USOO93701 64B2
(12) United States Patent (10) Patent No.: US 9,370,164 B2 Lewis et al. (45) Date of Patent: *Jun. 21, 2016
(54) BREEDING, PRODUCTION, PROCESSING 2013/0109747 A1 5, 2013 Whittle AND USE OF SPECIALTY CANNABS 2014/02434.05 A1 8/2014 Whalley 2014/0245494 A1 8, 2014 Cohen (71) Applicant: Biotech Institute LLC, Los Angeles, 2014/0245495 A1 8, 2014 Cohen CA (US) FOREIGN PATENT DOCUMENTS (72) Inventors: Mark Anthony Lewis, Los Angeles, CA GB 2459 125 10/2009 (US); Michael D Backes, Los Angeles, CA (US); Matthew W Giese, Los OTHER PUBLICATIONS Angeles, CA (US) No art cited. (73) Assignee: Biotech Institute, LLC, Westlake De Meijer et al., 2003, The Inheritance of Chemical Phenotype in Village, CA (US) Cannabis Sativa L. Genetics, 163:335-346. De Meijer et al., 2005. The Inheritance of Chemical Phenotype in (*) Notice: Subject to any disclaimer, the term of this Cannabis sativa L. (II) Cannabigerol Predominant Plants. Euphytica, patent is extended or adjusted under 35 1.45:189-198. U.S.C. 154(b) by 0 days. De Meijer et al., 2009. The Inheritance of chemical phenotype in Cannabis sativa L. (III) Variation in Cannabichromene Proportion This patent is Subject to a terminal dis- Euphytica, 165:293-311. claimer. De Meijer et al., 2009, The Inheritance of Chemical Phenotype in Cannabis Sativa L. (IV) Cannabinoid-Free Plants, Euphytica, (21) Appl. No.: 14/742,493 168:95-112. 2011, Taming THC: potential cannabis synergy and phytocan (22) Filed: Jun. 17, 2015 nabinoid-terpenoid entourage effects, British Journal of Pharmacol ogy, 163: 1344-1364. (65) Prior Publication Data Russo et al. (2006. A tale of two cannabinoids: the therapeutic ratio nale for combining tetrahydrocannabinol and cannabidiol, Medical US 2015/0359 188A1 Dec. 17, 2015 Hypothesis, 2006, 66:234-246). Hazekamp and Fischedick 2010. "Metabolic fingerprinting of Can O O nabis sativa L., cannabinoids and terpenoids for chemotaxonomic Related U.S. Application Data and drug standardization purposes” Phytochemistry 2058-73. (63) Continuation of application No. 14/216,744, filed on McPartland and Russo 2001 Cannabis and Cannabis Extracts: Mar 17, 2014, now Pat No. 9,095,554. Greater Than the Sum of Their Parts? Journal of Cannabis Therapeu r sw-- a-s tics vol. 1, No. 3/4.2001, pp. 103-132. (60) Provisional application No. 61/801,528, filed on Mar. RG Pertwee. 2008 “The diverse CB1 and CB2 receptor pharmacol 15, 2013, provisional application No. 61/897,074, ogy of three plant cannabinoids: delta 9 tetrahydrocannabinol, can filed on Oct. 29, 2013. nabidiol and delta 9 tetrahydrocannabivarin' Br. J Pharmacol. 153(2): 199-215. ( 51 ) Int. C. CBDCrew "About Us' provided as publishedon Apr. 10, 2012. URL: AOIH 5/02 (2006.01) http://cbdcrew.org/about-us/. AOIH 4/00 (2006.01) CBD Crew “Varieties” provided as published on Mar. 20, 2012. URL: AOIH 5/2 (2006.01) http://cbdcrew.org/varieties/. A6 IK36/85 (2006.01) Kojoma, M. et al., DNA polymorphisms in the tetrahydrocan AOIHL/04 (2006.01) nabinolic acid (THCA) synthase gene in "drug-type” and “fiber type” Cannabis sative L. Forensic Science International, Jun. 2, 2006, AOIG L/00 (2006.01) pp. 132-140, vole. 159, No. 2-3, Elsevier Scientific Publishers Ireland (52) U.S. Cl. Ltd. CPC A0IH 5/12 (2013.01); A0IGI/001 (2013.01); International PCT Search Report for PCT/US2014/046694, mailed A0IH I/04 (2013.01); A0IH 4/00 (2013.01); on Jan. 5, 2015. A0IH 5/02 (2013.01); A61K 36/185 (2013.01) Bertoli A. et al., “Fibre hemp inflorescences: From crop-residues to (58) Field of Classification Search essential oil production”, Industrial Crops and Products, Nov. 1, None 2010, pp. 329-337, vol. 32, No. 3. See application file for complete search history. (Continued) (56) References Cited Primary Examiner — Russell Kallis U.S. PATENT DOCUMENTS (74) Attorney, Agent, or Firm — Cooley LLP 4.279,824 A 7/1981 McKinney 6,403,126 B1 6, 2002 Webster et al. 6,630,507 B1 10/2003 Hampson et al. (57) ABSTRACT 7,968,594 B2 6/2011 Guy et al. 2004.0049059 A1 3, 2004 Mueller The invention provides compositions and methods for the 2008. O103193 A1 5, 2008 Castor et al. 2008/024.1339 A1 10/2008 Mitchell et al. breeding, production, processing and use of specialty Can 2009.0035396 A1 2/2009 De Meijer nabis. 2010/0216872 A1 8, 2010 Letzel et al. 2011/0098348 A1 4, 2011 De Meijer 2012,0311744 A1 12, 2012 Sirkowski 16 Claims, 19 Drawing Sheets US 9,370,164 B2 Page 2
(56) References Cited The Werc Shop Terpene Profiling Services, Aug. 26, 2012. http:// web.archive.org/web/2012082607 1723/http://thewercshop.com/ser OTHER PUBLICATIONS vicesterpene-profiling-services. G. of Vancouver Island Seed Company, “How to make Clones', Cannabis Culture Magazine published on Tuesday, Apr. 29, 2009. Solon, Olivia, “Medicial Marijuana Without the High”. May 7, 2012, Available online at http://www.cannabisculture.com/content/how retrieved from the internet: www.wired.com. make-clones. Harm Van Bakel, et al. “The draft genome and transcriptome of Fishedick, J. et al., Phytochemistry 2010, vol. 71. pp. 2058-2073. Cannabis sativa”. Genome Biology, Oct. 20, 2011, p. R102, vol. 12, Russo, E.B., The British Journal of Pharmacology, 2011, pp. 1344 No. 10, Biomed Central Ltd., London, GB. 1364, vol. 613. International PCT Search Report for PCT/US14/30267, mailed on CBD Crew Web Pub; (Critical Mass sample available from North west Canna Connection; Feb. 26, 2014, pp. 1-5. http://analytical360. Nov. 7, 2014. com/m/expired 197158. Seedsman Listing of "Sweet and Sour Widow', on May 21, 2013, Satyal, Petal. Journal of Medicinally Active Plants, Dec. 2014, vol. http://web.archive.org/web/20130521.045347/http://www.seeds #3, Issue 1. pp. 9-16. man.com/en/cbd-Sweet-n-Sour-widow-regular-5-seeds. Analytical 360 Analysis of Critical Mass on Aug. 25, 2014. http:// Analytical 360 analysis of Sweet & Sour Widow CBD on Nov. 2, analytical360.com/m/expired 27.6599. 2013. http://analytical360.com/m/expired 131803. CBD Crew Analysis Report; (Critical Mass, Fundacion CANNA; CBD-crew front page on Nov. 22, 2014. www.cbdcrew.org. Mar. 21, 2012), pp. 1-2, http://cbdcrew.org/varieties/cbd-critical Agilent Technologies, Inc. “Consideration for Selecting GC/MS or mass. LC/MS for Metabiomics', Feb. 24, 2007. Analytical 360 Analysis of Sweet n' Sour Widow on May 8, 2014, Waksmundzka-Hajnos and Monika, “High Performance Liquid http://analytical360.com/m/expired/230612. Chromatography in Phytochemical Analysis (Chromatograhic Sci ence Series).” Published May 14, 2012. p. 582 provided. * cited by examiner
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How would you describe the aroma
How would you describe the flavor?
How would you rate the mind high?
How would you rate your level of mood enhancement
How would you rate your level of hunger?
How would you rate your level of physical comfort?
How would you rate your ability to function normally?
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US 9,370,164 B2 1. 2 BREEDING, PRODUCTION, PROCESSING the federal government would allow states to create a regime AND USE OF SPECIALTY CANNABS that would regulate and implement the legalization of Can nabis, including loosening banking restrictions for Cannabis CROSS-REFERENCE TO RELATED dispensaries and growers (Jacob Sullum “Eric Holder Prom APPLICATIONS ises To Reassure Banks About Taking Marijuana Money Very Soon” Forbes January 2014). This application is a continuation filing of U.S. non-provi In addition to these recent developments, the U.S. govern sional application Ser. No. 14/216,744 filed on Mar. 17, 2014, ment has already set a precedent for patenting Cannabis, and which itself claims priority to U.S. provisional application Cannabis-related inventions. For example, U.S. Pat. No. No. 61/801,528 filed on Mar. 15, 2013, and U.S. provisional 10 6,630.507 issued on Oct. 7, 2003 and assigned on the patent application No. 61/897,074 filed on Oct. 29, 2013, each of face to The United States of America, is directed to methods which are hereby incorporated by reference in their entireties, of treating diseases caused by oxidative stress by administer including all descriptions, references, figures, and claims for ing therapeutically effective amounts of a cannabidiol (CBD) all purposes. cannabinoid from Cannabis that has substantially no binding 15 to the N-methyl-D-aspartate (NMDA) receptor, wherein the FIELD OF THE INVENTION CBD acts as an antioxidant and neuroprotectant. A search of the U.S. PTO Patent Application Information Retrieval The invention relates to specialty Cannabis plants, com (PAIR) system also reveals the existence of thousands of positions and methods for making and using said Cannabis Cannabis related applications and issued patents including plants and compositions derived thereof. U.S. Pat. No. 8,034,843 (use of cannabinoids for treating nausea, vomiting, emesis, motion sickness), U.S. Pat. No. BACKGROUND OF THE INVENTION 7,698,594 (cannabinoid compositions for treatment of pain), and U.S. Pat. No. 8,632,825 (anti-tumoural effects of cannab Cannabis, more commonly known as marijuana, is a genus inoid combinations) among many others. of flowering plants that includes at least three species, Can 25 Thus, despite the official position of the U.S. Federal Gov nabis sativa, Cannabis indica, and Cannabis ruderalis as ernment, and as recognized by the states that have legalized it, determined by plant phenotypes and secondary metabolite Cannabis has been shown to provide substantial benefits for profiles. In practice however, Cannabis nomenclature is often medical and recreational uses. Cannabis is regularly used by used incorrectly or interchangeably. Cannabis literature can a wide cross-section of Society to treat a variety of maladies, be found referring to all Cannabis varieties as “sativas” or all 30 conditions and symptoms including, but not limited to, the cannabinoid producing plants as “indicas. Indeed the pro following: nausea, glaucoma, lack of appetite, mucous mem miscuous crosses of indoor Cannabis breeding programs brane inflammation, epilepsy, leprosy, fever, obesity, asthma, have made it difficult to distinguish varieties, with most Can urinary tract infections, coughing, anorexia associated with nabis being sold in the United States having features of both weight loss in AIDS patients, pain, and multiple Sclerosis. sativa and indica species. 35 Cannabis intoxication (i.e., euphoria, relaxation) can The use of Cannabis for Social and medical purposes has occur and other side effects may also accompany its use, been known for almost of all humanity's recorded history. particularly with higher doses, specific Cannabis varieties Cannabis is most commonly administered via inhalation or and/or over prolonged periods of usage. Undesirable side consumption of marijuana-infused food and drink. However, effects of using the available THC-predominant Cannabis since 1972 marijuana has been classified as a Schedule I drug 40 varieties can include, but are not limited to, the following: under the U.S. Controlled Substances Act because the U.S. decreased short-term memory, dry mouth, impaired visual Federal Government considers it to have “no accepted medi perception and motor skills, erectile dysfunction, lower fer cal use.” In stark contrast to this position, 20 of the 50 U.S. tility, red (i.e., blood shot) eyes, increased anxiety, occasional states and the District of Columbia have recognized the medi infarction, stroke, paranoia, acute psychosis, lowered mental cal benefits of Cannabis and have decriminalized its medical 45 aptitude, hallucinations, bizarre behavior, irrational panic use. The 20 U.S. states where medical marijuana has been attacks, irrational thoughts and various other cognitive and decriminalized as of the filing date of the present application Social problems. are as follows: Alaska, Arizona, California, Colorado, Con Some of the negative or undesirable side effects from using necticut, Delaware, Hawaii, Illinois, Maine, Massachusetts, available Cannabis varieties for medical and recreational pur Michigan, Montana, Nevada, New Hampshire, New Jersey, 50 poses are related to the plant's content of the chemical A-tet New Mexico, Oregon, Rhode Island, Vermont and Washing rahydrocannabinol (THC). A major hurdle to the more wide ton. The residency requirements, approved list of conditions/ spread acceptance of Cannabis and its legalization is that