Hemp Revitalization Challenges: A Multifaceted Crop
Throughout human history, hemp has been an important crop for food, fibre, and medicine. Despite significant advances made by the international research community, the basic biology of hemp plants is still poorly understood. To guide future research, clear objectives are required. Hemp, as a semi-domesticated plant, has many desirable traits that need to be improved, such as eliminating seed shattering, increasing the quantity and quality of stem fibre, and increasing phytocannabinoid accumulation. Manipulation of the sex of hemp plants will also be important for optimising seed, fibre, and cannabinoids yields. At the moment, research into trait improvement is hampered by a lack of molecular techniques tailored to hemp. In this section, we will discuss how addressing these constraints will help advance our understanding of plant biology and allow us to fully domesticate and maximise the agronomic potential of this promising crop.
Hemp: A Plant with Many Facets and Varieties
For over six millennia, the genus Cannabis (commonly classified into the species Cannabis indica, Cannabis sativa, and Cannabis ruderalis) has been used for food, fibre, and medicine.
Cannabis is classified into two types based on its intended use: marijuana and hemp. Marijuana, which is mostly used recreationally for its intoxicating properties, may have medicinal properties. Hemp, on the other hand, is valued for its medicinal compounds, fibre, and seed, which are used in over 25 000 known products. In comparison to marijuana, the medicinal compounds of interest found in hemp, such as cannabidiol, are non-intoxicating (CBD). To be legally classified as hemp in European and North American countries, the crop must contain less than 0.2 per cent or 0.3 per cent of the intoxicating compound 9-tetrahydrocannabinol (THC). This concentration of THC in cannabis is insufficient to cause intoxication. Differences in marijuana and hemp cultural practices result in minor differences in morphologies, allowing for some differentiation between these two crops.
Hemp has traditionally been grown for either seed or fibre. Hemp seeds have a protein content of about 30%, a starch content of 25%, and an oil content of 30%. When seeds are pressed, they produce oil that contains more than 90% polyunsaturated fatty acids. With a desirable lipid ratio of -6 to -3 hemp seed oil is an important addition to both human and animal diets.
Furthermore, the oil can be used in cooking or processed into cosmetics and fuels. The leftover seed cake can be used to make protein-rich animal feed. Bast fibres (see Glossary) are primarily used to produce high-quality papers, whereas the majority of hurd is used to produce animal beddings. Recent technological advancements have increased the use of hemp fibre and hurd in the production of carbon nanosheets, plastics, 3D-printer filaments, oil absorbent materials, and construction concrete. Furthermore, hemp produces over 100 known cannabinoids, the most notable of which is CBD. CBD is being studied in clinical trials in the United States for the treatment of 26 medical conditions. Furthermore, CBD has been designated as an orphan drug for eleven conditions.
Hemp production has recently expanded beyond Eurasia and Canada to include three additional countries: Greece, Malawi, and the United States. The rapid expansion in the United States may have a significant impact on the global hemp market. Several recent reviews have provided comprehensive information on the biochemistry, breeding, ecology, genetics, morphology, pathology, physiology, and production of Cannabis. Despite recent advances, there is still much to learn about this multifaceted and diverse plant. Importantly, there is a scarcity of information about key research challenges that must be addressed in order to improve this valuable crop. Thus, our goals are to briefly highlight the renewed interest in hemp, and illustrate strategic issues that researchers should address. While we are focusing on traits that will increase hemp yield, these target research topics will also reveal important information about basic plant biology and domestication in the long run.
The Global Hemp Industry
As an environmentally friendly and highly sustainable crop, the global market for hemp is expected to double between 2016 and 2020. Hemp is currently grown for commercial or research purposes in at least 47 countries, and indigenous populations of a few countries use it for textiles. There has been an increase in hemp tonnage and acreage worldwide since 2011. The Food and Agriculture Organization of the United Nations (FAO) has statistics on hemp production for 16 countries. The top hemp producers are currently Canada, China, Chile, France, India and North Korea.
The United States is the largest importer of hemp products, with the majority of its seed and fibre coming from Canada and China, respectively. The United States government authorised research into industrial hemp production in the Agricultural Act of 2014. As a result, hemp production and research have skyrocketed in a number of states. The establishment of a hemp industry in the United States may have an impact on global commerce by reducing hemp imports from exporting countries.
The global hemp market has the potential to grow significantly as consumer demand for organic and environmentally sustainable products rises. Currently, crop value varies greatly depending on product type; for example, the value of CBD far exceeds that of seed or fibre. Using 2015 market prices and excluding costs, revenue/ha is estimated to range between $625 and $25,000 USD. A focus on developing or improving products that can penetrate multibillion-dollar markets (e.g., livestock health, improved construction materials, or energy storage) should be encouraged to advance the industry. Increased demand for hemp-derived products will aid in the establishment of a long-term sustainable market.
