only 1.35% regeneration
from callus cultures. In their study on somatic embryogenesis and organogenesis from
stem and root tissues, Plawuszewski et al. [67] report successful regeneration in stem-
derived callus but did not specify how much regeneration was achieved. A subsequent
study published by this research group in 2008 included regeneration levels from stem
tissues, reporting 14% regeneration in the most successful treatments [68]. Other authors
have chosen to report callusing data, such as Farag [28] in their study of juvenile leaf
callus. However, they do not specify the percentage of callus that regenerated, reporting
only an average of 8.5 regenerants per callus. Movahedi et al. [86] also did not report
the regeneration percentage, describing it as “low” with an average of less than one
regenerant per seedling-derived callus culture. In a study on the regeneration potential
of floral tissues, Piunno et al. [74] report organogenic regeneration from florets but do
not specify the percentage of cultures that regenerated. They did not determine if this
was de novo regeneration or proliferation of existing meristems but hypothesized that it
was from pre-existing meristems and not de novo regeneration. As discussed above, the
latter’s hypothesis has since been supported by histological examinations that identified
the presence of quiescent vegetative meristems within Cannabis inflorescences, and the
use of the term “regeneration” is inaccurate [76]. These studies highlight the value of
more detailed reporting of results in regeneration experiments and showcase how, when
reported, regeneration rates are often low (Table 2).

6.2. Genotype and Tissue Specificity

In tissue culture, the commitment to regeneration is highly dependent on the genotype,
tissue type, and physiological state of the material. The range of genetic variability in
Cannabis can be seen by the physiological and chemical differences between hemp and
drug-type cultivars. Hemp produces negligible (<0.3%) levels of ∆9 -tetrahydrocannabinol
(THC; Government of Canada 2015) and has been bred for a high fiber and oil content [26].
In contrast, drug-type Cannabis can contain anywhere from 5% to over 20% THC and has
primarily been bred for indoor production, highlighting the biochemical and morphological
variability within the species [38]. This variability has also been demonstrated by the In
Vitro responses of Cannabis, which show genotypic and tissue-dependent regeneration
responses [68,81,82,87,88].
As previously discussed (see Section 2. Brief History of C. sativa in North America),
access to Cannabis for research purposes has historically been limited. As such, most
US-based federally funded research can only be conducted with Cannabis obtained from
the NIDA [10,12]. Recently, concerns have been raised that NIDA-supplied medical-grade
Cannabis is biochemically [109] and genetically homogenous and does not accurately
represent the diversity available commercially [12]. Vergara et al. [109] showed that NIDA-
supplied Cannabis has a limited cannabinoid profile and a lower concentration of THC
compared to legal, dispensary-supplied Cannabis in the United States. Genetic evidence has
also emerged which shows that NIDA-supplied Cannabis is more closely related to hemp
than most drug-type genotypes, despite containing moderately high levels of THC [12].
Regeneration studies have been conducted more frequently and with more cultivars in
hemp, and many of these studies have been more successful than studies using true drug-
type cultivars [67,75,81,82,87,88]. The finding that NIDA-supplied Cannabis may be more
genetically similar to hemp than commercially available drug types underscores the need
to test existing regeneration studies on a large, representative sample of drug-type cultivars
before findings from research using NIDA-supplied Cannabis can be assumed to have
general applicability.
Plants 2021, 10, 185 17 of 28

