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This includes the novel polyketide cyclase for the synthesis of olivetolic acid [ 11 , 12 ], and the prenyl transferase, also called cannabigerolic acid synthase, that produces the central precursor cannabigerolic acid CBGA [ 13 ]. There are published genomes for Cannabis , and the genetic diversity for the biosynthetic pathway of cannabinoids in both marijuana and hemp strains is under investigation [ 18 , 20 ].
This schema is derived from pathways reviewed in [ 2 ]. Based on the diversity of cannabinoid content, five unique chemotypes have been described [ 21 — 24 ]: In addition to the cannabinoids, medicinal Cannabis samples are also rich in bioactive terpenoids [ 25 ]; there appears to be a positive correlation between the accumulation of each of these classes of compounds [ 1 ]. The complexity of the terpenoid and cannabinoid composition in these samples should be routinely quantified to accurately and adequately determine the medicinal potential of a particular sample [ 26 ].
Inclusion of the terpenoid composition in a characterization of a medicinal marijuana sample also provides information about the provenance of the sample [ 1 ], as unique chemical abundances of specific terpenoids are predicted to be associated with chemotypes and species level taxa [ 5 , 27 ].
As the aroma of the plant is largely the result of the terpenoid composition [ 25 ], human selections for this plant may have been based in part on the monoterpenoid and sesquiterpenoid based aroma and on the cannabinoid psychoactivity.
Within the past several decades, significant scientific evidence for the medical utility of both the floral material and specific cannabinoids from these plants has been presented for a number of human health diseases and conditions [ 9 , 28 — 30 ]. In the United States of America, as of , more than half of the states license the use of medical marijuana http: As a result, there is very limited reliable data on the chemical composition of Cannabis strains, the variability within the plant for the production of these compounds and the interaction of the environment of production with the chemical quality of a specific strain.
Much of the material currently available for medical use was selected from the numerous strains available from the illicit drug growers; understandably, the provenance and genetic backgrounds of these strains are not easily traceable. Our past experience characterizing the sources of variability for secondary metabolite accumulation in medicinal plants like lavender [ 31 ] yerba mansa [ 32 ] or in Capsicum cultivars chile [ 33 ] lead us to investigate the variability for cannabinoid accumulation in medical marijuana produced by licensed growers in New Mexico.
There were several aims for the experiments reported here. We wanted to develop a rapid, reliable and inexpensive method to quantify the important bioactive compounds in medical marijuana, cannabinoids and terpenoids. We wanted to demonstrate the utility of the method using commercial material, available as medical marijuana in New Mexico. These studies were also designed to determine the consistency of total and relative cannabinoid and terpenoid accumulation within a given accession, and to see if leaf levels of cannabinoids could be used to predict eventual floral levels.
The terpenoid contents of selected medicinal strains are also presented. We also report here a reliable and sustainable gas chromatography method for the separation and quantification of these five neutral cannabinoids.
This method has superior limit of detection LoD and limit of quantitation LoQ capabilities for these metabolites over the more common liquid chromatographic separations [ 34 ].
We present this method as a way to easily monitor both the cannabinoid and terpenoid compositions of medical marijuana plant material https: All of the plant material characterized in this report will be referred to as Cannabis , without a species level description. For the developmental analyses, plants were grown in a commercial greenhouse in central New Mexico, under a medical marijuana producer license from the New Mexico Department of Health. Cuttings were propagated in Coco Air Max for 6 to 8 weeks with a 20 h light photoperiod.
Plants were then transferred into 38 L pots and moved to a 12 h photoperiod to induce flowering. Again, natural sunlight was supplemented with artificial lighting as needed. Leaf and flower samples were collected at the days indicated post floral induction. In some cases, samples were collected from the top upper third of plant or bottom lowest third of plant. Samples for RNA extraction were not dried but frozen in liquid nitrogen and stored frozen until extraction. A second source of plant material includes cured flowers from medicinal marijuana produced by licensed growers throughout the state of New Mexico.