the diseases, and the other laws/rules regarding the possession land races and commercially available Cannabis genotypes and cultivation of medical marijuana generally differ by state. (of drug varieties) contain relatively high concentrations of President Obama has publicly commented on the recre 55 THC. Indeed the average THC content of traditional recre ational legalization of Cannabis in Colorado and Washington ational Cannabis has risen over the years from an average of stating that “it’s important for it to go forward because its 0.74 in 1975, to 3.35% in the 1990's, and average of 6.4% in important for Society not to have a situation in which a large 2003 (Annual Reports (Nov. 9, 1999 to Nov. 8, 2003) of portion of people have at one time or another broken the law Mahmoud A. ElSohly, PhD, Director of the National Institute and only a select few get punished. Indeed in the same 60 on Drug Abuse (NIDA) Marijuana Project at the National interview, President Obama remarked about Cannabis “I Center for Natural Products Research, School of Pharmacy, don’t think it's more dangerous than alcohol. In fact, it is less University of Mississippi). There is a real need for Cannabis dangerous than alcohol in terms of its impact on the indi varieties for potential medical use that produce modulated vidual consumer.” (Conor Friedersdorf January 2014, THC concentrations and varying concentrations of other “Obama on Pot Legalization: It’s Important for it to go 65 pharmacologically active Substances that reduce the negative Forward’” The Atlantic). In line with the President’s com side effects of THC and increase the medical benefits realized ments the U.S. Attorney General Eric Holder announced that from its use. There is also a need for healthier Cannabis for US 9,370,164 B2 3 4 recreational use with reduced negative side effects from THC. In some embodiments, the present invention provides spe The inventions described herein meet that long-felt need. cialty Cannabis plants, plant parts, tissues and cells having a THC:CBs ratio greater than or equal to of 8:1. In other SUMMARY OF THE INVENTION embodiments, the specialty Cannabis of the present invention has THC:CBS ratios approaching 1:1, or lower. By compari According to the methods and compositions of the present son, the THC:CBs ratio of the currently available Cannabis invention, plants, plant parts, plant tissues and plant cells are varieties is 20:1 and approaches 25:1, 30:1, 35:1, 40:1 and produced to contain pentyl, propyl, C-4, C-1 and monometh higher. Thus, the specialty Cannabis plants, plant parts, plant ylether constituents of cannabinoid families, including but tissues and plant cells of the present invention will have a not limited to acidic and neutral forms of the cannabigerol, 10 THC:CBS ratio of less than 20:1, 15:1, 10:1, 9:1.8:1,7:1, 6:1, cannabichromene, cannabidiol, delta-9-tetrahydrocannab 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:9, or below. inol, delta-8-tetrahydrocannabinol, cannabielsoin, cannab The present invention provides Classes of Cannabis Vari inol and cannabinodiol cannabinoid classes; and, cis and trans eties developed by selection from landraces of mixed Can terpenoids, including but not limited to myrcene, limonene, nabis genotypes and resulting from further breeding, wherein 15 these Classes of Cannabis Varieties can provide useful linalool, ocimene, beta-pinene, alpha-pinene, beta-caryo patient treatment and also are used as breeding material to phyllene, alpha-caryophyllene, delta-3-carene, gamma-bis develop Specialty Cannabis plants and varieties according to abolene, alpha-farnesene, beta-fenchol, guajol, alpha the present invention. guaiene, terpinolene, beta-eudesmol, alpha-bergamotene, The present invention provides Specialty Cannabis plants epi-alpha-bisabolol and caryophyllene oxide ranging from and varieties with increased organoleptic appeal as a result of 0.1% of dry weight of inflorescences, plant parts, plant tissues having specified, predetermined terpene and sesquiterpene and plant cells to 35% of inflorescences and/or 95% of plant profiles and content. In some embodiments of the present parts, plant parts, plant tissues and plant cells. invention, the increased organoleptic appeal of the Specialty The present invention provides specialty Cannabis plants, Cannabis is inherited in-whole or in-part as a result of using plant parts, plant tissues and plant cells which provide a way 25 the Classes of Cannabis Varieties in the breeding program to to deliver a consistent and more tolerable and effective ratio of develop the Specialty Cannabis plants. For, example, in some cannabinoids by providing plants that comprise non-THC embodiments, Classes of Cannabis Varieties with specific cannabinoids (“CBs) to patients (e.g.,
OH
60
O Tetrahydrocannabinol (THC)
65 A-Tetrahydrocannabinol-C4 Known as delta-9-tetrahydrocannabinol (A9-THC), THC A-THC-C, is the principal psychoactive constituent (or cannabinoid) of US 9,370,164 B2 23 24
-continued -continued
10 A-Tetrahydrocannabiorcolic acid A-Tetrahydrocannabivarin A-Tetrahydrocannabiorcol A and for B A-THCV-C. A-THCO-C A-THCOA-CA and/or B
15
(-)-A-trans-(6aR.10aR)-A- A-Tetrahydrocannabinolic acid A Tetrahydrocannabinol
A-THCA-C5. A 25 A-THC-Cs
30
(-)-A-trans-(6aR.10aR)- 35 Tetrahydrocannabinolic acid A A-THCA-C5. A HO O A-Tetrahydrocannabinolic acid B A-THCA-Cs B 40
45 (-)-(6aS.10aR)-A- Tetrahydrocannabinol (-)-cis-A-THC-Cs
50 A-Tetrahydrocannabinolic acid-C4 OH A and or B A-THCA-C4A and/or B
55 OH
Cannabidiol (CBD)
60 CBD is a cannabinoid found in Cannabis. Cannabidiol has displayed sedative effects in animal tests (Pickens, 1981, “Sedative activity of Cannabis in relation to its delta'-trans A-Tetrahydrocannabivarinic acid A tetrahydrocannabinol and cannabidiol content”. Br. J. Phar 65 macol. 72 (4): 649-56). Some research, however, indicates A-THCVA-C. A that CBD can increase alertness, and attenuate the memory impairing effect of THC. (Nicholson et al., June 2004, “Effect US 9,370,164 B2 25 26 of Delta-9-tetrahydrocannabinol and cannabidiol on noctur nal sleep and early-morning behavior in young adults' J Clin Psychopharmacol 24 (3): 305-13; Morgan et al., 2010, “Impact of cannabidiol on the acute memory and psychoto mimetic effects of smoked Cannabis: naturalistic study, The 5 British Journal of Psychiatry, 197:258-290). It may decrease the rate of THC clearance from the body, perhaps by interfer ing with the metabolism of THC in the liver. Medically, it has been shown to relieve convulsion, inflammation, anxiety, and nausea, as well as inhibit cancer cell growth (Mechoulam, et 10 al., 2007, "Cannabidiol—recent advances”. Chemistry & ediol Biodiversity 4 (8): 1678-1692.) Recent studies have shown 5 cannabidiol to be as effective as atypical antipsychotics in treating schizophrenia (Zuardi et al., 2006, "Cannabidiol, as Cannabis sativa constituent, as an antipsychotic drug Braz. J. Med. Biol. Res. 39 (4): 421-429). Studies have also shown that it may relieve symptoms of dystonia (Consroe, 1986, “Open label evaluation of cannabidiol in dystonic movement disorders”. The International journal of neuroscience 30 (4): 20 277-282). CBD reduces growth of aggressive human breast cancer cells in vitro and reduces their invasiveness (McAllis ter et al., 2007, "Cannabidiol as a novel inhibitor of Id-1 gene expression in aggressive breast cancer cells'. Mol. Cancer Ther. 6 (11): 2921-7.) 25 Cannabidiol has shown to decrease activity of the limbic Cannabidiol momomethyl ether system (de Souza Crippa et al., “Effects of Cannabidiol CBDM-Cs (CBD) on Regional Cerebral Blood Flow”. Neuropsychop harmacology 29 (2): 417-426) and to decrease social isola tion induced by THC (Malonet al., “Cannabidiol reverses the 30 reduction in social interaction produced by low dose A9-tet rahydrocannabinol in rats'. Pharmacology Biochemistry and Behavior 93 (2): 91-96.) It's also shown that Cannabidiol reduces anxiety in Social anxiety disorder (Bergamaschietal. 35 2003, "Cannabidiol Reduces the Anxiety Induced by Simu lated Public Speaking in Treatment-Naive Social Phobia Patients. Neuropsychopharmacology 36 (6): 1219-1226). Cannabidiol has also been shown as being effective intreating Cannabidiol-C4 an often drug-induced set of neurological movement disor- 40 CBD-C ders known as dystonia (Snider et al., 1985, “Beneficial and Adverse Effects of Cannabidiol in a Parkinson Patient with Sinemet-Induced Dystonic Dyskinesia'. Neurology, (Suppl 1): 201.) Morgan et al. reported that strains of Cannabis which contained higher concentrations of Cannabidiol did 45 not produce short-term memory impairment VS. Strains which contained similar concentrations of THC (2010, “Impact of cannabidiol on the acute memory and psychotomimetic effects of Smoked Cannabis: naturalistic study: naturalistic study corrected.”. British Journal of Psychiatry 197 (4): 285-90) (-)-Cannabidivarin Cannabidiol acts as an indirect antagonist of cannabinoid CBDV-C, agonists. CBD is an antagonist at the putative new cannab inoid receptor, GPR55. Cannabidiol has also been shown to 55 act as a 5-HT1A receptoragonist, an action which is involved in its antidepressant, anxiolytic, and neuroprotective effects. Cannabidiol is also an allosteric modulator at the Mu and Delta opioid receptor sites. Cannabis produces CBD-carboxylic acid through the 60 same metabolic pathway as THC, until the last step, where CBDA synthase performs catalysis instead of THCA syn thase. See Marks et al. (2009, “Identification of candidate genes affecting A9-tetrahydrocannabinol biosynthesis in Cannabis sativa”. Journal of Experimental Botany 60 (13): 65 Cannabidiorcol 3715-3726.) and Meijer et al. I, II, III, and IV. Non-limiting CBD-C examples of CBD variants include: US 9,370,164 B2 27 28 -continued -continued OH
10 Cannabigerol monomethyl ether Cannabidiolic acid CBDA-Cs (E)-CBGM-CSA OH O
15 S OH
HO
Cannabinerolic acid A (Z)-CBGA-C5. A
OH Cannabidivarinic acid CBDVA-C, 25 S
HO OH 30
Cannabigerovarin (E)-CBGV-C, HO OH O Cannabigerol (CBG) 35 S OH CBG is a non-psychoactive cannabinoid found in the Can nabis genus of plants. Cannabigerol is found in higher con HO centrations in hemp rather than in varieties of Cannabis cul 40 tivated for high THC content and their corresponding psychoactive properties. Cannabigerol has been found to act as a high affinity C2-adrenergic receptor agonist, moderate Cannabigerolic acid A affinity 5-HT1A receptor antagonist, and low affinity CB (E)-CBGA-Cs A 45 receptor antagonist. It also binds to the CB receptor. Can OH O nabigerol has been shown to relieve intraocular pressure, which may be of benefit in the treatment of glaucoma (Craig et al. 1984, “Intraocular pressure, ocular toxicity and neuro toxicity after administration of cannabinol or cannabigerol 50 Experimental eye research 39 (3):251-259). Cannabigerol has also been shown to reduce depression in animal models (U.S. patent application Ser. No. 1 1/760.364). Non-limiting examples of CBG variants include: Cannabigerolic acid A monomethyl ether 55 (E)-CBGAM-Cs. A
OH OH O
60
HO HO
Cannabigerol 65 Cannabigerovarinic acid A (E)-CBG-Cs (E)-CBGVA-C. A US 9,370,164 B2 29 30 -continued -continued
OH OH O 5
OH
O 10 Cannabinolic acid A Cannabinol (CBN) CBNA-Cs A
CBN is a psychoactive substance cannabinoid found in 1. Cannabis sativa and Cannabis indica/afghanica. It is also a 15 metabolite of tetrahydrocannabinol (THC). CBN acts as a weak agonist of the CB1 and CB2 receptors, with lower affinity in comparison to THC. Non-limiting examples of CBN variants include
Cannabinol methyl ether CBNM-C5. A
OH 25
O 30 HO Cannabinol CBN-Cs Cannabichrimene (CBC)
CBC bears structural similarity to the other natural cannab 35 inoids, including tetrahydrocannabinol, tetrahydrocannabi OH Varin, cannabidiol, and cannabinol, among others. Evidence has suggested that it may play a role in the anti-inflammatory and anti-viral effects of Cannabis, and may contribute to the overall analgesic effects of Cannabis. Non-limiting examples 40 O of CBC variants include: Cannabinol-C4 CBN-C (RS) 45
OH
50 HO
O (E)-Cannabichromene CBC-Cs Cannabivarin CBN-C, (RS) 55
OH OH 60
O O O OH
Cannabinol-C2 Cannabiorcol 65 (E)-Cannabichromenic acid A CBCA-C5 A US 9,370,164 B2 31 32
-continued 21 (RS) O
HO (E)-Cannabivarichromene, 10 (E)-Cannabichromevarin Tetrahydrocannabivarin (THCV, THV) CBCV-C.
