GENETIC VARIATIONS OF CANNABIS SATIVA AND IMPLICATIONS ON INDUSTRIAL PRODUCTION 2015

Genetic Variations of Cannabis Sativa and Implications of Industrial Production
Jack Anderson

Abstract

In the United States, the debate about the relationship between hemp and marijuana is ongoing. The Cannabis plant is cultivated for numerous purposes. For centuries, it been used as an intoxicant, medicine, and for food, but also as a source of fiber and textile. Cannabis has a complex and varied taxonomy making it difficult to breed varieties specific for the intended purpose. Today, biochemical methods are used to classify variants by effective lineages. This report reviews agricultural genetics of the Cannabis sativa L. (C. sativa L.) and Cannabis indica (C. indica) cultivars. An electronic search from 1990 to October 2015 identified studies that evaluated classification, genetic testing, and evolution of the species C. sativa L. and C. indica for use in hemp agriculture. In addition, I pursued methods of genetic testing for the numerous varieties of hemp cultivars. The main outcome assessed from this review of literature determined that there are breeding techniques used to propagate a cannabis variety that embodies the desired characteristics of hemp without the intoxicant properties.

Introduction

The Cannabis plant in America has become more prominent in the last five years due to legalization of recreational marijuana in four states and medical marijuana in twenty-three others. In 2013, soon after the legalization of marijuana in Colorado, many farmers planted hemp crops. Several acres of industrial hemp were harvested, making it the first hemp crop in the United States to be produced in over half a century. Despite the usefulness of hemp (the stem fiber of the cannabis plant) and hempseed, many myths have been generated about this product and how it is not different than the psychoactive component, marijuana. Concerned about the intoxicant properties of Cannabis in the United States, hemp was made illegal to grow without a permit in 1970 under the Controlled Substances Act. Regrettably, this was fear-based legislation and little science was applied. There are many studies that indicate that Hemp can be used as a natural fiber source without psychotropic properties of the marijuana plant. This report considers the genetic differences in hemp versus marijuana and why hemp farming should be legalized in the United States.
This report reviews agricultural genetics of the Cannabis sativa L. (C. sativa L.) and Cannabis indica (C. indica) cultivars. In addition, it examines what properties the species contains in order to cultivate it with a potency meeting federal regulations. I performed an electronic search from 1990 to October 2015 to identify studies that evaluated classification, genetic testing, and evolution of the species C. sativa L. and C. indica for use in hemp agriculture. In addition, I pursued evidence of genetic testing for the numerous varieties of hemp cultivars. Other studies that were included in this literature review had interventions to improve the Cannabis cultivar for purposes of hemp production. I excluded studies focusing on increasing the potency of THC content in C. sativa L. and C. indica cultivars. The main outcome measures were to find hemp strains that are adaptable to various conditions found in the United States and explore the future of hemp genetics.

      Cannabis is a genus of flowering plants that includes three different species, Cannabis sativa L., Cannabis indica (C. indica) and Cannabis ruderalis (C. ruderalis) (Piluzza, 2013). Cannabis has long been used for hemp fiber, oils, medicinal purposes, and as a recreational drug. The most recognized and the identifiable Cannabis constituent that causes the intoxicant effects of marijuana is D9-tetrahydrocannabinol (THC) (Small, 2015). Cannabis strains are bred to either produce minimal levels of THC, such as industrial hemp products made from the plant fiber, or to produce maximum THC potency, obtained through the dried flowers of Cannabis plants, otherwise known as marijuana. This paper will focus on various genetics the Cannabis species sativa and indica as it is used primarily in hemp production. 

Literature Review

Many authors reference a continued debate over the taxonomic organization of the genus Cannabis (Piluzza, 2013). Gilmore (2007) stated, “The currently accepted opinion is that cannabis comprises a highly variable, highly hybridized and introgressed, panmictic (unstructured) species.” Some authors suggest a monotypic genus for Cannabis, C. sativa L.; while others state that three species can be distinguished, C. sativa, C. indica, and C. ruderalis (Piluzza, 2013). The genus Cannabis is composed of several variants. A study by Moteaa El-Deftar (2014), concluded, “For drug varieties, or cultivars, Cannabis is considered to be a single species, C. sativa L. However, physical differences or phenotypes vary widely depending on the seed source (or variety) and growing conditions.” One study, by Elsohly (2005), notes that there are several elements of C. sativa L. that interact with its environment to produce various cultivars including the cultivar for hemp products (ElSohly, 2005). Sawler (2015) claims that although many studies use C. sativa L. as the main cultivar for hemp, C. indica actually has more alleles in common with the hemp-type cannabis cultivar. With either species, THC, the psychoactive component of Cannabis, depends on the cultivar since individual cultivars have either high or low levels of THC. Industrial hemp products are traditionally harvested from variants low in THC.