This material reflects the medical marijuana commercially available, and represents a wide range of strains. Curing is the method used to properly age and dry cannabis flower prior to consumption. All of the plant material was processed at Rio Grande Analytics, a licensed medicinal Cannabis testing laboratory. The homogenates were placed on an orbital shaker for 1 h.
The samples were then centrifuged briefly to remove insoluble matter and analyzed using a Varian model GC-FID with an Rxi column 15 m x 0. Ultrapure nitrogen was employed as the carrier gas flow rate: The injection volume was 2. The regression equation parameters for each analyte standard were determined using at least 10 different concentrations between 0 and ng for the terpenoids and between 0 and ng for the cannabinoids. Peak ID is the code for each of the 21 terpenoids T1-T21 and six cannabinoids C1-C6 , for which a calibration curve was generated.
Flowers and leaves were harvested and immediately frozen in liquid nitrogen; RNA was isolated using methods described earlier [ 35 ]; the quality of the RNA was confirmed by formaldehyde agarose gel electrophoresis.
The primers and conditions for qRT-PCR analysis were developed based on methods described earlier [ 36 ]. Calibration curves using purified amplicons for each gene were generated to allow for quantification of transcripts with expression data represented as pg transcript per ng total RNA.
PCR cycling conditions for prenyl transferase were: Gas chromatography is a useful approach for quantifying terpenoids and the abundant cannabinoids; however, achieving base line separation of some of the minor cannabinoids, CBC and CBD, can be difficult.
Therefore, GC conditions were developed to improve the separation of these analytes. All of the cannabinoids were readily detected at 2 ng, and the peak area for these compounds increased linearly over the range from 2 ng to ng.
Our working range for terpenoid detection was between 0. The retention times for 19 terpenoid and 6 cannabinoid standards are presented in Table 1. This table also provides the linear regression parameters for the conversion of GC-FID peak areas to mass quantities of each of these specific metabolites. An example of the resolution of the terpenoids and cannabinoids in a cured Cannabis flower sample is presented in Fig 2.
All six of the cannabinoids were detected in this strain, and we were able to identify and quantify a number of terpenoid peaks. Peaks are labeled with the Peak ID code. The method described here for the analysis of cannabinoids and terpenoids differs from most published methods in that the cured plant samples were extracted with acetone.
Most other published methods utilize methanol, methanol-acetonitrile, or methanol-chloroform as the extraction solvent [ 5 , 26 , 37 ]. Acetone is additionally a less polar solvent, and therefore extracts fewer sugars and polysaccharides than does methanol. A second difference in this assay for quantification of terpenoids and cannabinoids was the use of a single chromatographic system, GC-FID. The use of nitrogen as the carrier gas was a key element in the method described here.
Helium gas, which is the more common carrier gas in GC methods for cannabinoids has become difficult and expensive to obtain. Nitrogen gas is readily and sustainably available; further the switch to nitrogen gas improved the separation of the CBC and CBD compounds on the chromatogram. These differences allow this method to recover and detect the lower abundance cannabinoids. One group has quantified terpenoids and cannabinoids using a single GC-FID run [ 1 ]; they also used nitrogen as a carrier gas.
This group extracted their plant samples sequentially three times with ethanol for a total of mL for a 1 g plant sample. In contrast we use only one acetone extraction, 1 g in 20 mL, homogenized and then shaken on an orbital shaker 1 h. This much simpler protocol recovers equivalent levels of terpenoids and cannabinoids as demonstrated below. There is a limitation to this method; we cannot detect the free acid forms of the cannabinoids, only the neutral forms of the cannabinoids.
