High Yielding CBD-type Cannabis in the High Country of North Carolina

BIO 2000 Botany, Appalachian State University

Kristopher M. Gupton

25 November 2017

High Yielding CBD-type Cannabis in the High Country of North Carolina

BIO 2000 Botany, Appalachian State University

Kristopher M. Gupton

25 November 2017

Introduction:

            Cannabis (Cannabis indica, sativa, and ruderalis) have long been cultivated for the production of fiber, seed, oil and food around the world. It has been cultivated in North America up to the 1937 Marihuana Tax Act, which required those who used it medicinally to buy a stamp and pay a tax. Illicit use continued and the tax was not paid by the users as this would be self- incriminating. This ultimately led to the 1970 congress ruling to place marijuana on the “Schedule 1” list where it has remained to this day (Chandra, 2017). In 2015 the North Carolina (NC) General Assembly passed Senate Bill 313 that allows the Industrial Hemp Commission to act within the federal laws to set in place rules and guidelines for the cultivation of what is labeled Industrial Hemp. According to the 

2016 NC House Bill 992, Industrial Hemp is characterized by different strains spanning up to 6 meters in height during a growing season and a reduced flower set. Here in North Carolina the legal threshold for the total concentration of THCA is 0.3% (NCAGR.GOV).

            There are two abundant and medicinal pertinent compounds found in Cannabis, THC (tetrahydrocannabinoid) and CBD (cannabinoid).  High yielding THC type Cannabis plants are classified as drug type. High yielding CBD type Cannabis plants are gaining traction as medicinal type. Of the one-hundred different natural occurring cannabinoids found in Cannabis, seven have been labeled as CBD type compounds. These seven types are provoking considerable interest due to neuroprotective, antiepileptic, anxiolytic, antipsychotic, and anti-inflammatory properties (Morales, 2017). 

 Lynch, et al. (2015) states there are seventeen strains identified as hemp through the use of high resolution melting (HRM). HRM is a specific technique used for the genotyping of single-nucleotide polymorphism (SNP) markers. SNP has numerous advantages and is the most popular system in genetic analyses. HRM differentiates drug type and medicinal type by the nucleotide

sequence changes that accrue between the active and inactive forms of Δ9-tetrahydrocannabinolic acid synthase (THCAS). “The sequencing results of the THCAS gene in drug and fiber accessions revealed two distinct forms and the THCA synthase one belonging to the drug type and the other related to fiber type accessions. These divergent THCA synthase sequences represent alleles coding for an active and inactive form of the THCAS” (Tahereh, 2017). 

            During the 1990s, GW Pharmaceuticals in the Netherlands successfully bred chemotypes with purity levels between 71% and 84% CBDV. Following 1999 GW Pharmaceuticals has enhance growing methods for indoor cultivation which has been recreated at the University of Mississippi (UM). Then in 2017, following the June 19 and July 13 planting at the Mountain Research Station/Center in Waynesville and Mills River, North Carolina State University successfully harvested fifteen different strains of Cannabis. Three of the fifteen were harvested for fiber, the remaining twelve were for grain. No CBD research was conducted in the high country of North Carolina. The purpose of this project is to determine the conditions needed to cultivate high yielding CBD-type cannabis here in the high country of North Carolina.

Literature Review:

                        The researchers at the University of Tehran, Iran acknowledge that the use of drug type C. sativa L. are outlawed in many countries while the cultivation of non-drug type C. sativa L. are advocated. The comparison between the two are established in the cannabinoids components: tetrahydrocannabinoid (THC), cannabidiol (CBD), and cannabinol (CBN). In this report, a protocol is determined to differentiate the drug type and non-drug type through the use of high resolution melting (HRM) of a single-nucleotide polymorphisms (SNP) in the THCAS gene. Based on the results of HRM one can conclude which C. sativa L. can legally be cultivated (Tahereh Borna, et al.).   In Genomic and Chemical Diversity in Cannabis, the researchers at the University of Colorado Boulder partnered with

many corporations across the country comment that the genetic identities and histories of the many different strains of C. sativa L. remain largely undisclosed. Through the analyses of SNP, 360 Cannabis varieties were described in three groups: hemp, narrow leaflet drug-type, and broad leaflet drug-type. Of the 360 varieties described, 17 are identified as hemp, allowing a cultivator to select a specific strain based on phylogenetic (Lynch, Ryan C, et al.).     