The historically clandestine nature of the Cannabis plant has made studying the genetic features difficult within the United States. Sawler (2015), who wrote, The Genetic Structure of Marijuana and Hemp, argues that due to complex sociopolitical matters, genetics and taxonomy are poorly understood. He discusses that errors in Cannabis speciation are conceivably secondary to the inaccuracy of reported genetics. He also believes that landraces, a geographic variety of a domesticated species of plant that has developed over time through adaptation to its natural and cultural environment of agriculture, plays a large role in speciation. There are noted cultivars of Cannabis that include both C. sativa L. and C. indica. For example, the common “Skunk” hybrid is a combination of C. indica and C. sativa L. cultivars based on landraces from Afghanistan, Mexico and Colombia (Pilluzza, 2012). Salentijn (2014) emphasizes that genetic differences in cannabis are mainly due to geographic isolation. The author goes on to say that the C. sativa and C. indica genetic variances are not restricted to genes involved in THC production and the origins of marijuana strains only partially illustrate the relationship of marijuana’s genetic structure and genetic distinctions between hemp and marijuana. A (2014) study by Goa, supports that the geographic distribution owes to “the coefficient of similarity”.
There are a variety of ways Cannabis has been studied over the years. A 2007 study by Gilmore (2007) used chloroplast and mitochondrial markers to study the evolutionary history of a panel of 188 plants derived from 76 populations and found that haplotypes, DNA variations that tend to be inherited together, were represented for the different geographical regions. A breakthrough finding was completed in 2011 when the first genome sequence of C. sativa was published (Salentijn, 2014). Goa (2014), who outlined the first large-scale development of SSR markers for cannabis, states that a variety of genetic markers have been used to study genetic diversity in Cannabis. Random amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLP), and inter simple sequence repeat amplification (ISSR) genetic markers had many limitations, poor repeatability, and dominance. He notes that other markers frequently used are Single nucleotide polymorphism (SNP) for studies of specific genes in Cannabis and microsatellite markers useful in applied breeding programs. Goa (2014) explains that microsatellites, recognized as simple sequence repeats (SSRs), are tandem repeats of short DNA sequences that occur throughout the entire genome of eukaryotic organisms. Most studies after 2012 incorporate microsatellite analysis as SSRs are among the most useful genetic markers in biology (Goa, 2014).

Cannabis is a multifaceted and versatile plant, which provokes the argument that hemp cannot be purified to produce exclusively fiber. A study by Elsohly (2005) reports that Cannabis has a very complex chemistry due, in part, to several various constituents and their interactions with one another. He separates these compounds into three broad categories: the intoxicant component, THC; cannabinoids (70 known), and over 400 “other” constituents. Elsohly (2005) goes on to discuss how these numerous components have potential to interact with different environmental factors. Weiblen (2015), remarks that THC and CBD are products from a common precursor, cannabigerolic acid. Weiblen remarks that, “Although recent work has identified the genes encoding the enzymes of the cannabinoid biosynthetic pathway, fundamental questions remain about the inheritance of these genes in relation to the diversity of cannabinoid phenotypes”. Small (2015), notes that some varieties bred for fiber production in temperate climates would transform into the intoxicant variety when transplanted into more arid regions. He calls this phenotypic plasticity, or the ability of individual genotypes to alter their growth and development in response to environmental changes. He notes that strains selected for fiber or narcotic characteristics preserve these features when their gene frequencies are sustained through modern agricultural methods (Small, 2015).

Many studies have shown that despite the complex nature of Cannabis, hemp cultivars would be free from the intoxicant effects of THC. In 2006, a study conducted by Datwyler, found that molecular markers distinguishing both lawful and illegal cultivars of Cannabis have great scientific benefit. At that time Datwyler (2006) noted that no direct comparison of hemp and marijuana amplified fragment length polymorphism (AFLP) has been made to date. She describes the AFLP as a fingerprint for different cannabis cultivars. She demonstrated the ability to separate cultivars using AFLP analysis to determine that there are fixed genetic differences between marijuana and nonpsychoactive hemp. Weiblen (2015) relates research that differentiates marijuana from hemp genetically. He used a simple Mendelian model involving co-dominant alleles at a single locus to explain the segregation of drug content among marijuana and hemp plants (Weiblen, 2015). He concluded that Marijuana is distinguished from hemp by a nonfunctional CBDA synthase that appears to have been positively selected to augment the psychoactive effects of the plant (Weiblen, 2015). Weiblen’s (2015) findings indicate that the total quantity of cannabinoids were significantly different between marijuana and hemp plants, as marijuana plants possessed 4.5 times more total cannabinoids per unit inflorescence biomass (Weiblen, 2015.) Research conducted by Sawler (2015), took 14,031 single-nucleotide polymorphisms (SNPs) genotyped in 81 marijuana and 43 hemp samples that demonstrated marijuana and hemp are significantly differentiated at a genome-wide level. He also uncovered that the difference between these populations is not restricted to genes concentrated on THC. Sawler’s observations are based on modern commercial strains and landraces and do not incorporate potential environmental influences and phenotypic plasticity that may affect the cannabis plant.