Hemp Research's Future Directions
Hemp is a genetically diverse and variable crop that yields three distinct raw products: seed/oil, fibre, and metabolites. Hemp can be improved through a variety of research avenues within each category. We highlight key research areas that help growers increase yield or improve product quality for processors. These topics are not exhaustive, but are intended to direct research to the most pressing issues.
Notably, due to the diverse nature of hemp-derived raw materials, research focusing on hemp yield traits will advance our understanding of basic plant biology. Seed and oil research will help us better understand grain yield and composition. Hemp fibre research will advance our understanding of stem development and composition, genetic regulation of fibre traits, and biofuel production. Studies focusing on metabolite yield will provide new insights into both Cannabis-specific and shared plant chemistries, biotic stress interaction, and trichome development. Research into the plasticity of hemp's sexual phenotype will help to identify the mechanisms underlying plant sex determination. Importantly, unlike previously domesticated crops increasing hemp yield provides a unique opportunity to study plant domestication for grain, fibre, and chemistry traits. Unlike most other crops, these valuable characteristics of hemp can be studied within a single species, which is critical to sustainable and profitable production.
Production of Grain and Oil
Many traits for hemp seed and oil yield require improvement as a semi-domesticated crop, including seed size consistency and improved shattering resistance. With the development of FIN-314 ('Finola,' an auto-flowering grain variety with short stature, adaptation to high latitudes and high yield, significant advances in hemp seed production occurred, and it is now the most popular cultivar grown in Canada. However, seed size varies greatly between hemp cultivars, and 'Finola' seeds are half the size of many commercial varieties. It will be critical to select genetically stable cultivars with larger seeds in order to increase hemp grain yields.
Hemp has exhibited little resistance to shattering during domestication. However, hemp field trials have revealed that significant grain is lost due to shattering prior to and during harvesting as a result of inconsistent inflorescence maturity, particularly if collected outside of the optimal harvest time windows. To address this issue, growers harvest seeds at 70 per cent maturity. Hemp inflorescences are large multi-seeded heads with each individual seed partially surrounded by a bract and an abscission zone connecting the hull to the pedicle. Selection for a thicker-walled abscission zone or prevention of bracts releasing seeds are two possible physiological traits to target to reduce hemp seed loss due to shattering.
Furthermore, due to incomplete embryo development, immature seeds are similar in size but weigh half as much as mature seeds. Immature seeds would fully mature without shattering, increasing yield by up to 15%. Thus, further domestication of non-shattering cultivars could greatly improve yield through a multifaceted mechanism that prevents harvesting loss and allows all seeds to mature.
Seed traits that broaden market options will be valuable as well. There has, for example, been little research into the differences in hemp seed flavours. Taste tests in our lab revealed varieties with weak to strong hazelnut (cv. 'Georgina') or walnut (cv. 'CRS-1') flavours, as well as one with a mild flavour (cv. 'Victoria'). More research has been conducted on modifying seed oil composition, despite the fact that hemp seeds already have valuable -3 properties. Hemp seed oil contains 85% polyunsaturated fatty acids, with 60% and 24% being -6 and -3 fats, respectively. Further increases in -3 fatty acid levels may favour hemp seed for human and animal dietary needs. Different flavours and oil compositions would broaden the use of hemp seed in human and animal food products.
Hemp Fiber Production and Quality
Hemp stalks contain two important fractions: bast fibre and hurd. The stalks must be retted in order to separate the bast fibres from the inner hurd. Retting relies on the environment's diverse microbial populations to break down pectin and other components that bind the fibres to the hurd tissue. Retting is influenced by crop maturity at harvest, retting method, environmental conditions, and the nature of the bacterial and fungal populations. Harvesting the crop at the beginning of flowering increases fibre yield, strength, and quality. Continued research into the biodiversity, relationships, and functions of microbial communities will improve our understanding of the retting process and increase the consistency with which high-quality products are produced.
Hemp is a fast-growing plant that can withstand high planting densities making it a viable biofuel crop. Hemp's total biomass per hectare is comparable to that of other energy crops such as giant miscanthus (Miscanthus giganteus), poplar (Populus sp.), switchgrass (Panicum virgatum), and willow (Salix sp). Hemp, on the other hand, may offer a significant advantage; its bast fibres contain 73–77 percent cellulose, 7–9 percent hemicelluloses, and 2–6 percent lignin, compared to 48 percent, 21–25 percent, and 17–19 percent in the hurd. As a result, hemp fibre contains more digestible cellulose and hemicellulose than other energy crops.