The regenerative potential of tissues varies from species to species and is usually
determined empirically [110]. One of the earliest studies on regeneration was conducted on
four tissue types (leaf, hypocotyl, cotyledon, and roots) in 12 commercial hemp cultivars.
In this study, Mandolino et al. [81] reported that callus formation from somatic tissues
was achieved in all 12 cultivars across all four tissues; however, callus morphology varied,
ranging from small amounts of brown callus to high amounts of white friable callus
depending on the cultivar and tissue tested. Despite the formation of callus in all cultivars,
only “occasional” regeneration from callus was obtained in one of the 12 tested cultivars.
Mandolino’s qualitative analysis of regeneration found that it was uneven across the tested
tissues and that hypocotyls were the most likely, and roots the least likely, to respond.
No regeneration was obtained from leaf tissues [81].
Following this early study by Mandolino et al. [81], subsequent studies tried to identify
which tissues in Cannabis are best suited to regeneration; however, no clear consensus
has emerged. Ślusarkiewicz-Jarzina et al. [82] assessed regeneration of young leaves,
petioles, internodes, and axillary buds in five hemp cultivars and found that callusing
and regeneration levels were cultivar- and tissue-dependent. The authors found that
petiole and leaf tissues were most responsive to regeneration; however, the magnitude
of this response varied among cultivars. In petioles, callusing ranged from 27% to 83%
depending on the cultivar tested [82]. Total regeneration by cultivar was low and ranged
from 0% to 6% [82]. Wielgus et al. [68] also reported a cultivar-specific response in their
study on direct organogenesis in three hemp cultivars. Of the three cultivars tested, two
showed a less than 2% regeneration response, and in the stem tissues of the third cultivar,
only 14% underwent direct organogenesis [68]. The study also compared the response
amongst three types of seedling-derived tissues: cotyledons, stems, and roots. Wielgus
and colleagues found that “seedling stem tissue” (assumed to be hypocotyl tissue) was
most responsive and cotyledon tissues were least responsive. Using seedling-derived leaf
tissues, Farag [28] reported successful callogenesis and subsequent regeneration with an
average of 8.5 regenerants per callus; however, the % regeneration was not stated.
A 2020 study by Galán-Ávila et al. on direct organogenesis of hypocotyl, cotyledon,
and true leaves in five hemp cultivars also found that hypocotyls were most responsive
across all five cultivars. In their study, 49.5% of hypocotyl tissues responded across all
treatments, compared with only 4.7% of cotyledon and 0.42% of true leaves [88]. The re-
generation response was tissue- and cultivar-dependent, ranging from 2% to 71% response
depending on the source tissue and cultivar [88]; however, within hypocotyl treatments,
this response range showed less variability (32–71% regeneration). The number of shoots
produced per explant was consistently between one and two [88]. The findings that
hypocotyl tissue is highly favorable for regeneration echo the early Cannabis micropropaga-
tion studies by Mandolino et al. [81]. While Wielgus and Galán-Ávila both reported low
regenerative capacity in cotyledons, a study by Chaohua et al. [87] has reported differing
results. In their study, Chaohua et al. [87] assessed the efficacy of regeneration in 1- to
6-day-old cotyledon tissues from eight commercial hemp cultivars. While regeneration
varied from 35.7% to 54.8% depending on the cultivar, these reported results highlight the
regeneration potential of hypocotyl tissues and the contradictory nature of the existing
studies on Cannabis regeneration with respect to tissue source [87].
In contrast to the several aforementioned reports on hemp, only one study examining
regeneration responses (Table 2) across multiple drug-type genotypes has been published
to date. This study by Piunno et al. [74] assessed the potential of floral tissue as a genesis for
explant regeneration in three commercial medicinal Cannabis genotypes. Their publication
reported that regeneration was only observed in only two of the three tested genotypes
and at “low levels” (which were not further specified) [74]. Further research has suggested
that this was not likely de novo regeneration [76], highlighting the absence of studies on
the de novo regeneration of drug-type Cannabis.
Plants 2021, 10, 185 18 of 28

6.3. Recalcitrance to Regeneration

Recalcitrance to regeneration has been reported throughout the existing literature
with very few exceptions. Recent Cannabis regeneration studies have struggled to obtain
high regeneration, shoot proliferation, and response rates. This challenge has already
been overcome in many species and overcoming it is a prerequisite for the effective use
of regeneration systems in many areas of plant biotechnology. In a regeneration study
of five hemp cultivars, Galán-Ávila et al. [88] found that only 54% of hypocotyl tissues
underwent organogenesis, reporting an average of 1.49 shoots/hypocotyl. A study of
epicotyl tissues conducted by Movahedi et al. [86] reported similarly low rates of shoot
multiplication of ~2 shoots per epicotyl, and the percent response was not specified. In
a regeneration study using juvenile leaves, Farag [28] also neglected to report the % of
callus cultures that underwent regeneration, reporting only a shoot proliferation of 8.5
shoots/callus culture. Chaohua et al. [87] found that across cultivars, 46.7% of 3-day-old
cotyledon tissues underwent regeneration on MS media supplemented with TDZ and
NAA, and the most responsive of the eight tested cultivars produced three shoots/explant.
The media used by this author resemble media used by Lata et al. [85]; however, the
media used differed in the levels of TDZ and NAA and the starting tissues were different,
highlighting the importance of tissue type and medium composition. These differences
are likely the reason that Chaohua et al. [87] reports lower regeneration rates than those
reported by Lata et al. [85] (3 vs. 12.3 shoots/explant, respectively). Medium composition
has been highlighted as a factor that could affect recalcitrance in Cannabis tissue culture
by Page et al. [77]. In this study, the authors found that commonly favored MS salts
supplemented with TDZ resulted in high levels of hyperhydricity (Figure 2E), poor explant
development, and occasional death in five commercially available cultivars of drug-type
Cannabis [77]. These phenotypes were largely reversed when the explants were cultured
on media using DKW basal salts rather than MS [77]. Future studies hoping to develop
a robust and replicable regeneration methodology are needed to determine the extent to
which recalcitrance is a species-wide phenomenon and identify genotypes of Cannabis that
are amenable to regeneration.

