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.