(RS) THCV, or THV is a homologue of tetrahydrocannabinol 21 (THC) having a propyl (3-carbon) side chain. This terpeno O 15 phenolic compound is found naturally in Cannabis, some times in significant amounts. Plants with elevated levels of propyl cannabinoids (including THCV) have been found in populations of Cannabis sativa L. ssp. indica (Cannabis O indica Lam.) from China, India, Nepal. Thailand, Afghani H stan, and Pakistan, as well as Southern and western Africa. THCV has been shown to be a CB1 receptor antagonist, i.e. it O OH blocks the effects of THC. Tetrahydrocannabinol has been (E)-Cannabichromewarinic shown to increase metabolism, help weight loss and lower acid A 25 cholesterol in animal models (U.S. patent application Ser. No. CBCVA-C. A 1 1/667,860)
OH OH 30
O 35 Cannabivarin (CBV) Cannabicyclol (CBL) Cannabicyclol (CBL) is a non-psychotomimetic cannab Cannabivarin, also known as cannabivarol or CBV. is a inoid found in the Cannabis species. CBL is a degradative non-psychoactive cannabinoid found in minor amounts in the 40 product like cannabinol. Light converts cannabichromene to hemp plant Cannabis sativa. It is an analog of cannabinol CBL. Non-limiting examples of CBL variants include: (CBN) with the side chain shortened by two methylene bridges (—CH2-). CBV is an oxidation product of tetrahy drocannabivarin (THCV. THV). 45
50 HO (+)-(1aS,3aR,8bR.8cR)-Cannabicyclol CBL-Cs
55 Cannabidivarin (CBDV) O CBDV is a non-psychoactive cannabinoid found in Can nabis. It is a homolog of cannabidiol (CBD), with the side 60 chain shortened by two methylene bridges (CH2 units). Can nabidivarin has been found reduce the number and severity of HO seizures in animal models (U.S. patent application Ser. No. 13/075,873). Plants with relatively high levels of CBDV have O OH been reported in feral populations of C. indica (FC. sativa ssp. 65 (+)-(1aS,3aR,8bR,8cR)-Cannabicyclolic acid A indica var. kafiristanica) from northwest India, and in hashish CBLA-Cs. A from Nepal.
US 9,370,164 B2 38 -continued olivetolate geranyltransferase (GOT) to form Cannabigerol acid (CBGA). Alternatively, GPP and divarine acid are con densed by GOT to form Cannabigerovarinic acid (CBGVA). S CBGA or CBGAV is transformed to (1) CBC by CBC syn 5 thase or CBCV by CBCV synthase; (2) THC by THC syn HO thase or THCV by THCV synthase; or (3) CBD by CBD synthase or CBDV by CBDV synthase. The genes coding for THC synthase and CBD synthase are found on the same B locus. Thus Cannabis plants can be categorized into THC Cannabigerovarin (CBGV) 10 CBD chemotypes based on the state of the Blocus B/B 8 9 OH (THC producing, chemotype I), B/B (CBD producing, chemotype III), and B/B, (producing both THC and CBD, 6 21 2 21 chemotype II). Additional information on the genetic regula CH7 15 tion of cannabinoids can be found in Meijer et al. I, II, III, and O IV (I: 2003, Genetics, 163:335-346; II: 2005, Euphytica, 145:189-198: III: 2009, Euphytica, 165:293-311; and IV: CBCV (Cannabichromevarin) 2009, Euphytica, 168:95-112). OH More details of cannabinoids synthesis and the properties and uses of these cannabinoids are described in Russo (2011, Taming THC: potential Cannabis Synergy and phytocannab inoid-terpenoid entourage effects, British Journal of Phar macology, 163: 1344-1364), Russo et al. (2006. A tale of two cannabinoids: the therapeutic rationale for combining tet 25 rahydrocannabinol and cannabidiol, Medical Hypothesis, 2006, 66:234-246), Celia et al. (Impact of cannabidiol on the CBGM (Cannabigerol Monomethyl Ether) acute memory and psychotomimetic effects of Smoked Can nabis: naturalistic study, The British Journal of Psychiatry, Biosynthetic pathway of cannabinoids has been studied. 30 201, 197:285-290), de Mello Schier et al., (Cannabidiol, a See Meijer et al. I, II, III, and IV (I: 2003, Genetics, 163:335 Cannabis sativa constituent, as an anxiolytic drug, Rev. Bras. 346; II: 2005, Euphytica, 145:189-198: III: 2009, Euphytica, Psiquiatr; 2012, 34(S1):5104-5117), and Zhornitsky et al. 165:293-311; and IV: 2009, Euphytica, 168:95-112), each of (Cannabidiol in Humans—the Quest for Therapeutic Targets, which is herein incorporated by reference in its entirety for all Pharmaceuticals, 2012, 5:529-552), each of which is herein purposes. According to the current model, phenolic precur- 35 incorporated by reference in its entirety for all purposes. sors such as geranyl pyrophosphate (GPP) and polyketide, Please see Table 1 for a non-limiting list of medical uses for olivetolic acid (OA) are condensed by geranyl pyrophosphate cannabinoids. TABLE 1 Non-limiting list of medical uses for cannabinoids.
MEDICALUSES CANNABINOID REFERENCES 1 Distonia, Akathisia CBD (a) Consroe, 1986, The International journal of neuroscience 30 (4): 277-282 (Anti convulsant) (b) Snider et al., 1985, Neurology, (Suppl 1): 201. 2 Glaucoma (lowers CBD (a) Colasantietal, Exp. Eye Res. 30: 251-259, 1984 intraocular CBG (b) Gen. Pharmac. 15:479-484, 1984 pressure) (c) Craig et al. 1984, Experimental eye research 39 (3): 251-259 3 Ischemic disease CBD (a) U.S. Pat. No. 6,630,507 (Alzheimer's, (b) Snider et al., 1985, “Beneficial and Adverse Effects of Cannabidiol in a Parkinson's, Down Parkinson Patient with Sinemet-Induced Dystonic Dyskinesia. Neurology, (Suppl 1): Syndrome, HIV, 2O1. Dementia) 4 Good for patients CBD (a) U.S. Pat. No. 6,630,507 treated with oxidant-inducing agents for chemotherapy, radiation. 5 Motion Sickness CBD (a) U.S. Pat. No. 8,034,843 GW Pharma experiments on Shrews (Anti-emetic) (b) Mechoulam, et al., 2007, Chemistry & Biodiversity 4 (8): 1678-1692. 6 Pain-Brachial THC (a) US 20060135599 GW Pharma plexus avulsion THC:CBD 7 Pain and CBD:THC (a) US2008O139667 inflammation- (b) Mechoulam, et al., 2007, Chemistry & Biodiversity 4 (8): 1678-1692. Arthritis 8 Anti Cancer-cell CBD:THC (a) US20080262099 movement CBD (b) Mechoulam, et al., 2007, Chemistry & Biodiversity 4 (8): 1678-1692. (c) McAllister et al., 2007, Moi. Cancer Ther: 6 (11): 2921-7. Anti Convulsant CBDV (a) US20120004251 (against seizures) CBD (b) US20120165402 (d) Mechoulam, et al., 2007, Chemistry & Biodiversity 4 (8): 1678-1692. US 9,370,164 B2 39 40 TABLE 1-continued Non-limiting list of medical uses for cannabinoids.
MEDICALUSES CANNABINOID REFERENCES (a) Carlini et al., J. Clin. Pharmacol. 21:417S-427S, 1981 (b) Karler et al., J. Clin. Pharmacol. 21: 437S-448S, 1981 (c) Consroe et al., J. Clin Pharmacol. 21:428S-436S, 1981 10 Neurological Pain THC:CBD (a) US20100035978 (MS related) 11 Weight loss THCV (b) US20090306221 (c) US20080119544 12 Anti-Depressant CBG (a) US20080031977 (b) US 60/813,814 13 Irritable Bowel THC:CBD (c) EP 1361864 Syndrome (d) EP 1542657 (Crohns) (e) US2010O286098 14 Type II diabetes THCV:CBD (a) US20110082195 (b) 15 Anti-Psychotic THCV:CBD (c) US20110038958 (d) Zuardi et al., 2006, Braz. J. Med. Bioi...Res. 39 (4): 421-429. 16 Cancer Pain THC:CBD (e) US20110230549 17 Anxiety Reduction CBD (a) Mechoulam, et al., 2007, Chemistry & Biodiversity 4 (8): 1678-1692. (b) Bergamaschi et al., 2003, Neuropsychopharmacology36 (6): 1219-1226
Terpenes and Terpenoids in Cannabis Plants (DMAPP) and isopentenyl pyrophosphate (IPP) via the Terpenes are a large and diverse class of organic com catalysis of GPP synthase. Alternatively, DMAPP and IPP are pounds, produced by a variety of plants. They are often strong 25 ligated by FPP synthase to produce farnesyl pyrophosphate Smelling and thus may have had a protective function. Terpe (FPP), which can be used to produce sesquiterpenoids. Gera nes are derived biosynthetically from units ofisoprene, which nyl pyrophosphate (GPP) can also be converted into monot has the molecular formula CHs. The basic molecular formu erpenoids by limonene synthase. lae of terpenes are multiples of that, (CH), where n is the 30 In addition to cannabinoids, Cannabis also produces over number of linked isoprene units. The isoprene units may be 120 different terpenes (Russo 2011, Taming THC: potential linked together "head to tail” to form linear chains or they Cannabis synergy and phytocannabinoid-terpenoid entou may be arranged to form rings. Non-limiting examples of rage effects, British Journal of Pharmacology, 163: 1344 terpenes include Hemiterpenes, Monoterpenes, Sesquiterpe 1364). Within the context and verbiage of this document the nes, Diterpenes, Sesterterpenes, Triterpenes, Sesquarterpe 35 terms terpenoid and terpene are used interchangeably. nes, Tetraterpenes, Polyterpenes, and Norisoprenoids. Cannabinoids are odorless, so terpenoids are responsible for Terpenoids, a.k.a. isoprenoids, are a large and diverse class the unique odor of Cannabis, and each variety has a slightly of naturally occurring organic chemicals similar to terpenes, different profile that can potentially be used as a tool for derived from five-carbon isoprene units assembled and modi identification of different varieties or geographical origins of fied in thousands of ways. Most are multicyclic structures that 40 samples (Hillig 2004. A chemotaxonomic analysis of terpe differ from one another not only in functional groups but also noid variation in Cannabis' Biochem System and Ecology in their basic carbon skeletons. Plant terpenoids are used 875-891). It also provides a unique and complex organoleptic extensively for their aromatic qualities. They play a role in profile for each variety that is appreciated by both novice traditional herbal remedies and are under investigation for users and connoisseurs. In addition to many circulatory and antibacterial, antineoplastic, and other pharmaceutical func 45 muscular effects, some terpenes interact with neurological tions. The terpene Linalool for example, has been found to receptors. A few terpenes produced by Cannabis plants also have anti-convulsant properties (Elisabetsky et al., Phy bind weakly to Cannabinoid receptors. Some terpenes can tomedicine, May 6(2):107-13 1999). Well-known terpenoids alter the permeability of cell membranes and allow in either include citral, menthol, camphor, Salvinorin A in the plant more or less THC, while other terpenes can affect serotonin Salvia divinorum, and the cannabinoids found in Cannabis. 50 and dopamine chemistry as neurotransmitters. Terpenoids are Non-limiting examples of terpenoids include, Hemiterpe lipophilic, and can interact with lipid membranes, ion chan noids, 1 isoprene unit (5 carbons); Monoterpenoids, 2 iso nels, a variety of different receptors (including both G-protein prene units (10C); Sesquiterpenoids, 3 isoprene units (15C); coupled odorant and neurotransmitter receptors), and Diterpenoids, 4 isoprene units (20C) (e.g. ginkgolides); Ses enzymes. Some are capable of absorption through human terterpenoids, 5 isoprene units (25C); Triterpenoids, 6 iso 55 skin and passing the blood brain barrier. prene units (30C) (e.g. sterols); Tetraterpenoids, 8 isoprene Generally speaking, terpenes are considered to be pharma units (40C) (e.g. carotenoids); and Polyterpenoid with a cologically relevant when present in concentrations of at least larger number of isoprene units. 0.05% in plant material (Hazekamp and Fischedick 2010. Terpenoids are mainly synthesized in two metabolic path "Metabolic fingerprinting of Cannabis sativa L., cannab ways: mevalonic acid pathway (a.k.a. HMG-CoA reductase 60 inoids and terpenoids for chemotaxonomic and drug stan pathway, which takes place in the cytosol) and MEP/DOXP dardization purposes’ Phytochemistry 2058-73; Russo 2011, pathway (a.k.a. The 2-C-methyl-D-erythritol 4-phosphate/1- Taming THC: potential Cannabis Synergy and phytocannab deoxy-D-xylulose 5-phosphate pathway, non-mevalonate inoid-terpenoid entourage effects, British Journal of Phar pathway, or mevalonic acid-independent pathway, which macology, 163: 1344-1364). Thus, although there are an esti takes place in plastids). Geranyl pyrophosphate (GPP), which 65 mated 120 different terpenes, only a few are produced at high is used by Cannabis plants to produce cannabinoids, is enough levels to be detectable, and fewer still which are able formed by condensation of dimethylallyl pyrophosphate to reach pharmacologically relevant levels. US 9,370,164 B2 41 42 For the purposes of this application, Cannabis terpene D-Linalool is a monoterpenoid with very well-known profile will be defined as the absolute and relative values of 17 anxiolytic effects. It is often associated with lavender, and of the most expressed terpenes: terpinolene, alpha phella frequented used in aromatherapy for its sedative impact. It drene, beta ocimene, carene, limonene, gamma terpinene, 5 acts as a local anaesthetic and helps to prevent Scarring from alpha pinene, alpha terpinene, beta pinene, fenchol, cam burns, is anti-nociceptive in mice, and shows antiglutamater phene, alpha terpineol, alpha humulene, beta caryophyllene, linalool, cary oxide, and myrcene. A Survey of the terpene gic and anticonvulsant activity. Its effects on glutamate and profiles of several Cannabis varieties has found that these GABA neurotransmitter systems are credited with giving it its sedative, anxiolytic, and anticonvulsant activities (Russo terpenes express at high enough levels so as to have their own 10 pharmacological effects and also to act in Synergy with can 2011, Taming THC: potential Cannabis synergy and phyto nabinoids. Both experts and consumers believe that there are cannabinoid-terpenoid entourage effects, British Journal of biochemical and phenomenological differences between dif Pharmacology, 163:1344-1364). ferent varieties of Cannabis, which are attributed to their C.