The heat in the protocol converts all free acid forms to neutral forms. In order to quantify the free acid forms of cannabinoids, the samples must be derivatized prior to GC separation [ 37 ]. The cannabinoids present in leaf and floral samples of 16 different medicinal marijuana plants were determined. The levels of six different cannabinoids in floral samples are listed in Table 3 and the levels in leaf samples are listed in Table 4. In both tables the levels are reported as percent of dry weight of the respective organ, these organs were collected from plants at 50 to 65 d post-light induction.
As expected the levels of cannabinoids are variable between strains, and the levels in floral tissues are much higher than in leaf tissues. The other important medical cannabinoid CBD, was barely detectable in leaf or floral tissue of most of the strains except Alien Blues, Thunderstruck, Love Lace and Juanita.
A third cannabinoid, CBC, was present at similar levels in leaves and floral tissues, and varied between strains. Platinum Buffalo had the highest leaf levels, 0. Thunderstruck had the highest CBC levels in floral tissues, 0. CBG levels in floral samples ranged between 2. The strain Platinum Buffalo had the highest levels in floral samples at 1. The higher levels of cannabinoids in floral versus leaf tissue is expected and has been described by many other investigators [ 2 , 9 , 22 ].
The induction of flowering in the clonally propagated plants is initiated by a shift to a short-day photoperiod. Floral and leaf samples were collected from two strains, Sour Willie and Bohdi Tree at multiple intervals following the floral induction. This plant material was collected from greenhouse grown plants. These are the averages of multiple flowers collected from different parts of the plant. When those flowers are separated into their locations on the plant, high, middle and low, a pattern of accumulation is detected.
This was observed in both Sour Willie and Bohdi Tree and is anecdotal knowledge among producers of this crop. This same pattern of accumulation was observed in two additional strains, increasing the biological replication of this observation. The observation that the cannabinoid content increases during floral development has been described by other investigators for plants cultivated in vitro as well as in greenhouse settings [ 22 , 39 ].
The quantification of the variability within the plant for floral content of cannabinoids has not been described in the literature previously.
For example, does the cannabinoid content of vegetative leaves, those on the plant prior to flower induction predict the cannabinoid content of the mature flowers that develop later on that plant. A comparison of the levels of CBD in vegetative leaves and mature flowers is plotted for samples from 16 strains Fig 4. There is a positive correlation between the CBD content of leaf and flower samples, with an R 2 value of 0. Conversely, if a strain is going to have appreciable levels of CBD in its flowers, then the vegetative leaves will have at least 0.
Testing vegetative leaves to determine if a medical marijuana strain predicted to have high CBD floral content will provide valuable information early on in the management of the plant. Flowers and leaves were collected from 16 Cannabis strains. A detailed analysis of the terpenoid and cannabinoid content of commercially generated medical Cannabis floral samples was conducted.
The growth conditions of the plant, the harvest of the samples and their curing process were all under the purview of the producer. The strain names for these samples are provided in the supporting information S1 Table , but we have somewhat limited confidence in the provenance of the strain name.
However, the chemical composition of this material used medicinally in New Mexico has been accurately determined. In the supplemental material we provide the abundances of 19 terpenoids and 6 cannabinoids determined on 72 strains of medical Cannabis.
The totals of these values were plotted to look for correlations in levels Fig 5. Cured trimmed flowers were extracted and the terpenoid and cannabinoid composition determined by GC-FID. The total values for cannabinoids and terpenoids in each sample were plotted. The total terpenoid content ranged between 0. There are limited biosynthetic interactions between these two biosynthetic pathways, so a strong correlation based on shared biochemical pathways is not predicted.
One group has reported a positive correlation between the terpenoid and cannabinoid content [ 1 ] of selected strains developed in the Netherlands for medicinal uses. Those authors do not consider the abundance of these metabolites to be linked metabolically either.
The positive association between the abundance of terpenoids and cannabinoids in Fig 5 probably reflects increased production of all metabolites, including oils in larger healthier floral buds. We also tested for chemotypes within the medical cannabis population based on terpenoid profiles. For that analysis we clustered the Cannabis strains using the relative abundance of 19 unique terpenoids in 72 different strains. The dendrogram that resulted from this analysis is presented in Fig 6.