6.4. Lack of Reproducibility

Reproducibility of existing methods remains a challenge in both regeneration [89]
and SM systems [80]. As previously discussed, with most contemporary US-based studies
presenting data from a single cultivar [28,78,85,86], it is likely that this lack of genotypic
diversity is contributing to the struggle with protocol reproducibility in Cannabis tissue
cultures. The lack of diversity in drug-type Cannabis studies is juxtaposed with regeneration
studies on commercial hemp varieties which frequently use between 5 and 12 cultivars,
offering findings that are more representative of the general biological responses across
industrial hemp cultivars rather than a single specific cultivar (Table 1) [81,82,87,88].
While many studies report signs of recalcitrance to regeneration, this is not universal.
The most successful report of regeneration from somatic tissues of medicinal Cannabis
was published in 2010 by Lata et al., in which they reported indirect regeneration in
96.6% of callus derived from young leaf material, with an average of 12.3 shoots per
culture. Callus induction was achieved on MS media with 0.5 µM NAA and 1.0 µM TDZ
followed by a transfer to MS media with 0.5 µM TDZ, which induced the high levels of
regeneration they reported [85]. This protocol reported unprecedentedly high levels of de
novo regeneration, making it an optimal protocol to be used in the application of plant
biotechnologies. Until recently, this decade-old protocol had not been replicated in any
publication by an independent research group or used for subsequent biotechnological
development by the original authors. A 2020 replication study testing this protocol across
10 simple sequence repeats (SSR) characterized Cannabis cultivars found that callus was
formed but no regeneration was observed [89]. The contradictory findings between these
two studies raise concerns about the applicability of existing tissue culture methods across
genotypes and/or their reliability.
Plants 2021, 10, 185 19 of 28

Strong cultivar-specific responses to treatments have been noted in Cannabis tissue

culture and this likely contributes to the lack of reproducibility in both shoot proliferation
and regeneration-based systems [55,74,77,79,87,89]. As Cannabis research becomes more ac-
cessible, the implications of drawing sweeping conclusions based on single-cultivar studies
have come under scrutiny [12,89], and evidence has called into question the genotype-
independent responses implicit in many of these single-cultivar studies [77]. It will be the
goal of future studies to establish complete and detailed methodologies applied across
a broad genotypic sample to overcome recalcitrance in the species or at least to iden-
tify amenable cultivars to overcome the challenges currently faced in the tissue culture
of Cannabis.

7. Genetic Stability and Preservation

One of the main applications of micropropagation is the preservation of genetics in
a safe environment, free from biotic pressure. Tissue sources used for micropropagation
carry varying probabilities of experiencing mutations, referred to as somaclonal variation,
which can be problematic for the maintenance of clonal lines of plants [111]. Micropropa-
gation through the proliferation of existing meristems is generally considered to have a
lower mutation load than the use of de novo regeneration, especially for indirect de novo
regeneration in which there is a callusing phase [111,112]. As such, shoot proliferation is
often preferred for genetic preservation, although there is still a risk of somaclonal variation
occurring [112].
In general, micropropagated Cannabis has been shown to produce plants that are
morphologically and chemically similar to the parent material [69]. Furthermore, authors
have reported that it appears to be genetically stable, with a low occurrence of muta-
tions [56,69,72,75,113]. However, most studies that have assessed the genetic fidelity of
micropropagated Cannabis did not assess it over long-term maintenance with many sub-
cultures. Additionally, they used specific genetic markers such as Inter Simple Sequence
Repeats (ISSR), resulting in only the detection of mutations that occurred at those specific
sequences, which does not quantify the mutation rate across the whole genome [113]. As
such, the actual mutation rate in Cannabis plants, including both In Vitro and ex vitro, has
not been reported and there is significant potential for mutations to occur in both systems.
Further research using more advanced sequencing techniques is needed to determine
the relative mutation rate of plants growing in the greenhouse/indoors/outdoors, micro-
propagated plants produced through shoot proliferation, and plants produced through de
novo regeneration.
While the actual mutation rate in these various settings has not been quantified,
several approaches are known to mitigate somaclonal variation, some of which have been
explored in Cannabis. The first approach is using low-temperature cultures in which plant
growth is slowed down by maintaining them at lower-than-normal temperatures. This was
first reported by Lata et al. [113], who encapsulated axillary buds and were able to store
them for 6 months in 5 ◦ C storage before planting. The ISSR profiles of the cold-stored
plants were comparable to the mother plant, making this approach suitable for maintaining
genetic lines [113]. A logical extension of this is the use of cryopreservation to store tissues
at cryogenic temperatures. Since this process essentially halts plant metabolism and cell
division, it can be used to store plant genetics indefinitely and eliminate the accumulation
of genetic mutations during the storage period [114,115].
An important aspect of genetic preservation is to recognize that all plants mutate
as they grow, including plants in ex vitro conditions, making cryopreservation one of
the only approaches to prevent mutations from occurring in clonal lines over time [116].
While cryopreservation requires a significant up-front cost and more sophisticated facilities,
studies in other species have demonstrated that it is often more cost-effective than in situ
preservation over the long term [117]. As such, cryopreservation represents the most
effective approach for maintaining genetic fidelity of clonal lines over long periods and
may be economically advantageous. Uchendu et al. [118] have begun the development of a
Plants 2021, 10, 185 20 of 28