-Pinene is a monoterpene common in nature, also with a unique relative cannabinoid and terpenoid ratios. This is 15 plethora of effects on mammals and humans. It acts as an known as the entourage effect and is generally considered to acetylcholinesterase inhibitor which aids memory and coun result in plants providing advantages over only using the teracts the short-term memory loss associated with A9-THC natural products that are isolated from them (Russo 2011, Taming THC: potential Cannabis Synergy and phytocannab intoxication, is an effective antibiotic agent, and shows some inoid-terpenoid entourage effects, British Journal of Phar activity against MRSA. In addition, C-pinene is a bronchodi macology, 163:1344-1364). lator in humans and has anti-inflammatory properties via the These advantages include synergy with THC, the primary prostaglandin E-1 pathway (Russo 2011, Taming THC: active ingredient, and also mitigation of side effects from potential Cannabis Synergy and phytocannabinoid-terpenoid THC (McPartland and Russo 2001 "Cannabis and Cannabis 25 entourage effects, British Journal of Pharmacology, 163: Extracts: Greater Than the Sum of Their Parts?” Hayworth 1344-1364). Press). Terpenoids can be extracted from the plant material by B-Caryophyllene is often the most predominant sesquiter steam distillation (giving you essential oil) or vaporization, penoid in Cannabis. It is less Volatile than the monoterpe however the yield varies greatly by plant tissue, type of 30 noids, thus it is found in higher concentrations in material that extraction, age of material, and other variables (McPartland has been processed by heat to aid in decarboxylation. It is very and Russo 2001 "Cannabis and Cannabis Extracts: Greater interesting in that it is a selective full agonist at the CB2 Than the Sum of Their Parts?” Hayworth Press). Typically the receptor, which makes it the only phytocannabinoid found yield of terpenoids in Cannabis is less than 1% by weight on analysis; however it is thought that they may comprise up to 35 outside the Cannabis genus. In addition, it has anti-inflam 10% of the trichome content. Monoterpenoids are especially matory and gastric cytoprotective properties, and may even Volatile, thus decreasing their yield relative to sesquiterpe have anti-malarial activity. noids (Russo 2011, Taming THC: potential Cannabis synergy Caryophylene oxide is another sesquiterpenoid found in and phytocannabinoid-terpenoid entourage effects, British Cannabis, which has antifungal and anti-platelet aggregation Journal of Pharmacology, 163: 1344-1364). 40 properties. As an aside, it is also the molecule that drug D-Limonene is a monoterpenoid that is widely distributed sniffing dogs are trained to find (Russo 2011, Taming THC: in nature and often associated with citrus. It has strong anxi potential Cannabis Synergy and phytocannabinoid-terpenoid olytic properties in both mice and humans, apparently entourage effects, British Journal of Pharmacology, 163: increasing serotonin and dopamine in mouse brain. D-li 45 1344-1364). monene has potent anti-depressant activity when inhaled. It is Nerolidol is a sesquiterpene that is often found in citrus also under investigation for a variety of different cancer treat peels that exhibits a range of interesting properties. It acts as ments, with some focus on its hepatic metabolite, perillic a sedative, inhibits fungal growth, and has potent anti-ma acid. There is evidence for activity in the treatment of der 50 larial and antileishmanial activity. It also alleviated colon matophytes and gastro-oesophageal reflux, as well as having adenomas in rats (Russo 2011, Taming THC: potential Can general radical scavenging properties (Russo 2011, Taming nabis synergy and phytocannabinoid-terpenoid entourage THC: potential Cannabis synergy and phytocannabinoid-ter effects, British Journal of Pharmacology, 163:1344-1364). penoid entourage effects, British Journal of Pharmacology, Phytol is a diterpene often found in Cannabis extracts. It is a 163: 1344-1364). 55 B-Myrcene is a monoterpenoid also found in Cannabis, degradation product of chlorophyll and tocopherol. It and has a variety of pharmacological effects. It is often asso increases GABA expression and therefore could be respon ciated with a Sweet fruit like taste. It reduces inflammation, sible the relaxing effects of green tea and wild lettuce. It also aids sleep, and blockShepatic carcinogenesis, as well as act prevents vitamin-A induced teratogenesis by blocking the ing as an analgesic and muscle relaxant in mice. When 60 conversion of retinol to its dangerous metabolite, all-trans B-myrcene is combined with A9-THC it could intensify the retinoic acid (Russo 2011, Taming THC: potential Cannabis sedative effects of A9-THC, causing the well-known "couch synergy and phytocannabinoid-terpenoid entourage effects, lock' effect that some Cannabis users experience (Russo British Journal of Pharmacology, 163: 1344-1364). 2011, Taming THC: potential Cannabis synergy and phyto 65 Some of the most commonly found terpenoids in Cannabis cannabinoid-terpenoid entourage effects, British Journal of are summarized in Table 2, with their individual organoleptic Pharmacology, 163:1344-1364). properties as well as their basic pharmacology. US 9,370,164 B2 44 TABLE 2 A non-limiting list of the medical effects of Some of the most common terpenes found in Cannabis Terpenoid Odor Description Flavor Description Suggested Pharmacology -pinene Herbal, piney Woody, piney, Anti-inflammatory, camphoraceous bronchodilator, stimulant camphene Woody, piney Camphoraceous, Reduces plasma cholesterol and cooling, minty triglycerides, Antioxidant and free radical scavenger '-pinene Herbal, cooling, piney Fresh, piney, woody Strong antimicrobial myrcene Spicy, herbaceous Woody, vegetative, Anti-inflammatory, sedative, citrus antibiotic, analgesic '-phellandrene Terpenic, citrus Terpenic, citrus, lime Antinociceptive C8tle Citrus, Sweet None given CNS depressant, anti-inflamatory -terpinene Woody, citrus, medicinal Terpenic, woody, piney Antioxidant limonene Citrus, fresh Sweet, orange, citrus Anxiolytic, antidepressant, immunostimulant '-ocimene Floral, green Green, tropical, woody Possible anti-bacterial 8-terpinene Terpenic, woody Terpenic, citrus, lime Antioxidant like terpinolene Herbal, woody Sweet, fresh, piney, Comforting, calming, anti-oxidant, citrus antifungal linalool Floral, citrus Citrus, orange, lemon, Sedative, anxiolytic, floral immunostimulant fenchol Camphor, piney Fresh, piney Possible stimulant -terpineol Floral, piney None given Sedative, AChE inhibitor, antioxidant -caryophyllene Spicy, woody Spicy, clove, rosemary Selective agonist of CB2 receptor, anti-inflammatory, antimalarial -humulene Woody None given An i-inflammatory caryophyllene Woody, Sweet None given An ifungal, stimulant oxide
30 Cannabis Plants of flowering in Cannabis is the appearance of undifferenti Cannabis is an annual, dioecious, flowering herb. The ated flower primordial along the main stem of the nodes. At leaves are palmately compound or digitate, with Serrate leaf this stage, the sex of the plants are still not distinguishable. As lets. Cannabis normally has imperfect flowers, with stami the flower primordia continue to develop, female (pistillate), nate “male' and pistillate “female flowers occurring on 35 and male (staminate) flowers can be distinguished. separate plants. It is not unusual, however, for individual For most cannabinoid producing purposes, only female plants to separately bear both male and female flowers (i.e., plants are desired. The presence of male flowers is considered have monoecious plants). Although monoecious plants are undesirable as pollination is known to reduce the cannabinoid often referred to as “hermaphrodites, true hermaphrodites yield, and potentially ruin a crop. For this reason, most Can (which are less common in Cannabis) bear staminate and 40 nabis is grown “sinsemilla' through vegetative (i.e., asexual) pistillate structures on individual flowers, whereas monoe propagation. In this way, only female plants are produced and cious plants bear male and female flowers at different loca no space is wasted on male plants. tions on the same plant. In breeding new varieties of Cannabis, there are many The life cycle of Cannabis varies with each variety but can phenotypic and morphological characteristics one must con be generally Summarized into germination, vegetative 45 sider. For example, plants should produce high amounts of growth, and reproductive stages. Because of heavy breeding cannabinoids. Cannabinoid levels can be measured via and selection by humans, most Cannabis seeds have lost chemical analysis of mature plants, but can also be estimated dormancy mechanisms and do not require any pre-treatments in the field by the number and size of the trichomes produced or winterization to induce germination (See Clarke, RC et al. by a plants flower clusters. Plants with dense trichome pat “Cannabis: Evolution and Ethnobotany’ University of Cali 50 terns are said to be "frosty', and selected for further breeding. fornia Press 2013). Seeds placed in viable growth conditions The types of cannabinoids can also be determined in the field are expected to germinate in about 3 to 7 days. The first true via thin layer chromatography (TLC) analysis (see "Can leaves of a Cannabis plant contain a single leaflet, with Sub nabis Inflorescence & Leaf QC from The American Herbal sequent leaves developing in opposite formation, with Pharmacopeia 2013). The absolute cannabinoid and terpene increasing number of leafletts. Leaflets can be narrow or 55 contents are calculated based on weight of cannabinoid or broad depending on the morphology of the plant grown. Can terpene present in a sample divided by the dried weight of the nabis plants are normally allowed to grow vegetatively for the dried trimmed inflorescence. Dried inflorescences are har first 4 to 8 weeks. During this period, the plant responds to vested inflorescence tissue dried to ~10% moisture level. The increasing light with faster and faster growth. Under ideal terp trimmed inflorescence as used herein refers to inflores conditions, Cannabis plants can grow up to 2.5 inches a day, 60 cences with Sun leaves cut off such that only the calyx and and are capable of reaching heights of up to 20 feet. Indoor reproductive buds remain. Frosty leaves are left on the inflo growth pruning techniques tend to limit Cannabis size rescence. Trimming can be performed manually, through through careful pruning of apical or side shoots. careful manicuring of harvested tissue, or via automated Although, some Cannabis varieties will flower without the mechanical methods. need for external stimuli, most varieties have an absolute 65 Another important aspect of Cannabis breeding is the ter requirement for inductive photoperiods in the form of short pene profile of a plant. In some embodiments, the present days or long nights to induce fertile flowering. The first sign invention teaches the preference for Cannabis plant material US 9,370,164 B2 45 46 with diverse terpene profiles which are not dominated by 6.2%, 6.4%, 6.6%, 6.8%, 7%, 7.2%, 7.4%, 7.6%, 7.8%, or 8% myrcene. In other embodiments, the present invention based on dry weight of inflorescence. Thus in some embodi teaches Cannabis plants with high terpene essential oil con ments the absolute content of any one of myrcene is between tents. For the purposes of this application, a Cannabis plants about 0.05% and about 0.85%. terpene profile is defined in absolute or relative contents of 17 5 In some embodiments the terpene content of the specialty key terpenes including: terpinolene, alpha phelladrene, beta Cannabis of the present invention is described in relative ocimene, carene, limonene, gammaterpinene, alpha pinene, terms as a % composition of the total terpene profile. Thus for alpha terpinene, beta pinene, fenchol, camphene, alpha terpi example a specialty Cannabis with 1.2% absolute terpinolene neol, alpha humulene, beta caryophyllene, linalool, cary content and 1.2% myrcene content and no other terpenes oxide, and myrcene. A myrcene dominant terpene is used to 10 would said to have 50% terpinolene and 50% myrcene rela refer to terpene profiles in which myrcene is the most abun tive content. In some embodiments, the specialty Cannabis of dant terpene in the terpene profile (i.e., myrcene relative or the present invention has a relative content of any one of the absolute content is content of any single one of the 16 other 17 terpenes in the terpene profile that is greater than or less terpenes in the terpene profile). Terpene essential oil contents than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, are measured by adding the absolute contents by weight of the 15 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 17 terpenes from the terpene profile as defined above. The 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, absolute terpene content is measured as w/w % value based 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, on dry inflorescences. The present invention is based in part 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, on the discovery that consumers prefer specialty Cannabis 52%. 