Of the various types of plant cell culture available, cell suspension
cultures are the most commonly used due to their scalability and
relatively rapid growth rates
(Santos et al.,
2016). The use of cell suspension cultures involves growing
dedifferentiated plant cells in liquid medium supplemented with hormones
to induce culture proliferation
(Mustafa et al.,
2011). However, the use of suspension cultures for these purposes faces
several barriers to successful execution. Firstly, due to their genetic
instability, cultures can often lose their ability to produce valuable
compounds over time, while low productivity rates sometimes require
large volumes of biomass to be grown, therefore increasing costs
relative to field-grown plants
(Moon et al., 2020;
Weathers et al., 2010). Cell suspension cultures tend to form
heterogeneous cell clusters rather than proliferating as single cells in
culture, leading to increased difficulty of use and inconsistent growth
kinetics and product yield. In addition, the scale-up of cell suspension
cultures from laboratory to commercial production scale is often
associated with a decline in cell productivity (James and Lee, 2006).
In relation to Cannabis, the use of in vitro bioprocessing
techniques has the potential to allow the synthesis of high yields of
cannabinoids in a manner that satisfies good manufacturing practice
guidelines and guarantees a high-quality product. Metabolic engineering
offers the possibility of developing plant or microbial cell lines which
exclusively produce a desired cannabinoid, thus circumventing the high
costs associated with purifying a desired product during downstream
processing. However, achieving these aims poses a number of challenges
to which researchers must still find answers.
A study described by
Pacifico et al.,
(2008) assessed the cannabinoid content of callus cultures (which are
metabolically identical to and often constitute the starting material
for suspension cultures) derived from five different Cannabis varieties . The calli did not show any detectable levels of
phytocannabinoids at any time during culture, irrespective of the
presence or absence of hormones or the phytocannabinoid content of the
original plants from which the cultures were derived. As such, cell
suspension cultures are unlikely to be an effective biofactory for the
production of cannabinoids without some form of intervention.
One method which may overcome the lack of phytocannabinoid production in Cannabis suspension cultures is the use of elicitors. These are
compounds or a mixture thereof which can be added to the culture medium
to stimulate the transient production of a desired secondary metabolite.
Elicitors can be either biotic (animal, plant or microbial extracts) or
abiotic (metal ions, organic compounds or electric current) and have
been used previously with varying degrees of success
(Weathers et al.,
2010 and references therein). However, since many elicitors are either
toxic or stress-inducing, their addition to a suspension culture often
leads to a reduction in the vitality of the culture and can even be
fatal. Such an approach was used by
Flores-Sanchez et al.
(2009) in order to try to stimulate phytocannabinoid production in
suspension cultures of Cannabis. However, no detectable levels of
phytocannabinoids were found in response to any of the treatments used,
which included a range of biotic and abiotic stimuli. As such, the
search for an elicitor which can induce phytocannabinoid production
remains ongoing.
One other point worth noting is that phytocannabinoids are known to be
toxic to plant cells when they accumulate at high enough concentrations,
which is why Cannabis plants use trichomes to compartmentalise
these compounds into storage cavities outside the plant. THCA and its
precursor molecule CBGA are highly toxic to both Cannabis and
tobacco cell suspension cultures, inducing 100% apoptotic-like
programmed cell death after 24 hours in culture at a concentration of 50
μM (Sirikantaramas et
al., 2005). This negative feedback phenomenon is observed in many plant
species which produce secondary metabolites and is the reason why many
metabolites are sequestered in specialised structures.
However, the development of cell culture methods to avoid cell toxicity
is one area where researchers have relative success. Strategies such as
two-phase cultures have been shown to enhance the production of
secondary metabolites in a range of species
(Malik et al., 2013).
In these systems, an aqueous phase is used to support cell growth while
a non-aqueous phase, typically consisting of a solvent or resin, is
employed to act as a sink for the accumulation of the desired product
and in some cases facilitates its subsequent extraction. Two-phase
systems have been shown to greatly increase metabolite yield in both
plant cell suspension and hairy root culture cultures
(Chiang and Abdullah,
2007; Malik et al., 2013; Rudrappa et al., 2004; Sykłowska-Baranek et
al., 2019; Wu and Lin, 2003), although to the best of our knowledge
such an approach has not yet been attempted in Cannabis.