Fiber hemp (C. sativa L.) is a desirable crop as it can grow in a variety of climates and soil types, requires less water and fertilizer than other crops like cotton, does not require pesticides, needs less fertilizer, and can be grown in several consecutive years in the same fields (Karus, 2013). Hemp is believed to have originated in Central Asia, and several advocate for two centers of diversity, India and European–Siberian (Elsohly, 2005). China, Europe, and Canada are currently the main hemp producing regions in the world. New cultivars of hemp are steadily increasing for these regions as each area has their own unique cultivar to fulfill various purposes. (Salentijn, 2014).

Hemp cultivation in legalized countries is illustrated by the numerous cultivars and traditional landraces. It is wind-pollinated and is usually a dioecious annual crop, where female and male flowers are on different individuals, indicating that hemp is naturally a cross-pollinator, which can create much variation in plants. Since this variation can lead to a change in the hemp phenotype, the regulation of pollination is a critical matter. Approaches to quality hemp propagation have changed throughout the years. Originally, mass selection was used to choose the most valuable cultivars, however, pollination cannot be regulated and progress in fiber content is very gradual (Salentijn, 2014). According to Salentijn (2014), the Bredemann method that began in 1942 greatly impacted stem fiber content improvement. This method consisted in the individual selection of male plants on the basis of the fiber content, measured on a longitudinal section of the stem. Much breeding work has been conducted in countries where hemp horticulture is legalized. For example, breeding work conducted by Von Segenbuch, a German researcher, produced the first cultivars, comprised of both the male and female reproductive organs, which produced hermaphroditic cultivars that yield a high fiber content (Salentijn, 2014). Today the methods frequently used in hemp breeding are either through mass selection, crossbreeding, inbreeding, and hybrid breeding (Salentijn, 2014). Within the last 10 years there are some farms that use molecular markers to assisted breeding (Salentijn, 2014).

There are many traits geneticists are working toward for hemp development. Salentijn (2014) explains that at this time a large mapping panel including wild material, landraces, and cultivars are being genotyped by whole genome re-sequencing in the ‘Multi-hemp’ project conducted in Europe. Phenotypes of this panel are also being mapped based on three different environments (Salentijn, 2014). This genome wide association mapping (GWAS) is being implemented to provide information about essential alleles, quantitative trait loci, and linked DNA polymorphisms in the underlying genes (Salentijn, 2014). Elshire (2011), proposes that future genetic analysis using genotyping-by- sequencing (GBS) for breeding, conservation, and taxonomic surveys will allow hemp breeders to select their crop based on genetics. Weiblen (2015) purports that due to the current Cannabis legislation, consumers are requesting more diverse phenotypes for improved product. Weiblen (2015) believes his research on the enzymes involved in cannabinoid biosynthesis is a clear contender for selective breeding efforts and other various genetic engineering (Weiblen, 2015).

Conclusion

The genetic study of cannabis has advanced industrial production of hemp through identification of specific biomarkers and alleles specifying ideal properties of the hemp plant. In this review, it was found that Cannabis has a genetic structure that is intricate and highly influenced by the environmental conditions. Only two articles reviewed discussed the phenotypic plasticity found in Cannabis. All the articles pursued did not suggest that hemp could be used as an intoxicant despite the phenotypic plasticity. This should be discussed more frequently in articles relating to hemp production as the alteration of the phenotype in hemp could produce higher THC content than currently accepted by the Drug Enforcement Agency (DEA). Overall, many authors agree that hemp in the United States is highly regulated and a phenotypic alteration is unlikely to occur due to current agricultural standards for industrial hemp production. Further more there has been an increasingly vast amount of new genetic data that can link this phenotypic switch to specific alleles and enzymatic stimulus. Once the taxonomy of the genetics and phenotypic relationships with hemp cultivars is established, it will make selecting strains meeting specific criteria easier.

Currently, there is enough available information on the genetic structure of cannabis, on the methodology to determine what cultivars would be best for specific environments, and on the breeding practices of hemp cultivars that it is safe to produce hemp without producing the intoxicant properties associated with marijuana. Breeding efforts should be focused on organized propagation of the hemp cultivars to minimize cross-pollination and in order to produce a more dense fiber rich plant.

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