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, varieties with diverse terpene profiles not dominated by 20 62%, 63%, 64%. 65%, 66%, 67%, 68%, 69%, 70%, 71%, myrcene and with high terpene essential oil contents. 72%, 73%, 74%, 75%, 76%, 77%, 79%, 79%, 80%, 81%, In some embodiments, the specialty Cannabis of the 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, present invention has greater than 0.1%, 0.2%, 0.3%, 0.4%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Thus 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, in some embodiments the relative content of any one of the 2%, 2.2%, 2.4%, 2.6%, 2.8%, 3%, 3.2%, 3.4%, 3.6%, 3.8%, 25 terpenes is between 0% and 100%. 4%, 4.2%, 4.3%, 4.4%, 4.6%, 4.8%, 5%, 5.2%, 5.4%, 5.6%, In Some embodiments, the specialty Cannabis of the 5.8%, 6%, 6.2%, 6.4%, 6.6%, 6.8%, 7%, 7.2%, 7.4%, 7.6%, present invention has a relative myrcene content of less than 7.8%, or 8% terpene essential oil content by dry weight. Thus 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, in some embodiments the essential oil content of the specialty 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, Cannabis varieties of the present invention is between about 30 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 0.5% and about 8% by dry weight. In other embodiments the 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, essential oil contents of the specialty Cannabis varieties of 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, the present invention is between about 1.5% and about 5% by 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, dry weight. 63%, 64%. 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, In some embodiments, the specialty Cannabis of the 35 73%, 74%, 75%, 76%, 77%, 79%, 79%, 80%, 81%, 82%, present invention has an absolute content of any one of the 17 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, terpenes in the terpene profile that is 0%, 0.01%, 0.02%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Thus in 0.04%, 0.06%, 0.08%, 0.1%, 0.12%, 0.14%, 0.16%, 0.18%, Some embodiments the specialty Cannabis of the present 0.2%, 0.22%, 0.24%, 0.26%, 0.28%, 0.3%, 0.32%, 0.34%, invention has less than 60% relative myrcene content. 0.36%, 0.38%, 0.4%, 0.42%, 0.44%, 0.46%, 0.48%, 0.5%, 40 Another important breeding phenotype is flower color. The 0.52%, 0.54%, 0.56%, 0.58%, 0.6%, 0.62%, 0.64%, 0.66%, accumulation of anthocyanins, carotenoids, or other color 0.68%, 0.7%, 0.72%, 0.74%, 0.76%, 0.78%, 0.8%, 0.82%, producing compounds in leaves and flowers of Cannabis can 0.84%, 0.86%, 0.88%, 0.9%, 0.92%, 0.94%, 0.96%, 0.98%, have an effect on consumer visual appeal and flavor. Iconic 1%, 1.02%, 1.04%, 1.06%, 1.08%, 1.10%, 1.12%, 1.14%, examples of the appeal of color are the popular “Purple 1.16%, 1.18%, 1.2%, 1.22%, 1.24%, 1.26%, 1.28%, 1.3%, 45 Kush”, “Purple Haze', and “PurpleTrainwreck” varieties that 1.32%, 1.34%, 1.36%, 1.38%, 1.4%, 1.42%, 1.44%, 1.46%, express anthocyanins in their late maturation stages to pro 1.48%, 1.5%, 1.6%, 1.7% 1.8%, 1.9%, 2%, 2.2%, 2.4%, duce dark purple leaves. Color selections can also be based on 2.6%, 2.8%, 3%, 3.2%, 3.4%, 3.6%, 3.8%, 4%, 4.2%, 4.3%, (but not limited to) unique coloration of stem, leaf inflores 4.4%, 4.6%, 4.8%, 5%, 5.2%, 5.4%, 5.6%, 5.8%, 6%, 6.2%, cence, calyx, stamen, trichome bodies and finished products 6.4%, 6.6%, 6.8%, 7%, 7.2%, 7.4%, 7.6%, 7.8%, 8%, or 50 including extracts and hash. greater based on dry weight of inflorescence. Thus in some Yield is another important factor in breeding. Cannabis embodiments the absolute content of any one of the terpenes yield is measured by pounds (lbs), grams (g) or kilograms is between about 0.05% and about 0.85%. (Kg) of dried (10% moisture), and trimmed flowers. Yield can In some embodiments, the specialty Cannabis of the be expressed interms of yield perplant, yield per watt of light, present invention has a myrcene absolute content of less than 55 and yield per squared meter of growing area among others. 0.02%, 0.04%, 0.06%, 0.08%, 0.1%, 0.12%, 0.14%, 0.16%, Cannabis yield is also dependent on the growing environ 0.18%, 0.2%, 0.22%, 0.24%, 0.26%, 0.28%, 0.3%, 0.32%, ment. For example yields for a particular Cannabis strain will 0.34%, 0.36%, 0.38%, 0.4%, 0.42%, 0.44%, 0.46%, 0.48%, vary between outdoor growth long season, outdoor growth 0.5%, 0.52%, 0.54%, 0.56%, 0.58%, 0.6%, 0.62%, 0.64%, short season, or indoor growth. Yield may also be affected by 0.66%, 0.68%, 0.7%, 0.72%, 0.74%, 0.76%, 0.78%, 0.8%, 60 growing conditions such as type of lighting, Soil, fertilizer 0.82%, 0.84%, 0.86%, 0.88%, 0.9%, 0.92%, 0.94%, 0.96%, use, size of growing pot, etc. 0.98%, 1%, 1.02%, 1.04%, 1.06%, 1.08%, 1.10%, 1.12%, In Some embodiments, the specialty Cannabis of the 1.14%, 1.16%, 1.18%, 1.2%, 1.22%, 1.24%, 1.26%, 1.28%, present invention produces, 0.1 g, 0.2g, 0.3g, 0.4g, 0.5g, 0.6 1.3%, 1.32%, 1.34%, 1.36%, 1.38%, 1.4%, 1.42%, 1.44%, g, 0.7g, 0.8 g. 0.9 g, 1.0g, 1.1 g, 1.2g, 1.3g, 1.4g, 1.5g, 1.6 1.46%, 1.48%, 1.5%, 1.6%, 1.7% 1.8%, 1.9%, 2%, 2.2%. 65 g, 1.7g, 1.8g, 1.9 g, 2.0g, 2.1 g, 2.2g, 2.3 g, 2.4g, 2.5g, 2.6 2.4%, 2.6%, 2.8%, 3%, 3.2%, 3.4%, 3.6%, 3.8%, 4%, 4.2%, g, 2.7g, 2.8 g., 2.9 g, 3.0 g, 3.1 g, 3.2g, 3.3g, 3.4g, 3.5g, 3.6 4.3%, 4.4%, 4.6%, 4.8%, 5%, 5.2%, 5.4%, 5.6%, 5.8%, 6%, g, 3.7g, 3.8 g., 3.9 g, 4.0 g. 4.1 g, 4.2g, 4.3g, 4.4g, 4.5g, 4.6 US 9,370,164 B2 47 48 g, 4.7g, 4.8 g., 4.9 g, or 5.0 g of dried flowers per watt of light. breeding, Wiley-Blackwell, 2007, ISBN 1405136464, In some embodiments, the specialty Cannabis of the present 9781405136464, which is hereinincorporated by reference in invention produces 10g, 15g, 20g, 25 g, 30 g, 35 g, 40 g, 45 its entity). g, 50 g, 55 g. 60 g. 65g, 70 g, 75 g, 80 g, 85 g, 90 g, 95 g, 100 The present invention also relates to a mutagenized popu g, 105 g, 110 g, 115 g, 120g, 125 g, 130 g, 135g. 140 g, 145 lation of the Cannabis plants of the present invention, and g, 150 g. 155 g, 160 g, 165 g, 170 g, 175 g, 180g, 185 g, 190 methods of using Such populations. In some embodiments, g, 195g, 200g, 210g, 220g, 230g, 240g, 250g, 260g, 270 the mutagenized population can be used in Screening for new g, 280g, 290 g, 300 g, 310 g, 320 g, 330 g., 340 g, 350 g., 360 Cannabis lines which comprises one or more or all of the g, 370 g. 380 g. 390 g, 400 g, 410 g, 420 g, 430 g., 440 g, 450 morphological, physiological, biological, and/or chemical 10 characteristics of Cannabis plants of the present invention. In g, 460 g. 470 g. 480 g, 490 g, 500g, 550 g. 600 g. 650 g. 700 Some embodiments, the new Cannabis plants obtained from g, 750 g., 800 g, 850 g,900 g,950 g, 1000 g, 2000 g, 3000 g, the screening process comprise one or more or all of the or 5000 g of dried flowers per plant. morphological, physiological, biological, and/or chemical Desirable yield phenotypes include: characteristics of Cannabis plants of the present invention, High Yield Natural Light Production Long Season Se 15 and one or more additional or different new morphological, lection based on yield performance for early ripening variet physiological, biological, and/or chemical characteristic. ies during long seasons. The mutagenized population of the present invention can High Yield Natural Light Production Short Season Se be used in Targeting Induced Local Lesions in Genomes lection based on yield performance of late ripening varieties (TILLING) screening method, which combines a standard during long season and/or yield of plants that ripen in winter and efficient technique of mutagenesis with a chemical months and at low light levels. mutagen (e.g., Ethyl methanesulfonate (EMS)) with a sensi High Yield Indoor Production Selection based solely on tive DNA screening-technique that identifies single base plant yield performance in artificial lighting (e.g., HID). mutations (also called point mutations) in a target gene. Another important phenotype that is important for Cannabis Detailed description on methods and compositions on TILL production is structural features for easy harvesting. 25 ING(R) can be found in Till et al. (Discovery of induced point Other important breeding phenotypes include: mutations in maize genes by TILLING, BMC Plant Biology Structure for Manual Trim/Market—Selections are based 2004, 4:12), Weil et al., (TILLING in Grass Species, Plant on the relative ratio by weight of finished flower. This usually Physiology January 2009 vol. 149 no. 1158-164), Comai, L. is directly related to dense trichome morphology with very and S. Henikoff (“TILLING: practical single-nucleotide few sun leaves. 30 mutation discovery.” Plant J 45(4): 684-94), McCallum et al., Structure for Automated Trimming Selection based on (Nature Biotechnology, 18:455-457, 2000), McCallum et al., flower morphology that is more kola (continuous long bud) (Plant Physiology, 123: 439-442, 2000), Colbert et al., (Plant with many Sun leaves protruding from large inflorescences. Physiol. 126(2): 480–484, 2001), U.S. Pat. No. 5,994,075, Overall flower size is typically large, but trichomes are less U.S. Patent Application Publication No. 2004/0053236A1, densely packed and overall inflorescence is less dense than 35 and International Patent Application Publication Nos. WO what is traditionally selected for manual trim. 2005/055704 and WO 2005/048692, each of which is hereby Root Structure Positive root selection is marked by over incorporated by reference for all purposes. all root vigor and adventitious root growth, ease of transplant, The present invention also provides any compositions or rate of root development on clonal propagations, and root any products made from or isolated from the plants of the shooting from tissue culture samples. Root selections can also 40 present invention. In some embodiments, the compositions/ be based on resistance to soil and hydroponic pathogens products comprises extract of the plants, wherein the extract including pythium. contains more than 2% CBD and less than 98% THC. In some Vigor Selection for plant vigor are marked by tremen embodiments, the extract contains higher percentage ofter dous grow rates and robust stem/stalk infrastructure. Often penes/terpenoids compared to extract isolated from a control times, selection display morphologies that are very much 45 Cannabis plant variety (e.g., an existing variety, Such as a enlarged compared to sibling progeny. recreational Cannabis plant variety). Fungal Resistance—Selections based on plant that exhibit Methods of Using Cannabis Plants immunity or partial immunity to fungal diseases and patho The present invention provides methods of using the Can gens including but not limited to powdery mildew, botrytis, nabis plants or any parts, any compositions, or any chemicals downy mildew among others. 50 derived from said plants of the present invention. For a non-limiting list of cannabinoid phenotypes, please In some embodiments, the plants can be used for medical See Marijuana Botany, An Advanced study: The Propagation purpose. In other embodiments, the specialty Cannabis plants and Breeding of Distinctive Cannabis by Robert Connell of the present invention can be used for recreational purposes. Clarke. In some embodiments, the plants can be used by patients The present invention also relates to variants, mutants and 55 having a disease. In some embodiments, the diseases modifications of the seeds, plant parts and/or whole plants of includes, but are not limited to. Acquired Hypothyroidism, the Cannabis plants of the present invention. Variants, Acute Gastritis, Agoraphobia, AIDS Related Illness, Alcohol mutants and trivial modifications of the seeds, plants, plant Abuse, Alcoholism, Alopecia Areata, Alzheimer's Disease, parts, plant cells of the present invention can be generated by Amphetamine Dependency, Amyloidosis, Amyotrophic Lat methods well known and available to one skilled in the art, 60 eral Sclerosis (ALS), Angina Pectoris, Ankylosis, Anorexia, including but not limited to, mutagenesis (e.g., chemical Anorexia Nervosa, Anxiety Disorders, any chronic medical mutagenesis, radiation mutagenesis, transposon mutagen symptom that limits major life activities, any Chronic Medi esis, insertional mutagenesis, signature tagged mutagenesis, cal Symptom that Limits Major Life Activities, Arterioscle site-directed mutagenesis, and natural mutagenesis), knock rotic Heart Disease, Arthritis, Arthritis (Rheumatoid), Arthr outs/knock-ins, antisense and RNA interference. For more 65 opathy, gout, Asthma, Attention Deficit Hyperactivity information of mutagenesis in plants, such as agents, proto Disorder (ADD/ADHD), Autism/Asperger's, Autoimmune cols, see Acquaah et al. (Principles of plant genetics and Disease, Back Pain, Back Sprain, Bell's Palsy, Bipolar Dis US 9,370,164 B2 49 50 order, Brain Tumor, Malignant, Bruxism, Bulimia, Cachexia, Some embodiments, the specialty Cannabis of the present Cancer, Carpal Tunnel Syndrome, Cerebral Palsy, Cervical invention can reduce the effect of Cannabis use on fetal brain Disk Disease, Cervicobrachial Syndrome, Chemotherapy development by providing CBS and terpenes which attenuate Chronic Fatigue Syndrome, Chronic Pain, Chronic renal fail the activation of CB1 receptor by THC (Tortoriello et al., ure, Cocaine