The cellulose and hemicellulose content of hemp hurd, on the other hand, is comparable to that of giant miscanthus, poplar, switchgrass, and willow stems. Importantly, 20–30% of hemp stem biomass is high cellulose fibre; thus, the ratio of digestible sugars to lignin in hemp is higher than in other similar-yielding biofuel crops. Because of these characteristics, hemp is a superior energy crop for some biochemical-based biofuel production and greenhouse gas abatement applications 59, 60. Hemp's acceptance as a biofuel crop would benefit the industry by increasing demand for hurd and fibre.
Other Metabolites and Phytocannabinoids
Hemp produces a wide range of nonintoxicating phytocannabinoids, terpenes, and phenolic compounds that have potential pharmaceutical applications as drugs or supplements 3, 61, 62. Although the biosynthesis of terpenophenolic phytocannabinoids in Cannabis is well understood, several early steps in the pathway have yet to be identified. 63 and 64. Understanding the regulation of phytocannabinoid biosynthesis is critical for developing varieties that are optimised for the production of desirable metabolites while retaining low THC levels. Endogenous and environmental regulation of phytocannabinoids is poorly understood. Abscisic acid, ethylene, and gibberellic acid all influence phytocannabinoid production. However, the factors that control the epigenetic, transcriptional, and post-transcriptional regulation of phytocannabinoid biosynthesis are still unknown.
Trichomes in hemp are classified as bulbous, capitate-sessile, capitate-stalked, or non-glandular. The capitate-stalked glandular trichomes are the sites of phytocannabinoid production and accumulation. Marijuana's increased production of phytocannabinoids is due, at least in part, to the presence of larger glandular trichomes. Understanding the hormonal and other signalling cascades that regulate the development and size of specific types of hemp trichomes will also be critical in maximising phytocannabinoid production in hemp.
It is also necessary to investigate the effects of agronomic practices and nutrients on phytocannabinoid production. Anecdotal evidence from marijuana growers suggests that pollinating Cannabis flowers reduces phytocannabinoid yield, which is consistent with lower essential oil levels. Further research into this issue is required in order to maximise the production of CBD and other desired phytocannabinoids.
Hemp Breeding Restrictions
Plant breeding and research rely heavily on germplasm collections for genetic and phenotypic diversity. Due to the lack of a core Cannabis germplasm collection, access to and utility of accession collections is currently limited. Because THC levels may limit germplasm utility in many regions, accessions containing less than 0.3 per cent THC should be used to create a hemp-only germplasm core collection. The creation of a core collection that encompasses the entire spectrum of hemp genetic and phenotypic diversity would increase the utility of germplasm resources and be invaluable for breeding and genetic analyses. Comparisons of accessions in existing collections are required to assist in the establishment of such a core collection. Similarly, there are no centralised or curated collections of hemp mutants. The creation of mutant germplasm collections will provide a rich source of genetic variation for research into gene function and improved breeding traits.
Hemp is an anemophilous crop with pollen that can travel long distances. Cannabis pollen was found in atmospheric samples collected in southern Spain from over 100 kilometres away from Morocco's extensive marijuana fields. Long-distance pollen dispersal complicates breeding programmes that rely on spatial or mechanical methods to isolate germplasm. Cost-effective and efficient methods are required to allow breeders to develop multiple new hemp varieties at the same time in a small growing area.
Expression of Hemp Sex
Hemp is a dioecious plant, and male and female hemp plants are valued differently depending on the product. A pure female population is ideal for phytocannabinoid production. To maximise yield, a female-dominant population with a limited number of male plants for pollination, or a monoecious variety, is preferred as a seed crop. Males and females are both used for fibre production, though males are preferred. As a result, one of the primary goals of hemp growers and breeders is to quickly and easily determine or manipulate plant sex, preferably prior to planting.
Hemp's sex is determined by a pair of heteromorphic sex chromosomes; females have a XX chromosome pair, while males have an XY chromosome pair. Environmental conditions (e.g., photoperiod and temperature) and phytohormones, on the other hand, can affect sexual phenotype implying that other overriding regulatory mechanisms are involved in determining sex in hemp. Although monoecious cultivars have XX chromosomes, they produce flower clusters with male flowers at the bottom and females at the top of each inflorescence. Male flowers appear when the plant transitions from rapid growth to flowering. Gibberellin levels are associated with stem elongation and fibre development. Gibberellins are linked to plant masculinity and increased fibre number, length, and diameter in hemp. As a result, a gradient of gibberellin and other hormone concentrations may dictate inflorescence sex.