cryopreservation protocol for Cannabis by testing different plant vitrification solutions on

shoot tips. Their highest regrowth rate was 63%, providing a viable approach for Cannabis
germplasm conservation.

8. Future Directions
As highlighted in Tables 1 and 2, most Cannabis micropropagation studies have fo-
cused on evaluating the effects that PGRs and explant type have on regeneration. However,
other factors that affect the efficiency of regeneration protocols are often neglected. In this
section, we highlight these overlooked factors that affect In Vitro Cannabis propagation and
present promising new approaches studied in other species that could be used to overcome
the hurdles currently faced in Cannabis research. As stated previously, genetics is one of
the key factors influencing the regenerative capacity of a cultivar. This genotypic effect
is well documented in the micropropagation of Cannabis and is, in part, due to the differ-
ences in endogenous phytohormones’ concentrations specific to individual cultivars [119].
Unfortunately, understanding of the biochemistry and metabolism of Cannabis beyond
the cannabinoid pathways is limited and represents a necessary area of future study. To
date, several studies [120–124] showed that the gene expression pattern of endogenous
PGRs and the balance between endogenous and exogenous PGRs play an important role in
regeneration efficiency, especially in recalcitrant plants. For instance, Kumari et al. [120]
studied the endogenous level of PGRs via UHPLC–MS analysis and its effect on shoot
regeneration and somatic embryogenesis of Tulbaghia simmleri. They reported that endoge-
nous phytohormone signaling and transport affects In Vitro biological processes related to
auxin and cytokinin, and generally, high-frequency regeneration protocols can be achieved
when there is a balance between endogenous and exogenous PGRs. A similar approach was
recently taken by Smýkalová et al. [75], who carried out UPLC-MS-guided studies on the
effects of exogenous application of auxin, cytokinins, and their inhibitors in Cannabis. They
showed that ex vitro hypocotyl segments have insignificant endogenous concentrations
of aromatic and free forms of cytokinins but have high concentrations of O-glucoside and
riboside bases of endogenous cytokinins. These studies have highlighted strong apical
dominance in the species and represent a forward-thinking model for future studies. The
continued development of such biochemical and molecular studies will prove imperative
to overcoming recalcitrance of Cannabis to In Vitro regeneration.
The physiological condition of the mother plant and the type, position, size, and
orientation of the explant play a pivotal role in micropropagation [125]. The source of the
explant (i.e., ex vitro and In Vitro) also has an impact on regeneration [126]. Generally,
In Vitro explants have more regeneration potential than ex vitro explants due to their
juvenility and since they are already adjusted to In Vitro conditions [127]. There are,
however, no studies comparing the regeneration potential of ex vitro and In Vitro Cannabis
explants. The type of explant (e.g., cotyledon, leaf, node, root, etc.) also impacts the plant’s
ability to regenerate. This is mainly due to differences in their endogenous phytohormone
levels. Another overlooked factor is explant orientation, which can affect the initiation site,
polarity, and regeneration efficiency [127,128]. Generally, horizontally positioned explants
have higher regeneration rates than vertically positioned explants. This is likely due to
the explants having more surface area in contact with the medium. For instance, Jun-jie
et al. [128] reported that leaf segments oriented abaxially (lower surface facing down)
had significantly higher shoot production than those facing upwards (adaxial). However,
there are no reports in Cannabis that compare explant type, age, and orientation in In Vitro
propagation. These studies will help clarify how these factors can be optimized to improve
Cannabis regeneration protocols.
The incubation conditions, especially light and temperature, play an important role in
regeneration efficiency. Wavelength, photoperiod, and flux density have a significant im-
pact on In Vitro morphogenesis, photosynthesis, and phototropism [129] and require further
study in Cannabis. Different species have different responses to light conditions [129,130].
Some plants may respond positively to the addition of photosynthetic photon flux, par-
Plants 2021, 10, 185 21 of 28