Dependence, Colitis, Conjunctivitis, Constipa 2013 “Miswiring the brain: delta 9 tetrahydrocananbinol dis tion, Crohn's Disease, Cystic Fibrosis, Damage to Spinal rupts cortical development by inducing an SCG10/stathmin-2 Cord Nervous Tissue, Darier's Disease, Degenerative Arthri degradation pathway” EMBO 10 Dec. 2013). In some tis, Degenerative Arthropathy, Delirium Tremens, Dermato embodiments, the traditional recreational marijuana plant myositis, Diabetes, Diabetic Neuropathy, Diabetic Peripheral variety is the variety White Widow. In some embodiments, Vascular Disease, Diarrhea, Diverticulitis, Dysthymic Disor 10 der, Eczema, Emphysema, Emphysema, Endometriosis, Epi the traditional recreational marijuana plant variety contains at dermolysis Bullosa, Epididymitis, Epilepsy, Felty’s Syn least 98%, at least 98.5%, at least 99%, at least 99.5%, at least drome, Fibromyalgia, Friedreich's Ataxia, Gastritis, Genital 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% Herpes, Glaucoma, Glioblastoma Multiforme, Graves Dis THC in the cannabinoid accumulation in the plant. ease, Cluster Headaches, Migraine Headaches, Tension 15 In some embodiments, the plants can be used for non Headaches, Hemophilia A, Henoch-Schonlein Purpura, medical purposes. In some embodiments the specialty Can Hepatitis C, Hereditary Spinal Ataxia, HIV/AIDS, Hospice nabis plants of the present invention can be used for recre Patients, Huntington's Disease, Hypertension, Hypertension, ational purposes. In some embodiments, the specialty Hyperventilation, Hypoglycemia, Impotence, Inflammatory Cannabis plants of the present invention can be used for autoimmune-mediated arthritis, Inflammatory Bowel Dis industrial purposes. In some embodiments, the plants are ease (IBD). Insomnia, Intermittent Explosive Disorder (IED), used for producing food, oil, wax, resin, rope, cloth, pulp. Intractable Pain, Intractable Vomiting, Lipomatosis, Lou fiber, feed for livestock, construction material, plastic and Gehrig's Disease, Lyme Disease, Lymphoma, Major Depres composite materials, paper, jewelry, water and soil purifica Sion, Malignant Melanoma, Mania, Melorheostosis, tion materials, weed control materials, cultivation materials, Meniere's Disease, Motion Sickness, Mucopolysaccharido 25 textiles, clothing, biodegradable plastics, body products, sis (MPS), Multiple Sclerosis (MS), Muscle Spasms, Muscu health food and biofuel. lar Dystrophy, Myeloid Leukemia, Nail-Patella Syndrome, Cannabis Breeding Methods Nightmares, Obesity, Obsessive Compulsive Disorder, Opi In some embodiments, the plants of the present invention ate Dependence, Osteoarthritis, Panic Disorder, Parkinson's can be used to produce new plant varieties. In some embodi Disease, Peripheral Neuropathy, Peritoneal Pain, Persistent 30 ments, the plants are used to develop new, unique and Superior Insomnia, Porphyria, Post Polio Syndrome (PPS), Post-trau varieties or hybrids with desired phenotypes. matic arthritis, Post-Traumatic Stress Disorder (PTSD), Pre menstrual Syndrome (PMS), Prostatitis, Psoriasis, Pulmo In some embodiments, selection methods, e.g., molecular nary Fibrosis, Quadriplegia, Radiation Therapy, Raynaud's marker assisted selection, can be combined with breeding Disease, Reiter's Syndrome, Restless Legs Syndrome (RLS). 35 methods to accelerate the process. Additional breeding meth Rheumatoid Arthritis, Rheumatoid Arthritis, Rheumatoid ods have been known to one of ordinary skill in the art, e.g., Arthritis, Rosacea, Schizoaffective Disorder, Schizophrenia, methods discussed in Chahal and Gosal (Principles and pro Scoliosis, Sedative Dependence, Seizures, Senile Dementia, cedures of plant breeding: biotechnological and conventional Severe Nausea, Shingles (Herpes Zoster), Sinusitis, Skeletal approaches, CRC Press, 2002, ISBN 084931321X, Muscular Spasticity, Sleep Apnea, Sleep Disorders, Spastic 40 9780849313219), Taji et al. (In vitro plant breeding, Rout ity, Spinal Stenosis, Sturge-Weber Syndrome (SWS), Stutter ledge, 2002, ISBN 156022908X, 9781560229087), Richards ing, Tardive Dyskinesia (TD), Temporomandibular joint dis (Plant breeding systems, Taylor & Francis US, 1997, ISBN order (TMJ), Tenosynovitis, Terminal Illness, Thyroiditis, 0412574500, 9780412574504), Hayes (Methods of Plant Tic Douloureux, Tietze's Syndrome, Tinnitus, Tobacco Breeding, Publisher: READ BOOKS, 2007, Dependence, Tourette's Syndrome, Trichotillomania, Viral 45 ISBN 1406737062, 9781406737066), each of which is incor Hepatitis, Wasting Syndrome. Whiplash, Wittmaack-Ek porated by reference in its entirety for all purposes. Cannabis bom's Syndrome, Writers' Cramp, nausea, vomiting, pre genome has been sequenced recently (Bakel et al., The draft menstrual syndrome, unintentional weight loss, insomnia, genome and transcriptome of Cannabis sativa, Genome Biol and lack of appetite, spasticity, painful conditions, especially ogy, 12(10):R102, 2011). Molecular makers for Cannabis neurogenic pain, movement disorders, asthma, glaucoma, 50 plants are described in Datwyler et al. (Genetic variation in adrenal disease, inflammatory bowel disease, migraines, hemp and marijuana (Cannabis sativa L.) according to ampli fibromyalgia, and related conditions, multiple Sclerosis, spi fied fragment length polymorphisms, J Forensic Sci. 2006 nal cord injuries. It exhibits antispasmodic and muscle-relax March; 51(2):371-5), Pinarkara et al., (RAPD analysis of ant properties as well as stimulates appetite. Other studies seized marijuana (Cannabis sativa L.) in Turkey, Electronic state that Cannabis or cannabinoids may be useful in treating 55 Journal of Biotechnology, 12(1), 2009), Hakki et al., (Inter alcohol abuse, amyotrophic lateral Sclerosis, collagen-in simple sequence repeats separate efficiently hemp from mari duced arthritis, asthma, atherosclerosis, bipolar disorder, col juana (Cannabis sativa L.), Electronic Journal of Biotechnol orectal cancer, HIV-Associated Sensory Neuropathy, depres ogy, 10(4), 2007), Datwyler et al., (Genetic Variation in Hemp Sion, dystonia, epilepsy, digestive diseases, gliomas, hepatitis and Marijuana (Cannabis sativa L.) According to Amplified C. Huntington's disease, leukemia, skin tumors, methicillin 60 Fragment Length Polymorphisms, J Forensic Sci, March resistant Staphylococcus aureus (MRSA), Parkinson's dis 2006, 51(2):371-375), Gilmore et al. (Isolation of microsat ease, pruritus, posttraumatic stress disorder (PTSD), psoria ellite markers in Cannabis sativa L. (marijuana), Molecular sis, sickle-cell disease, sleep apnea, and anorexia nervosa. Ecology Notes, 3(1):105-107, March 2003), Pacifico et al., In some embodiments, the plants of the present invention (Genetics and marker-assisted selection of chemotype in provide one or more medical benefits to a person in need 65 Cannabis sativa L.), Molecular Breeding (2006) without any side effects, or with reduced side effects com 17:257-268), and Mendoza et al., (Genetic individualization pared to a traditional recreational marijuana plant variety. In of Cannabis sativa by a short tandem repeat multiplex sys US 9,370,164 B2 51 52 tem, Anal Bioanal Chem (2009)393:719-726), each of which when expressed lead to desired phenotypes. The most com is herein incorporated by reference in its entirety for all pur mon method for the introduction of new genetic material into poses. a plant genome involves the use of living cells of the bacterial In some embodiments, molecular markers are designed pathogen Agrobacterium tumefaciens to literally inject a and made, based on the genome of the plants of the present piece of DNA, called transfer or T-DNA, into individual plant application. In some embodiments, the molecular markers are cells (usually following wounding of the tissue) where it is selected from Isozyme Electrophoresis, Restriction Fragment targeted to the plant nucleus for chromosomal integration. Length Polymorphisms (RFLPs), Randomly Amplified Poly There are numerous patents governing Agrobacterium medi morphic DNAs (RAPDs), Arbitrarily Primed Polymerase ated transformation and particular DNA delivery plasmids Chain Reaction (AP-PCR), DNA Amplification Fingerprint 10 designed specifically for use with Agrobacterium—for ing (DAF), Sequence Characterized Amplified Regions example, U.S. Pat. No. 4,536,475, EP0265556, EP0270822, (SCARs). Amplified Fragment Length Polymorphisms WO8504899, WO8603516, U.S. Pat. No. 5,591,616, (AFLPs), and Simple Sequence Repeats (SSRs) which are EP0604662, EP0672752, WO8603776, WO9209696, also referred to as Microsatellites, etc. Methods of developing WO9419930, WO9967357, U.S. Pat. No. 4,399.216, molecular markers and their applications are described by 15 WO8303259, U.S. Pat. No. 5,731,179, EP068730, Avise (Molecular markers, natural history, and evolution, WO9516031, U.S. Pat. No. 5,693,512, U.S. Pat. No. 6,051, Publisher: Sinauer Associates, 2004, ISBN 0878930418, 757 and EP904362A1. Agrobacterium-mediated plant trans 9780878930418), Srivastava et al. (Plant biotechnology and formation involves as a first step the placement of DNA molecular markers, Publisher: Springer, 2004, fragments cloned on plasmids into living Agrobacterium ISBN1402019114,9781402019111), and Vienne (Molecular cells, which are then Subsequently used for transformation markers in plant genetics and biotechnology, Publisher: Sci into individual plant cells. Agrobacterium-mediated plant ence Publishers, 2003), each of which is incorporated by transformation is thus an indirect plant transformation reference in its entirety for all purposes. method. Methods of Agrobacterium-mediated plant transfor The molecular markers can be used in molecular marker mation that involve using vectors with no T-DNA are also assisted breeding. For example, the molecular markers can be 25 well known to those skilled in the art and can have applica utilized to monitor the transfer of the genetic material. In bility in the present invention. See, for example, U.S. Pat. No. Some embodiments, the transferred genetic material is a gene 7,250,554, which utilizes P-DNA instead of T-DNA in the of interest, Such as genes that contribute to one or more transformation vector. favorable agronomic phenotypes when expressed in a plant Direct plant transformation methods using DNA have also cell, a plant part, or a plant. 30 been reported. The first of these to be reported historically is Details of existing Cannabis plants varieties and breeding electroporation, which utilizes an electrical current applied to methods are described in Potter et al. (2011, World Wide a solution containing plant cells (M. E. Fromm et al., Nature, Weed: Global Trends in Cannabis Cultivation and Its Con 319, 791 (1986); H. Jones et al., Plant Mol. Biol., 13, 501 trol), Holland (2010, The Pot Book: A Complete Guide to (1989) and H. Yang et al., Plant Cell Reports, 7,421 (1988). Cannabis, Inner Traditions/Bear & Co, ISBN 1594778981, 35 Another direct method, called “biolistic bombardment, uses 9781594778988), Green I (2009, The Cannabis Grow Bible: ultrafine particles, usually tungsten or gold, that are coated The Definitive Guide to Growing Marijuana for Recreational with DNA and then sprayed onto the surface of a plant tissue and Medical Use, Green Candy Press, 2009, ISBN with sufficient force to cause the particles to penetrate plant 1931160589, 9781931160582), Green II (2005, The Can cells, including the thick cell wall, membrane and nuclear nabis Breeder's Bible: The Definitive Guide to Marijuana 40 envelope, but without killing at least some of them (U.S. Pat. Genetics, Cannabis Botany and Creating Strains for the Seed No. 5,204,253, U.S. Pat. No. 5,015,580). A third direct Market, Green Candy Press, 1931160279, 9781931160278), method uses fibrous forms of metal or ceramic consisting of Starks (1990, Marijuana Chemistry: Genetics, Processing & sharp, porous or hollow needle-like projections that literally Potency, ISBN 0914171399,9780914171393), Clarke (1981, impale the cells, and also the nuclear envelope of cells. Both Marijuana Botany, an Advanced Study: The Propagation and 45 silicon carbide and aluminum borate whiskers have been used Breeding of Distinctive Cannabis, Ronin Publishing, ISBN for plant transformation (Mizuno et al., 2004; Petolino et al., 091417178X, 9780914171782), Short (2004, Cultivating 2000; U.S. Pat. No. 5,302,523 US Application 20040197909) Exceptional Cannabis: An Expert Breeder Shares His and also for bacterial and animal transformation (Kaepler et Secrets, ISBN 1936807122, 9781936807123), Cervantes al., 1992: Raloff, 1990; Wang, 1995). There are other methods (2004, Marijuana Horticulture: The Indoor/Outdoor Medical 50 reported, and undoubtedly, additional methods will be devel Grower's Bible, Van Patten Publishing, ISBN 187882323X, oped. However, the efficiencies of each of these indirect or 9781878823236), Franck et al. (1990, Marijuana Grower's direct methods in introducing foreign DNA into plant cells Guide, Red Eye Press, ISBN 0929349016, 9780929349015), are invariably extremely low, making it necessary to use some Groteinhermen and Russo (2002, Cannabis and Cannab method for selection of only those cells that have been trans inoids: Pharmacology, Toxicology, and Therapeutic Poten 55 formed, and further, allowing growth and regeneration into tial, Psychology Press, ISBN 07890 15080,97807890 15082), plants of only those cells that have