                       With continuing research finding more and more favorable results for the use of cannabidiol in the treatment of many disorders, GW Pharmaceuticals and the University of Mississippi realize large scale production is needed to continue research and development. GW Pharmaceuticals has identified eight chemotypes in cannabinoids, two of which are currently supporting epilepsy clinical trials. University of Mississippi has set in place in vitro breeding protocol with no detectable differences comparable to the mother plant. Together as an assemblage, these guidelines allows the cultivator to clone strains season after season identical to the parent generation that contain the compounds desired  in medical research (Chandra, et al.). Two researchers, Morales and Paula, at University of North Carolina Greensboro understand that CBD-type functional groups have become increasingly of interest in medical chemistry and have composed this overview. They provide the reader with a review of the naturally occurring and synthetic CBD derivatives to include each associated molecular targets. Armed with this general comprehension a cultivator can assess which CBD-type compound to select for (Morales, Paula, et al.).   

                        North Carolina’s Industrial Hemp Pilot Program portal is full of information for the cultivator of C. sativa L. From history and committee ruling to rules and applications, this site gives its reader reliable information from the commanding authorities.  Since the Drug Enforcement Administration still has C. sativa L as a schedule one narcotic, it is very important to stay within the legal frame of one’s governing state body. To insure proper information is received, a direct link is mandatory (NCAGR.GOV.).  To be able to compare cannabinoids ratios found in one strain to that of another, a

standard must be established; Dr. Karl Hillig and Dr. Paul Mahlberg at the Indiana University do this in 2004. They collected samples from 157 Cannabis accessions of known geographic origin and performed gas chromatography along with starch gel electrophoresis to each. By doing so they were able to assign the various strains to one of three chemotypes and support a two-species concept of Cannabis (Hillig, K. W., and P. G. Mahlberg). When it comes to the cultivation of Industrial Hemp no one has done more research in the United States then the University of Kentucky. Much of their agronomic findings have been applied throughout the county to include North Carolina State University. By following the recommendation set forth, failure to grow cannabis will fall not on the plant’s adaptability, but to the farmer (UK Agronomic Industrial Hemp Research).      

Methods:

            High Country Herbs LLC (HCH) will cultivate a 12,250 square foot plot of  AOSCA certified  selected strains purchased from Bio-Regen Innovations Cooperative. Rows will be hilled three feet wide with a two foot spacing between each row. A three foot row is chosen so alternating double rowing can be performed in each row for maximizing biomass yield. Soil impediments of lime at 1.9 tons per-acre and nitrogen at 100lbs per-acre will be implemented as recommend by NCDA&CS Soil Report and the University of Kentucky (UK Agronomic Industrial Hemp Research). Plants will be grown on black plastic to control weed growth and promote the maximizing of nutrients reaching the crop.  Drip line irrigation will also be incorporated to maintain hydration throughout the growing season and reduce influencing factors. Seeds will be germinated in seed pods in a greenhouse/high tunnel until the height of 6-8in tall. Plants will be spaced between 2-4ft alternating in each three foot row.  After transplanting into the field, close observation is made to determine males from females. Males will be removed and the androecium frozen to later fertilize randomly selected females within respective strains. These fertilized females will undergo a separate harvest when 80% of the seeds have reached maturity. The seeds will then be collected and frozen for strain preservation. Around the 1st of

September, harvesting of the remaining females will commence once the trichrome have turned white. The cola and leaves will be removed

Sample names and FLOCK assignment of 195 varieties to three groups, represented with different cell colors. Green (left) are BLDT, blue (center) are NLDT, and yellow (lower right) are hemp.