ticularly under mixotrophic/photoautotrophic conditions (CO2 -rich and low sugar level).
Temperature also affects different biological processes such as photosynthesis and respira-
tion [131]. Although the growth chamber temperature commonly ranges from 20 to 27 ◦ C,
the optimal temperature can vary based on the genotype. Despite the importance of light
and temperature conditions, there are no studies on the effects these conditions have on
in-vitro-grown Cannabis. It is essential to optimize these conditions to improve Cannabis
micropropagation.
The composition of the culture medium, including gelling agents, carbohydrates,
additives (e.g., PGRs, activated charcoal, phloroglucinol, and nanoparticles), basal salts,
and vitamins, is the most important element of a tissue culture protocol and is often the
focus of micropropagation and regeneration studies, including those previously discussed
(Tables 1 and 2). Generally, a culture medium can be categorized as a semi-solid or
liquid medium. Although the concentration of gelling agents plays a conspicuous role
in regeneration efficiency [132], there are no reports for Cannabis comparing the different
types and concentrations of gelling agents. Carbohydrates are essential for many species in
culture and there are many different sources (sucrose, glucose, fructose, maltose, glycerol,
etc.). Different cultivars or species may react differently depending on the carbohydrate
source, and some sources can be used for certain In Vitro processes depending on their
roles in metabolism [133]. No existing Cannabis micropropagation and regeneration studies
have compared the effects of carbohydrate sources on regeneration or explant growth.
Currently, sucrose remains the carbohydrate source of choice when preparing media for
In Vitro studies of Cannabis [70,77,78,80,89]. Hence, it seems that studying the effects of
carbohydrate sources on Cannabis micropropagation may be an avenue for improving
available In Vitro regeneration systems.
Adjusting PGRs and additives, especially balancing auxins and cytokinins, is a com-
mon experiment in plant tissue culture systems because the auxin/cytokinin ratio is often
required for callogenesis, organogenesis, embryogenesis, and rhizogenesis. Most of the
Cannabis micropropagation studies have investigated the effects of common auxins (e.g.,
2,4-D, NAA, IBA, and IAA) and cytokinins (e.g., BAP, Kinetin, mT, and TDZ) on In Vitro
regeneration (see Table 2). However, there are some lesser-used additives such as nitric
oxide (NO) [134], polyamines [135], and nanoparticles [136] that have shown promising
outcomes in other plant species. NO is categorized as a new phytohormone that plays a
pivotal role in different biological processes, especially in cell division [137], morphogene-
sis [138], organogenesis, rhizogenesis [139], and plant defense mechanisms [134]. Several
studies [140–143] have recently demonstrated the positive role of exogenous NO and/or
sodium nitroprusside on callogenesis, shoot regeneration, and root initiation in differ-
ent plants. Other studies [144–146] have shown that polyamines act as a key signal in
organogenesis and shoot regeneration. In addition, nanoparticles (e.g., TiO2 , Ag, Zn, ZnO,
graphite, graphene, carbon nanotubes, quantum dots, and polymer dendrimers) have been
successfully used in different In Vitro processes from the first step, through decontam-
ination, to callogenesis, organogenesis, somatic embryogenesis, shoot regeneration, the
study and production of secondary metabolites, and somaclonal variation [147–150]. For
instance, Sarmast et al. [151] showed that adding 60 mg/l Ag nanoparticles to MS medium
containing 0.1 mg/l IAA and 2.5 mg/l BAP increased shoot regeneration frequency, shoot
number, and length of Tecomella undulata. Generally, the positive impact of nanoparticles in
plant tissue cultures might be due to their role in delaying senescence through downreg-
ulation of genes such as aminocyclopropane-1-carboxylic acid synthase ACS gene [151]
and microRNAs such as miR408 and miR398, related to ethylene production [152], and by
decreasing the content of proline, hydrogen peroxide, and malondialdehyde through the
improvement of antioxidant enzymes’ activity [153]. While micropropagation of Cannabis
is a relatively new area in the established field of tissue culture, recent advances in nan-
otechnology and phytohormone signaling could be readily applied to the field of Cannabis
tissue culture.
Plants 2021, 10, 185 22 of 28