The specific composition and strain identifications are presented in supporting information S1 Table. Pie charts for selected strains are presented along with the color code for the terpenoid composition in S1 Table. As demonstrated in Fig 6 , there were three major clades or chemotypes: The biosynthetic pathway for the monoterpenoids and sesquiterpenoids present in the Cannabis samples are highly interrelated and positive and negative correlations between the accumulations of specific compounds are expected.
Terpenoid types are identified: Several other groups around the world have used terpenoid profiles to categorize Cannabis strains [ 1 , 22 , 26 , 40 ]. In these cases the authors used a principal components analysis approach to identify metabolites important in either clustering or discriminating specific strains.
Recently, a PCA and hierarchical analysis of 30 cultivars from a medical Cannabis dispensary in California also identified five major groups based on the abundance of 16 terpenoids in these samples [ 40 ]. Our approach of agglomerative clustering and then inspection of pie chart displays of terpenoid composition, allowed us to identify the clades in the trees with their most abundant terpenoid.
Plant samples for these studies were collected from greenhouse grown plants. Transcript levels for these three genes were quantified in leaves Fig 7 and three different development stages in floral tissues Fig 8A—8C. RNA was isolated from flowers collected from the indicated strains at early blue , middle orange or late purple after the floral induction phase. Transcripts for all three genes were detected in leaves of all four strains.
The levels of transcripts for prenyl transferase and THCA synthase were similar within a strain. The researchers expect the map will speed up breeding efforts to create new strains with desired medical properties as well as varieties that can be grown more sustainably, or with increased resistance to diseases and pests.
Hughes, Page and van Bakel first got together in when they released the first draft of cannabis genome which was too fragmented to reveal gene position on chromosomes.
The new map reveals how hemp and marijuana, which belong to the same species Cannabis sativa, evolved as separate strains with distinct chemical properties. Cannabis plants grown for drug use "marijuana" are abundant in psychoactive tetrahydrocannabinol, or THC, whereas hemp produces cannabidiol, or CBD, popular of late for its medicinal potential. Some people use CBD to relieve pain and it is also being tested as a treatment for epilepsy, schizophrenia and Alzheimer's.
Both are found on chromosome 6 of the ten chromosomes the cannabis genome is packaged into. There, the enzyme genes are surrounded by vast swathes of garbled DNA which came from viruses that colonized the genome millions of years ago. This viral DNA, or retroelements as it is known, made copies of itself that spread across the genome by jumping into other sites in the host cell's DNA. The combination of a genetic map and PacBio sequencing technology allowed us to increase the size of the puzzle pieces and find enough distinguishing features to facilitate the assembly process and pinpoint the synthase genes.
The researchers believe that gene duplication of the ancestral synthase gene and expanding retroelements drove ancient cannabis to split into chemically distinct types. Humans subsequently selected for plants containing desirable chemistry such as high THC. The gene sequences for the THCA and CBDA synthases are nearly identical supporting the idea that they come from the same gene which was duplicated millions of years ago.
Over time, one or both gene copies became scrambled by invading retroelements, and by evolving separately, they eventually came to produce two different enzymes - CBDA synthase found in hemp fibre-type , and THCA synthase in drug-type marijuana.
How ancient viruses got cannabis high
We know sativa plants are tall, feature slim leaves and are usually Ruderalis is often lower in THC in its resin and is known for its high cannabidiol (CBD) content . Ruderalis genetics are often bred into strains to control certain factors All of these fantastic autoflowering strains, and more, have won well. All autoflowering strains contain ruderalis genetics. Plants can deliver high doses of CBD-rich buds in just several weeks And even though THC is already a great painkiller, CBD makes it even better. Stress Killer won't grow as fast. Still, it's not all bad for hemp: The plant produces another lucrative compound These genetic maps showed that the THC and CBD genes are.