from the stems and branches.  The stems and branches will be sold at market for the production of fiber. The cola and leaves will be air dried in a well-ventilated room on screens. A 50mg sample will be ground and combined with 1ml of chloroform. After one hour the compound will be sonicated and a 20 μL extract will be place in a round bottom flask to undergo fractional distillation. A 1mL distillated sample will be analyzed using the Vernier Mini GC Plus Gas Chromatogram instrument to determine the retention times of the present compounds. The Vernier LabQuest2 will be used to graphically represent the data obtained for the Mini GC Plus. Peak area will be calculated to mediate purity percentage and retention times will be compared to known values. This procedure will be repeated until sufficient data is collected to generated histograms, scatter plots and a Student T distribution for each strain. The values will finally be used to develop a difference of proportions. An independent third party will conduct the same procedure to eliminate basis results.   

 

 

 

 

 

 

 

 

 

 

 

 

Expected Results & Discussion:

By applying the methods already established by former researchers of C. sativa L. presented above, the ability to cultivate high yielding CBD-type Industrial Hemp in the high country of  North Carolina will not only probable but plausible . The percentages of these functional groups will be found within a 99% confidence interval of the standard deviations (SD) and mass percentages as determined by Dr. Hillig and Dr. Mahleberg (Fig 2). It is expected that by meeting these standards the market for Industrial Hemp will no longer be limited to fiber production but also medical research. A possible ripple effect could lead to medical research contacted at the University of North Carolina and/or Duke Medical.

Dr. Davis produced between 1000-2000lbs per-acre of grain in 2017 at the Mills River Research Center (NCAGR.GOV) and the same is expected for this experiment. Therefore since the mass percentage of 4.01% presumably is achieved an amount of 40-80lbs of CBD extract could be cultivatedper-acre. To put this in retrospect, HCH is projected to harvest 1400lbs of biomass and 56.14lbs of CBD.

Conclusion:

                        Based on the information gathered, the cultivation of Industrial Hemp for CBD extraction in North Carolina can meet the demands of the world market. Research on the remaining legalized strains will be conducted over the next few years. The percentages of each strain will be determined and appropriate rank will be established. In addition the amount of land needed to cultivate industrial hemp for this product is not massive and therefore could be an alternative for the small scale farmer.  Much remains too been understood from this newly legalized crop but it is now clear North Carolina can be a major producer. 

Works Cited

Hillig, K. W., and P. G. Mahlberg. “A Chemotaxonomic Analysis of Cannabinoid Variation in Cannabis (Cannabaceae).” American Journal of Botany, vol. 91, no. 6, Jan. 2004, pp. 966–975., doi:10.3732/ajb.91.6.966.

Tahereh Borna, Seyed Alireza Salami & Majid Shokrpour (2017) High resolution melting curve analysis revealed SNPs in major cannabinoid genes associated with drug and non-drug types of cannabis, Biotechnology & Biotechnological Equipment, 31:4, 839-845, DOI: 10.1080/13102818.2017.1333456

Lynch, Ryan C, et al. “Genomic and Chemical Diversity in Cannabis.” BioRxiv, Cold Spring Harbor Laboratory, Jan. 2015, www.biorxiv.org/content/early/2015/12/13/034314.

Chandra, Suman, Hemant Lata, Mahmoud A. Elsohly, Larry A. Walker, and David Potter. "Cannabis Cultivation: Methodological Issues for Obtaining Medical-grade Product." Epilepsy & Behavior 70 (2017): 302-12. Print.

Morales, Paula, et al. “An Overview on Medicinal Chemistry of Synthetic and Natural Derivatives of Cannabidiol.” Frontiers in Pharmacology, Frontiers Media S.A., 2017, www.ncbi.nlm.nih.gov/pmc/articles/PMC5487438/.

“NCAGR.GOV.” North Carolina Department of Agriculture & Consumer Services, www.ncagr.gov/hemp/.

“UK Agronomic Industrial Hemp Research.” UK Agronomic Industrial Hemp Research, hemp.ca.uky.edu/.

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