been transformed. Rosenthal (2007. The Big Book of Buds: More Marijuana For efficient plant transformation, a selection method must Varieties from the World's Great Seed Breeders, ISBN be employed Such that whole plants are regenerated from a 1936807068,9781936807062), Clarke, RC (Cannabis: Evo single transformed cell and every cell of the transformed plant lution and Ethnobotany 2013 (In press)), King, J (Cannabible 60 carries the DNA of interest. These methods can employ posi Vols 1-3, 2001-2006), and four volumes of Rosenthal's Big tive selection, whereby a foreign gene is Supplied to a plant Book of Buds series (2001, 2004, 2007, and 2011), each of cell that allows it to utilize a substrate present in the medium which is herein incorporated by reference in its entirety for all that it otherwise could not use. Such as mannose or Xylose (for purposes. example, refer U.S. Pat. No. 5,767,378; U.S. Pat. No. 5,994, Plant Transformation 65 629). More typically, however, negative selection is used Plants of the present invention can be further modified by because it is more efficient, utilizing selective agents such as introducing into the plants one or more transgenes which herbicides or antibiotics that either kill or inhibit the growth US 9,370,164 B2 53 54 of nontransformed plant cells and reducing the possibility of was invented in 1983-1984 at Cornell University by John chimeras. Resistance genes that are effective against negative Sanford, Edward Wolf, and Nelson Allen. It and its registered selective agents are provided on the introduced foreign DNA trademark are now owned by E. I. du Pont de Nemours and used for the plant transformation. For example, one of the Company. Most species of plants have been transformed most popular selective agents used is the antibiotic kanamy using this method. cin, together with the resistance gene neomycin phospho Agrobacterium tumefaciens is a naturally occurring bacte transferase (nptII), which confers resistance to kanamycin rium that is capable of inserting its DNA (genetic informa and related antibiotics (see, for example, Messing & Vierra, tion) into plants, resulting in a type of injury to the plant Gene 19: 259-268 (1982); Bevan et al., Nature 304:184-187 known as crown gall. Most species of plants can now be (1983)). However, many different antibiotics and antibiotic 10 transformed using this method, including cucurbitaceous resistance genes can be used for transformation purposes species. A transgenic plant formed using Agrobacterium (refer U.S. Pat. No. 5,034.322, U.S. Pat. No. 6,174,724 and transformation methods typically contains a single gene on U.S. Pat. No. 6,255,560). In addition, several herbicides and one chromosome, although multiple copies are possible. herbicide resistance genes have been used for transformation Such transgenic plants can be referred to as being hemizy purposes, including the bar gene, which confers resistance to 15 gous for the added gene. A more accurate name for Such a the herbicide phosphinothricin (White et al., NuclAcids Res plant is an independent segregant, because each transformed 18: 1062 (1990), Spencer et al., Theor Appl Genet 79: 625 plant represents a unique T-DNA integration event (U.S. Pat. 631(1990), U.S. Pat. No. 4,795,855, U.S. Pat. No. 5,378,824 No. 6,156,953). A transgene locus is generally characterized and U.S. Pat. No. 6,107,549). In addition, the dhfr gene, by the presence and/or absence of the transgene. A heterozy which confers resistance to the anticancer agent methotrex gous genotype in which one allele corresponds to the absence ate, has been used for selection (Bourouis et al., EMBO J. of the transgene is also designated hemizygous (U.S. Pat. No. 2(7): 1099-1 104 (1983). 6,008,437). Genes can be introduced in a site directed fashion using General transformation methods, and specific methods for homologous recombination. Homologous recombination transforming certain plant species (e.g., maize) are described permits site specific modifications in endogenous genes and 25 in U.S. Pat. Nos. 4,940,838, 5,464,763, 5,149,645, 5,501,967, thus inherited or acquired mutations may be corrected, and/or 6,265,638, 4,693,976, 5,635,381, 5,731,179, 5,693,512, novel alterations may be engineered into the genome. 6,162,965, 5,693,512, 5,981,840, 6,420,630, 6,919,494, Homologous recombination and site-directed integration in 6,329,571, 6,215,051, 6,369,298, 5,169,770, 5,376,543, plants are discussed in, for example, U.S. Pat. Nos. 5,451, 5,416,011, 5,569,834, 5,824,877, 5,959,179, 5,563,055, and 513; 5,501,967 and 5,527,695. 30 5,968.830, each of which is incorporated herein by reference Methods of producing transgenic plants are well known to in its entirety for all purposes. those of ordinary skill in the art. Transgenic plants can now be Non-limiting examples of methods for transforming Can produced by a variety of different transformation methods nabis plants and Cannabis tissue culture methods are including, but not limited to, electroporation; microinjection; described in Zweger (The Biotechnology of Cannabis sativa, microprojectile bombardment, also known as particle accel 35 April 2009); MacKinnon (Genetic transformation of Can eration or biolistic bombardment; viral-mediated transforma nabis sativa Linn: a multi purpose fiber crop, doctoral thesis, tion; and Agrobacterium-mediated transformation. See, for University of Dundee, Scotland, 2003), MacKinnon et al. example, U.S. Pat. Nos. 5,405,765; 5,472,869; 5,538,877; (Progress towards transformation of fiber hemp, Scottish 5,538,880; 5,550,318; 5,641,664; 5,736,369 and 5,736,369; Crop Research, 2000), and US 20120311744, each of which and International Patent Application Publication Nos. 40 is herein incorporated by reference in its entirety for all pur WO/2002/038779 and WO/2009/117555; Lu et al., (Plant poses. The transformation can be physical, chemical and/or Cell Reports, 2008, 27:273-278); Watson et al., Recombinant biological. DNA, Scientific American Books (1992); Hinchee et al., Bio/ Breeding Methods Tech. 6:915-922 (1988); McCabe et al., Bio/Tech. 6:923-926 Classical breeding methods can be included in the present (1988); Toriyama et al., Bio/Tech. 6:1072-1074 (1988); 45 invention to introduce one or more recombinant expression Fromm et al., Bio/Tech. 8:833-839 (1990); Mullins et al., cassettes of the present invention into other plant varieties, or Bio/Tech. 8:833-839 (1990); Hiei et al., Plant Molecular other close-related species that are compatible to be crossed Biology 35:205-218 (1997): Ishida et al., Nature Biotechnol with the transgenic plant of the present invention. ogy 14:745-750 (1996); Zhang et al., Molecular Biotechnol In some embodiments, said method comprises (i) crossing ogy 8:223-231 (1997); Ku et al., Nature Biotechnology 50 any one of the plants of the present invention comprising the 17:76-80 (1999); and, Raineri et al., Bio/Tech. 8:33-38 expression cassette as a donor to a recipient plant line to (1990)), each of which is expressly incorporated herein by create a F1 population; (ii) selecting offspring that have reference in their entirety. Other references teaching the expression cassette. Optionally, the offspring can be further transformation of Cannabis plants and the production of cal selected by testing the expression of the gene of interest. lus tissue include Raharjo et al 2006, "Callus Induction and 55 In some embodiments, complete chromosomes of the Phytochemical Characterization of Cannabis sativa Cell Sus donor plant are transferred. For example, the transgenic plant pension Cultures’, Indo. J. Chem 6 (1) 70-74; and “The with the expression cassette can serve as a male or female biotechnology of Cannabis sativa” by Sam R. Zwenger, elec parent in a cross pollination to produce offspring plants, tronically published April, 2009. wherein by receiving the transgene from the donor plant, the Microprojectile bombardment is also known as particle 60 offspring plants have the expression cassette. acceleration, biolistic bombardment, and the gene gun (Bi In a method for producing plants having the expression olistic(R) Gene Gun). The gene gun is used to shootpellets that cassette, protoplast fusion can also be used for the transfer of are coated with genes (e.g., for desired traits) into plant seeds the transgene from a donor plant to a recipient plant. Proto or plant tissues in order to get the plant cells to then express plast fusion is an induced or spontaneous union, such as a the new genes. The gene gun uses an actual explosive (.22 65 Somatic hybridization, between two or more protoplasts (cells caliber blank) to propel the material. Compressed air or steam in which the cell walls are removed by enzymatic treatment) may also be used as the propellant. The Biolistic(R) Gene Gun to produce a single bi- or multi-nucleate cell. The fused cell US 9,370,164 B2 55 56 that may even be obtained with plant species that cannot be naturally (e.g., by wind) or by hand or by bees (commonly interbred in nature is tissue cultured into a hybrid plant exhib Apis mellifera L. or MegaChile rotundata F.) with plants from iting the desirable combination of traits. More specifically, a other populations. Selection is applied to improve one (or first protoplast can be obtained from a plant having the Sometimes both) population(s) by isolating plants with desir expression cassette. A second protoplast can be obtained from able traits from both sources. a second plant line, optionally from another plant species or There are basically two primary methods of open-polli variety, preferably from the same plant species or variety, that nated population improvement. First, there is the situation in comprises commercially desirable characteristics, such as, which a population is changed en masse by a chosen selection but not limited to disease resistance, insect resistance, valu procedure. The outcome is an improved population that is able grain characteristics (e.g., increased seed weight and/or 10 indefinitely propagatable by random-mating within itself in seed size) etc. The protoplasts are then fused using traditional protoplast fusion procedures, which are known in the art to isolation. Second, the synthetic variety attains the same end produce the cross. result as population improvement but is not itself propagat Alternatively, embryo rescue may be employed in the able as Such; it has to be reconstructed from parental lines or transfer of the expression cassette from a donor plant to a 15 clones. These plant breeding procedures for improving open recipient plant. Embryo rescue can be used as a procedure to pollinated populations are well known to those skilled in the isolate embryos from crosses wherein plants fail to produce art and comprehensive reviews of breeding procedures rou viable seed. In this process, the fertilized ovary or immature tinely used for improving cross-pollinated plants are pro seed of a plant is tissue cultured to create new plants (see vided in numerous texts and articles, including: Allard, Prin Pierik, 1999, In vitro culture of higher plants, Springer, ISBN ciples of Plant Breeding, John Wiley & Sons, Inc. (1960); 079235267x. 9780792352679, which is incorporated herein Simmonds, Principles of Crop Improvement, Longman by reference in its entirety). Group Limited (1979); Hallauer and Miranda, Quantitative In some embodiments, the recipient plant is an elite line Genetics in Maize Breeding, Iowa State University Press having one or more certain desired traits. Examples of desired (1981); and, Jensen, Plant Breeding Methodology, John traits include but are not limited to those that result in 25 Wiley & Sons, Inc. (1988). increased biomass production, production of specific chemi Mass Selection. cals, increased seed production, improved plant material In mass selection, desirable individual plants are chosen, quality, increased seed oil content, etc. Additional examples harvested, and the seed composited without progeny testing of desired traits includes pest resistance, vigor, development to produce the following generation. Since selection is based time (time to harvest), enhanced nutrient content, novel 30 growth patterns, flavors or colors, salt, heat, drought and cold on the maternal parent only, and there is no control over tolerance, and the like. Desired traits also include selectable pollination, mass selection amounts to a form of random marker genes (e.g., genes encoding herbicide or antibiotic mating with selection. As stated herein, the purpose of mass resistance used only to facilitate detection or selection of selection is to increase the proportion of Superior genotypes transformed cells), hormone biosynthesis genes leading to 35 in the population. the production of a plant hormone (e.g., auxins, gibberellins, Synthetics. cytokinins, abscisic acid and ethylene that are used only for A synthetic variety is produced by crossing interse a num selection), or reporter genes (e.g. luciferase, B-glucuronidase, ber of genotypes selected for good combining ability in all chloramphenicol acetyltransferase (CAT, etc.). The recipient possible hybrid combinations, with Subsequent maintenance plant can also be a plant with preferred chemical composi 40 of the variety by open pollination. Whether parents are (more tions, e.g., compositions preferred for medical use or indus or less