Vitamins and basal salts are the main components of medium compositions. MS basal
medium has been widely used for Cannabis micropropagation. Recently, Page et al. [77]
demonstrated that nodal explants of Cannabis cultivated on MS medium resulted in abnor-
mal morphology and that the DKW medium was better at supporting shoot growth and
promoting callogenesis in leaf disk cultures. However, Page et al. [77] also reported that
the explants cultured on DKW medium still had some physiological defects, suggesting
that further optimization is needed. Formulating and optimizing a new basal medium is
time-consuming and expensive due to the number of essential elements and vitamins and
their interactions that need to be considered. To solve this challenge, machine learning
algorithms as a statistical and computational approach have been suggested as a solu-
tion [154]. Recently, machine learning algorithms have been used for developing and
optimizing medium in multiple species such as Pistacia vera [155], Centella asiatica [156],
pear rootstock [157], and chrysanthemum [158]. Therefore, these methods can be used for
designing and optimizing Cannabis-specific medium and environmental conditions.
As highlighted in this review, micropropagation is a complex, multi-variable, and
non-linear process that can be influenced by many factors, especially incubation condi-
tions, medium composition, explant, genotype, and their interactions (Figure 5). The
application of new computational approaches such as machine learning algorithms for
analyzing, predicting, and optimizing In Vitro processes represents a forward-looking
and novel approach to solving the challenges faced in Cannabis tissue culture. Recently,
different machine learning algorithms have been successfully used for predicting and
optimizing different micropropagation systems such as shoot regeneration, embryogenesis,
androgenesis, and rhizogenesis, underscoring that these in silico predictions are viable and
effective In Vitro [154]. Advances in machine learning, the synthesis of novel PGRs, and
the application of cutting-edge nanoparticle technologies to tissue culture can open a new
window for the comprehensive study of Cannabis micropropagation and pave the way to
tackling the persistent recalcitrance and poor replicability affecting current In Vitro tissue
culture studies of C. sativa.

9. Conclusions
The body of Cannabis micropropagation and regeneration literature is poised to un-
dergo substantial growth as regulations around the globe begin to relax. While several
existing publications report high rates of MSCs, there have been challenges in replicating
the results of these studies across genotypes and research groups. Reports of de novo
regeneration are even more limited; their success has been mixed and positive outcomes
have been difficult to replicate. These challenges are highlighted by the fact that there
are no published reports of regeneration of transgenic plants obtained using traditional
molecular and genome editing approaches. In drug-type Cannabis, micropropagation
and regeneration protocols suffer from low multiplication rates, poor replicability, and
a vast array of starting tissues to choose from, coupled with high diversity in genotypic
responses and underwhelming robustness resulting from protocols conceived using single
genotypes. Precise methods using multiple genotypes are necessary to develop protocols
that can be reliably replicated by other research groups, and innovative new approaches
to Cannabis micropropagation are required if developments in Cannabis tissue culture and
plant biotechnologies are to keep pace with the needs of the producers and consumers in
this burgeoning industry.

Author Contributions: Conceptualization, A.S.M., S.R.P., M.H. and A.M.P.J.; writing—original draft
preparation, A.S.M., S.R.P., M.H. and A.M.P.J.; writing—review and editing, A.S.M., S.R.P., M.H. and
A.M.P.J.; visualization, A.S.M., S.R.P., and M.H.; supervision, A.M.P.J.; funding acquisition, A.M.P.J.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Natural Sciences and Engineering Research Council of
Canada, grant number RGPIN-2016-06252.
Institutional Review Board Statement: Not applicable.
Plants 2021, 10, 185 23 of 28

Informed Consent Statement: Not applicable.

Data Availability Statement: No new data were created or analyzed in this study. Data sharing is
not applicable to this article.
Acknowledgments: The authors express their gratitude to Avicanna™ for making photos of their
outdoor operations available for use in this review.
Conflicts of Interest: The authors declare no conflict of interest. The funder had no role in the writing
of the manuscript, or in the decision to publish.

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