Hairy root cultures are generated by the infection of plant tissues with Agrobacterium rhizogenes, a species which can modify the plant’s
genome by introducing a segment of DNA known as T-DNA which codes for a
number of genes affecting the production and regulation of plant
hormones (Ono and
Tian, 2011). This results in the development of extensive root networks
which can be easily cultured in vitro, are genetically identical
to the mother organ from which they were derived and can also produce
the same phytochemicals. Like cell suspension cultures, hairy root
cultures have already attracted attention as a means of producing
secondary metabolites such as flavonoids
(Gai et al., 2015),
isoflavonoids (Jiao et al., 2014),
artemisinin (Patra and
Srivastava, 2014) and lignans
(Wawrosch et al.,
2014), albeit less commonly than cell suspension cultures due to their
increased difficulty of use.
THCA has previously been produced from tobacco hairy root cultures which
were transformed to express the enzyme responsible for its production,
THCA synthase, under the transcriptional control of the cauliflower mosaic virus 35S promoter
(Sirikantaramas et
al., 2004). When these hairy roots were cultured in liquid medium
supplemented with CBGA, the precursor molecule to THCA, 8.2% of this
CBGA was converted to THCA after two days of culture, approximately half
of which was then secreted into the surrounding medium, thus
demonstrating that CBGA uptake and THCA release from these transgenic
roots was possible in vitro, albeit at low levels.
In Cannabis, a protocol for the production of hairy root cultures
has already been described which shows that cultures are best
established from the hypocotyl of intact seedlings by piercing the
epidermis with a syringe and inoculating with A. rhizogenes(Wahby et al., 2013).
Five varieties of Cannabis (three hemp-type and two marijuana
drug-type) were used and all were shown to be responsive to infection by A. rhizogenes, although with varying morphological responses.
Similarly, all eight A. rhizogenes strains used could induce a
hairy root morphology, albeit with varying degrees of frequency
(43-98%, depending on the strain).
An alternative approach described by
Farag and Kayser
(2015) outlines how hairy root cultures can be developed from Cannabis callus cultures without the use of A. rhizogenes by growing them in B5 medium supplemented with 4 mg/ml of the auxin NAA.
Under these conditions, cannabinoid contents peak at 1.04 μg/g dry
weight for THCA, 1.63 μg/g dry weight for CBGA and 1.67 μg/g dry weight
for CBDA after 28 days of culture. These low yields highlight the fact
that while phytocannabinoids can be produced from hairy root cultures,
significant improvements in yield will need to be achieved before this
methodology is commercially viable for phytocannabinoid production.
Recent studies have attempted to demonstrate the production of
cannabinoids in non-native hosts, with a particular focus on yeast due
to the relative ease with which metabolic engineering can be achieved in
this model organism. A landmark paper by
Luo et al. (2019)
described the complete biosynthesis of CBGA, THCA and CBDA and several
unnatural analogues in yeast via engineering of the native mevalonate
pathway and the introduction of a heterologous hexanoyl-CoA biosynthetic
pathway as well as the Cannabis genes responsible for the
biosynthesis of complete cannabinoids. However, the cannabinoid yields
achieved from this system were found to be approximately 100-fold less
than those produced by Cannabis plants. As such, the efficient
production of cannabinoids in either plant or microbial cell culture
remains a work in progress.
12. Conclusions: Stay tuned, there is more to come!
The recent progress in Cannabis research has been remarkable, and
has revealed exciting challenges ahead: The evolution and genetic
diversity of phytocannabinoid synthases has proven to be a complex field
of research, as is the genetic and environmental control of sex
determination and flowering time. The increasing availability of genomic
resources will undoubtedly facilitate progress in all those areas, but
we predict that experimental analyses, including detailed morphological,
molecular genetics and phenotyping studies will be equally important to
understand the developmental and physiological intricacies of Cannabis.
Unusual in that it is a multipurpose crop, the full sustainability
potential of Cannabis can only be fulfilled if it is used as
such. Thus, one major challenge will be to design crop ideotypes that
harmonise traits of medicinal relevance with those important for carbon
sequestration. This will not be an easy task, as the genetic control of
different traits is currently unclear. However, the production of e.g.
large fibre varieties that do nevertheless develop a dense inflorescence
with high CBD content seems not too farfetched. Even if those
hypothetical cultivars may not be able to provide the high yields of
specialized CBD cultivars they may provide farmers focusing on fibre
production with a second source of income.
In summary, the genetic and morphological diversity of Cannabis is a treasure trove that we are only beginning to explore. It is
important that we capitalise on this treasure to construct a
multipurpose swiss knife, and not a series of highly specialised tools.