To differentiate sex in hemp plants, genetic markers have been developed; however, such a method is not viable for commercial plantings. Recently, quantitative trait loci (QTLs) for sex expression in dioecious and monoecious hemp were discovered. Cloning the responsible genes from these QTLs will help us understand the genetic control of sex in hemp. Identification of genes on the sex chromosomes, particularly those outside the pseudoautosomal recombinant region , will be critical for understanding sex-linked traits. Continued molecular marker development is required to improve QTL mapping resolution and marker-assisted selection of desirable traits in breeding programmes.
Molecular Biology of Hemp
The organic food industry is a key player in the promotion of hemp food and CBD products. As a result, widespread public acceptance of genetically modified hemp is unlikely. It is also unclear whether the public will accept hemp that has been modified using gene-editing techniques, which lack non-plant or plant-pest DNA sequences. As a result, many improvements to hemp will most likely be accomplished through traditional breeding methods. However, for research purposes, the development of applicable molecular biology techniques is required to further investigate the molecular mechanisms that determine important hemp traits.
Publication of a high-quality draught Cannabis genome research and other genetic studies have shed some light on the distinction between marijuana and hemp. The Cannabis draught genome (Appendix Aiii) has been compared to draught genome sequences of its closest relative, the common hop (Humulus lupulus; Cannabaceae), as well as more distant species such as breadnut (Artocarpus camansi; Moraceae) and mulberry (Morus notabilis; Moraceae). Recently, 54 (11 hemp and 43 marijuana) and 325 (55 hemp and 213 marijuana) distinct cultivars were subjected to low coverage (4–6X) whole-genome sequencing and genotyping-by-sequencing, respectively. However, because only raw data files are available, the utility of these draft-level genome sequences is limited due to a lack of websites with simple graphical user interfaces for data analysis.
Transcriptome assemblies are also available (Medicinal Plants Genomes Resource and PhytoMetaSyn databases), but they are primarily used to study phytocannabinoid metabolism. When marijuana and hemp are compared, the expression of phytocannabinoid biosynthetic genes is higher in marijuana, implying that transcriptional regulation of the pathway is one-factor controlling cannabinoid production. A transcriptome for hemp bast fibres at various growth stages was recently generated, providing insight into fibre development. Less is known about the evolution of genetic differences between seed/oil, fibre, and dual-purpose cultivars. In order to identify the specific genes and mechanisms controlling important yield traits, in-depth genetic comparisons of diverse seed/oil, fibre, and phytocannabinoid cultivars are required. To reap the full benefits of these and other cross-species studies, the genome sequence must be
improved beyond draught quality, and websites with user-friendly graphical user interfaces must be created.
Methods to manipulate gene expression (e.g., via gene knockout or overexpression) are urgently needed to characterise hemp gene functions. There are protocols for developing transformed hairy roots and cell suspension cultures, but their utility is limited because neither tissue produces seed, fibre, or phytocannabinoids. A whole-plant regeneration protocol for marijuana has been developed, implying that the development of transgenic hemp plants is feasible. Virus-induced gene silencing methods would be useful for studying gene function as well but have so far been unsuccessful. Isolation of mutants from chemical mutagenesis screens is also possible, but extremely difficult due to hemp's anemophilous and dioecious nature. Currently, taking advantage of hemp's natural genetic diversity may be the most straightforward way to study gene functions.
Hemp is an unusually diverse crop with potential applications in seed/oil, fibre, and medicinal product markets. The global market for hemp-derived products is expected to double by 2020, owing largely to growth in the US market. Developing seed shattering resistance; increasing seed size; selecting for grain flavours; understanding the microbial populations involved in retting; characterising and improving hemp properties useful for biofuel applications; elucidating environmental, hormone, and nutritional impacts on production and accumulation of CBD and other valuable metabolites; establishment of a core hemp germplasm collection; identification of methods to specifically man. Traditional plant breeding can achieve many much-needed crop improvements. However, research into the underlying biology of hemp seed, fibre, and metabolite production is limited (see Outstanding Questions). Hemp research requires the immediate establishment of molecular biology techniques. Improvements in hemp genomics will help us better understand key agronomic traits. While many scientific advances are required to resurrect hemp production, we have highlighted target areas that we have identified as top research priorities.
We would like to thank Rich Mundell and Dr David Williams for providing us with hemp seed for research and for their invaluable insights into hemp research. Doris Hamilton and the Kentucky Department of Agriculture are also thanked for providing production statistics. We appreciate Dr David Zaitlin's critical comments on the manuscript. This research is funded in part by L.Y.'s Harold R. Burton Endowed Professorship and the National Science Foundation under Cooperative Agreement No. 1355438.