Abstract

Introduction

Trichomes are formed on the plant surface across a range of taxonomically disparate species, providing a variety of functions and benefits to the plant. These can include simple tasks, such as affecting leaf temperature and photosynthesis, or more complicated functions, such as pest-deterrence via their physical structures or production of compounds (Wagner, 1991; Hare et al., 2003). Glandular trichomes are of particular commercial interest as they are one of the key plant structures that produce essential oils – an industry valued at 18.62 billion USD in 2020 (Grand View Research, 2020). Other oil-producing plant structures are internal glands and other trichome types, some of which are capable of producing resinous secretions. Trichome morphology is highly variable both among plant species and within the plant itself (Sangwan et al., 2001). In Cannabis sativa L. (hereafter, cannabis), stalked glandular trichomes are the trichome morph that produces substances of economic value (Fairbairn, 1972; Sirikantaramas et al., 2005). These trichomes develop a secretory cavity between secretory disk cells and the cuticle where secondary metabolites, including cannabinoids and terpenes, are deposited and stored (Kim and Mahlberg, 1991, 1997; Sirikantaramas et al., 2005; Marks et al., 2009). Though there are a variety of other trichome morphs found across the cannabis plant, they are beyond the scope of this review.

While male plants produce small amounts of cannabinoids, in cannabis cultivation, the primary products are the female flowers clustered in inflorescences (Ohlsson et al., 1971). Stalked glandular trichomes are primarily concentrated on the calyces and bracts (Figure 1A; Spitzer-Rimon et al., 2019; Leme et al., 2020) with populations extending to the inflorescence “sugar leaves”; these are the sites of accumulation for secreted metabolic products. These valuable secretions include tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), terpenes, and flavonoids (ElSohly and Slade, 2005; Flores-Sanchez and Verpoorte, 2008). Cannabis plant morphology and cannabinoid profiles are influenced by genetics and the cultivation environment, highlighting the importance of controlled conditions for cannabis cultivation (Magagnini et al., 2018; Danziger and Bernstein, 2021a, b). With the gradual global increase in social and legal acceptance of cannabis, there has been considerable interest in producing consistent high-quality yields. In addition, as medicinal uses for cannabinoids are supported by peer-reviewed research and clinical trials, the global demand for medicinal cannabis products will continue to increase. This will create further pressure on growers to improve control over the concentration of specific cannabis metabolites and the associated cannabis genotypes. However, the genotypes and environmental conditions needed to obtain this level of precision remain poorly characterized. Ultimately, these elusive methods need to be centered around trichomes as the “factories” of the plant. Current efforts have focused on the effects of breeding and cultivar selection, industrial growing conditions, and fertilization methods on flower yield and cannabinoid profiles (Vanhove et al., 2011; Campiglia et al., 2017; Tang et al., 2017; Hawley et al., 2018; Janatová et al., 2018; Burgel et al., 2020; Saloner and Bernstein, 2021). However, as undefined cannabis plant material in pioneering research papers formed the backbone for future cannabis/cannabinoid research, comparing data with uniform standards is impossible. Thus, the need for systematically validating results of these papers and cannabis production “folklore” is paramount yet challenging due to the impact of genotype and growing environment. Regardless of these challenges, since trichomes are ultimately responsible for yield and quality control, it is necessary to advance our understanding of how they, specifically, are affected by these efforts, as well as to investigate new approaches to broaden the scope of possible cost-effective applications for improving yield.

Trichome Profiles

Cannabis Glandular Trichomes

Previously, three types of glandular trichomes on cannabis flowers were described – referred to as capitate-sessile, capitate-stalked, and bulbous – based on structural assessments by scanning electron microscopy (Hammond and Mahlberg, 1973). The trichomes were differentiated based on their morphology, where bulbous trichomes were small and low, sessile trichomes were comprised of a globular head on a very short stalk, and stalked trichomes had a larger globular head on a long stalk; of the three trichome types, stalked trichomes produce the greatest amount of cannabinoids (Hammond and Mahlberg, 1973; Mahlberg and Kim, 2004; Livingston et al., 2020). Unfortunately, this non-specific differentiation between trichome types led to misidentification of trichomes due to the similar appearance of sessile and stalked morphs (Dayanandan and Kaufman, 1976; Livingston et al., 2020). However, a recent study on trichome anatomy revealed that sessile trichomes on vegetative leaves consistently have exactly eight secretory disk cells while stalked glandular trichomes on mature flowers have 12–16; these numbers were consistent across hemp and drug-type varieties (Livingston et al., 2020). As sessile-presenting trichomes on immature cannabis flowers can contain more than eight disk cells and emit fluorescence at intermediate wavelengths, which true sessile trichomes cannot, sessile-presenting trichomes are now thought to be a precursor developmental stage of immature stalked trichomes (Livingston et al., 2020). These discoveries allow for improved accuracy of trichome classification during plant development, may provide more precise estimates of plant maturity and allow for identification of optimal points of metabolite production. This understanding further allows for greater accuracy when assessing the density of stalked glandular trichomes and the ability to predict mature flower trichome densities.

The causes of variable metabolite profiles found among varieties/genotypes and plant organs are genetic and environmental. For example, flowers sampled from the upper region of the plant produce significantly greater quantities of cannabinoids and terpenes than lower positions; light source and plant maturity are believed to be important factors influencing the concentration and/or amounts (Namdar et al., 2018; Eichhorn Bilodeau et al., 2019). Abiotic factors that influence cannabis growth are the same as those affecting other plant species, such as temperature, fertilization, photoperiod, and light intensity (Taschwer and Schmid, 2015; Conant et al., 2017; Pagnani et al., 2018; Bernstein et al., 2019; Eichhorn Bilodeau et al., 2019; Taghinasab and Jabaji, 2020). However, knowledge regarding how these factors influence growth and trichome formation is limited, with much work needed to produce scientific evidence to support links between metabolite production and environmental factors (Taghinasab and Jabaji, 2020). Research on cannabis is in the early stages, and future work is necessary to investigate signaling pathways that mediate the effect of external factors on metabolite production. Attention toward developing this area of cannabis research is increasing (Mudge et al., 2019; Aliferis and Bernard-Perron, 2020; Conneely et al., 2021).

Conclusion and Future Prospects

Cannabis was left behind in the agricultural research boom of the last century because of its illegal status in most jurisdictions. While many of the advancements in plant science for a wide range of other species are applicable to cannabis, multiple species-specific traits require dedicated research both to gain fundamental insights and to provide evidence-based data to the growing industry. Since industrial agriculture practices became globally established and genomic studies became possible in the 20^th century, researchers have been able to elucidate novel agricultural applications derived from molecular-scale understanding, while cannabis applications remain centered on breeding and environmental conditions; cultivation protocols were largely based on anecdotal rather than scientific evidence. For example, the soybean genome has been unraveled to identify genetic markers related to nematode resistance and this has been exploited to support precise breeding strategies (Kim et al., 2016); meanwhile, the simple taxonomy of cannabis remains controversial (Koren et al., 2020). The cannabis research field is slowly catching up to the level of investigation that is observed for other valuable crop species, with one example being a recent study demonstrating a high-throughput assay using genetic markers to identify sex and chemotype of cannabis germplasm (Toth et al., 2020). However, this study was primarily focused on THC:CBD ratios to determine chemotype and when modeling “total potential cannabinoids” only THC, CBD, CBG, and CBC were included, highlighting the limits of current genetic studies (Toth et al., 2020). Regardless of their limitations, these studies signal the beginning of cannabis truly entering 21^st century agricultural research.

Trichomes and essential oils in other plant species have been well characterized in recent decades, and it is important that our understanding of cannabis trichomes reach similar levels of comprehension. The increasingly widespread legalization and public acceptance of cannabis suddenly brings a once-shunned plant into a position of intense interest and high demand in a time of exceptional experimental standards, raising expectations that questions surrounding it be answered much more quickly than for previous crops. Simple breeding and agricultural production techniques for influencing metabolite profiles are not precise nor always consistent, leading to a host of potential complications for both producer and consumer. An example of this complication is the growing medicinal and recreational consumer demand for products with greater THC levels, causing a trend referred to as “lab shopping” that is observed where producers will test their products at several laboratories until they receive the desired cannabinoid concentration analysis for their products (Swider, 2021; Zoorob, 2021). The resulting lack of reliability in the identification might potentially lead to health complications and distrust by those who use cannabis for pain mitigation and as an appetite stimulant/anti-emetic. These issues highlight the need for not just a more reliable and ethical approach to cannabis product quality, but also for methods to reliably tailor metabolite production at the trichome source. New approaches, such as phytomicrobiome manipulation and exploitation, present interesting possibilities, as root inoculums have demonstrated similar effects on THC and CBD contents to nitrogen application (Pagnani et al., 2018; Lyu et al., 2019). If methods can be developed to consistently replicate specific metabolite concentrations and combinations within small ranges across cannabis plants at the trichome level, and if these methods were to become standard across the industry, the benefits for both producers, medical practitioners, and consumers would be great.

From a scientific perspective, multiple interesting questions are associated with the glandular trichomes. Primarily, these questions center around differences related to genotype and growing conditions. How changes to soil composition, light, nutrients, water levels, and other environmental factors affect trichome densities remain largely unknown for cannabis. Our knowledge on how the metabolite profiles themselves differ among varieties is limited and primarily based on poor reporting from growers that are incomplete beyond the major cannabinoids and terpenes, leaving 100 of metabolites unknown. Our lack of knowledge in these areas of cannabis metabolism and composition make it difficult to directly hypothesize exactly where and how differences occur, stressing the need for rigorous uniform standards to allow unbiased and scientifically sound data comparisons. The more we understand about trichomes, the more applicable our knowledge of this plant will be to those along the chain of production and consumption.

Author Contributions

CT compiled the literature and prepared the manuscript. AG and DS provided the conceptual context. RB, DS, and AG provided the revision and feedback. All authors contributed to the article and approved the submitted version.

Funding

Funding for this work was provided by the NSERC (CREATE 543319-2020) and the RGPIN/04714-2018, RGPIN-2020-07047.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Acknowledgments

References