inbred) seed-propagated lines, as in Some Sugar beet trial applications. and beans (Vicia) or clones, as in herbage grasses, clovers and Classical breeding methods can be used to produce new alfalfa, makes no difference in principle. Parents are selected varieties of Cannabis according to the present invention. on general combining ability, Sometimes by test crosses or Newly developed F1 hybrids can be reproduced via asexual 45 toperosses, more generally by polycrosses. Parental seed reproduction. lines may be deliberately inbred (e.g. by selfing or sib cross Open-Pollinated Populations. ing). However, even if the parents are not deliberately inbred, The improvement of open-pollinated populations of Such selection within lines during line maintenance will ensure crops as rye, many maizes and Sugar beets, herbage grasses, that some inbreeding occurs. Clonal parents will, of course, legumes Such as alfalfa and clover, and tropical tree crops 50 remain unchanged and highly heterozygous. Such as cacao, coconuts, oil palm and some rubber, depends Whether a synthetic can go straight from the parental seed essentially upon changing gene-frequencies towards fixation production plot to the farmer or must first undergo one or two of favorable alleles while maintaining a high (but far from cycles of multiplication depends on seed production and the maximal) degree of heterozygosity. Uniformity in Such popu scale of demand for seed. In practice, grasses and clovers are lations is impossible and trueness-to-type in an open-polli 55 generally multiplied once or twice and are thus considerably nated variety is a statistical feature of the population as a removed from the original synthetic. whole, not a characteristic of individual plants. Thus, the While mass selection is sometimes used, progeny testing is heterogeneity of open-pollinated populations contrasts with generally preferred for polycrosses, because of their opera the homogeneity (or virtually so) of inbred lines, clones and tional simplicity and obvious relevance to the objective, hybrids. 60 namely exploitation of general combining ability in a syn Population improvement methods fall naturally into two thetic. groups, those based on purely phenotypic selection, normally The numbers of parental lines or clones that enter a syn called mass selection, and those based on selection with prog thetic vary widely. In practice, numbers of parental lines eny testing. Interpopulation improvement utilizes the concept range from 10 to several hundred, with 100-200 being the of open breeding populations; allowing genes to flow from 65 average. Broad based synthetics formed from 100 or more one population to another. Plants in one population (cultivar, clones would be expected to be more stable during seed strain, ecotype, or any germplasm source) are crossed either multiplication than narrow based synthetics. US 9,370,164 B2 57 58 Pedigreed Varieties. impairment caused by THC. See Morgan, Celia JA, et al. A pedigreed variety is a Superior genotype developed from “Impact of cannabidiol on the acute memory and psychoto selection of individual plants out of a segregating population mimetic effects of smoked Cannabis: naturalistic study.” The followed by propagation and seed increase of self pollinated British Journal of Psychiatry 1974 (2010): 285-290. Other offspring and careful testing of the genotype over several non-THC cannabinoids (CBs) have also been demonstrated generations. This is an open pollinated method that works to have extensive medicinal uses (Table 1). well with naturally self pollinating species. This method can THC is produced primarily by narrow and broad-leafleted be used in combination with mass selection in variety devel drug Cannabis varieties. CBD is produced primarily by nar opment. Variations in pedigree and mass selection in combi row and broad leafleted fiber Cannabis varieties, commonly nation are the most common methods for generating varieties 10 in self pollinated crops. known as hemp. Other non-THC CBs such as THCV and Hybrids. CBDV can also be found in natural varieties (Meijer and A hybrid is an individual plant resulting from a cross Hammond 2005, Euphytica 145:189-198). CBC production between parents of differing genotypes. Commercial hybrids is associated with juvenile Cannabis and some natural vari are now used extensively in many crops, including corn 15 eties found in India (Meijer and Hammond 2009, Euphytica (maize), Sorghum, Sugarbeet, Sunflower and broccoli. 165:293–311). Hybrids can beformed in a number of different ways, includ Interbreeding drug and other natural varieties of Cannabis ing by crossing two parents directly (single cross hybrids), by can produce cultivars that produce both THC and other CBS, crossing a single cross hybrid with another parent (three-way in amounts that far exceed landrace Cannabis drug or fiber or triple cross hybrids), or by crossing two different hybrids varieties (See Clarke, R C et al. “Cannabis: Evolution and (four-way or double cross hybrids). Ethnobotany” University of California Press 2013). Unfortu Strictly speaking, most individuals in an outbreeding (i.e., nately, Such crosses have been rare, and have only produced open-pollinated) population are hybrids, but the term is usu Cannabis varieties lacking the terpenoid constituents respon ally reserved for cases in which the parents are individuals sible for the appealing aroma and flavor. Moreover, such whose genomes are sufficiently distinct for them to be recog 25 varieties, also lack the synergistic entourage effects of diverse nized as different species or subspecies. Hybrids may be terpene-cannabinoid combinations (2011, Taming THC: fertile or sterile depending on qualitative and/or quantitative potential Cannabis Synergy and phytocannabinoid-terpenoid differences in the genomes of the two parents. Heterosis, or entourage effects, British Journal of Pharmacology, 163: hybrid vigor, is usually associated with increased heterozy 1344-1364, Table 2). gosity that results in increased vigor of growth, Survival, and 30 Similar problems have been identified with oral adminis fertility of hybrids as compared with the parental lines that trations of Cannabis extracts such as Marinol R (dronabinol), were used to form the hybrid. Maximum heterosis is usually and SativeX(R), which have higher side effects, and lower achieved by crossing two genetically different, highly inbred consumer acceptance, partially due to the lack of terpene lines. entourage effects and lack of positive aroma/flavors (see This invention is further illustrated by the following 35 Hazenkamp et al 2013, “The Medicinal Use of Cannabis and examples which should not be construed as limiting. The Cannabinoids—An international Cross-Sectional Survey on contents of all references, patents and published patent appli Administration forms' Journal of Psychoactive drugs 45 (3) cations cited throughout this application, as well as the Fig 199-210; McPartland and Russo 2001 "Cannabis and Can ures and the Sequence Listing, are incorporated herein by nabis Extracts: Greater Than the Sum of Their Parts? Hay reference. 40 worth Press). Specialty Cannabis For example, all known varieties of chemotype II Cannabis The present invention is based in part on the discovery new (B/B, genotype) exhibit terpene profiles dominated by specialty Cannabis varieties with unique terpene and cannab myrcene. That is, these Cannabis varieties produce myrcene inoid profiles can be bred to produce Cannabis with reduced at higher levels than any other terpene. As such, these variet THC side effects and increased medicinal uses. 45 ies do not exhibit diverse terpene profiles and lack the varied Contemporary “recreational marijuana cultivars that are aroma, organoleptic feel of the specialty Cannabis of the currently available have been bred and selected primarily for present invention. The aroma and flavors for myrcene domi their THC content, without much regard for their terpenoid nant varieties tend to be “single tone', with the high myrcene aroma and flavor chemistry, or for their for their production of levels dominating the flavor and aroma profile. Moreover, as the other cannabinoids (CBs), such as CBD, THCV, CBC, 50 myrcene is associated with the Cannabis "couch lock' effect, CBG, etc. Indeed, almost 99% of Cannabis sold by dispen these varieties have produce less functional highs, with higher saries in California for medical purposes contains less than sedation. 1% non-THCCBs. (personal communication with SC Labo The present invention provides specialty Cannabis plants ratories and Halent Laboratory, 2013). with THC and CBS, and desirable terpene profiles. In some While THC has considerable medicinal value, it can be 55 embodiments, the CBs (e.g. CBD, or CBDV) level in dried responsible for a range of poorly tolerated side effects includ Cannabis plants of the present invention is higher compared ing anxiety, dizziness, tachycardia, asthenia, etc. It has to that of a dried recreational Cannabis plants, such as the recently been discovered that administration of CBD reduces strain White Widow. In some embodiments, the THC level or ameliorates some undesirable effects of THC including in the dried Cannabis plants of the present invention is lower intoxication, sedation and tachycardia, while contributing 60 compared to that of a dried recreational Cannabis plants, such analgesic, anti-emetic, and anti-carcinogenic properties as the strain White Widow. In some embodiments the spe (Russo and Guy, 2006, Medical Hypotheses (2006) 66, 234 cialty Cannabis of the present invention is a chemotype II 246). Evidence has also emerged that CBD may contribute plant. In some embodiments, the specialty Cannabis of the anti-anxiety effects to Cannabis varieties with THC. See present invention produces more than 1.5% of any one CBS. “Cannabidiol, a Cannabis sativa constituent, as an anxiolytic 65 In some embodiments, the specialty Cannabis plants of the drug” (Rev Bras Psiquiatr. 2012:34(Sup11):S104-S117) Also present invention also have terpene profiles that are not domi evidence has emerged that CBD can ameliorate the memory nated by myrcene. In some embodiments, the specialty Can US 9,370,164 B2 59 60 nabis of the present invention have higher terpene oil contents The instrumentation includes a Clarus 680 gas chromato which overcome high myrcene profiles. graph (GC) equipped with an autosampler, an Elite-5 column In some embodiments, the specialty Cannabis varieties of (PerkinElmer (Waltham, Mass.), 30 m length, 0.25 mm inter the present invention have been bred to produce high terpene nal diameter, 0.25um thickness film diameter) and a flame oil contents. In currently available Cannabis cultivars, 5 ionization detector (FID). Instrument control and data acqui increased terpene oil content is largely driven by increased sition and analyses was accomplished by TotalChrom Soft myrcene content, which can increase the “couch-lock' effect ware version 1.2.3.4 (Perkin Elmer, Waltham, Mass.). and overshadow the effects of the other terpenes. In contrast Calibration curves were generated by injecting each stan to current practice, the breeding programs of the present dard in triplicate and the RSDs provided the measure of invention were designed to produce specialty Cannabis vari 10 precision while the absolute accuracy was determined by eties with higher terpene oil content with terpene profiles in comparing the concentrations of the standards predicted by which myrcene has a relative terpene content of less than the calibration curve to their “known values determined by two-thirds of the terpene profile. In other embodiments, the dilution ratios. AOAC International standards for accuracy breeding programs of the present invention were designed to and precision were used as quality guidelines for every cali produce high terpene oil cultivars in which myrcene was not 15 bration. Check standards were run at the start, middle, and the dominant terpene. end of every analysis, and recalibration was performed when they varied more than +/-5% of their initial average response. EXAMPLES Levels that failed the acceptance criteria and analytes were not quantified at those levels until recalibration of the instru Example 1 ment corrected the deficiency. Most of the curves were linear to nearly two orders of magnitude and based on the sample Chemical Analysis of Cannabinoids and Terpenes mass extracted (500 mg) and the two possible extraction volumes (3x3 mL or 3x5 mL), this provided quantitation of Chemical analyses of the parental and progeny specialty terpene levels from 0.01-0.9% or 0.02-1.5% (typical) in the Cannabis varieties of the present invention was carried out 25 plant matrix. using standard chemical separation techniques well known to ii. Cannabinoids by GC-FID those skilled in the arts. Qualitative identification of cannab Cannabinoids were quantified by an analytical method inoids and terpenes was carried out by GCMS, while quanti developed and run on a PerkinElmer (Waltham, Mass.) GC tative analysis was done by GC-FID and/or HPLC-PDA FID instrument also. This method was developed to separate (Photo Diode Array). Initial field analyses of cannabinoids 30 six neutral cannabinoids, CBD, CBG, CBN, THC, A8-THC, was performed using thin layer chromatography as described and CBC. The cannabinoids are each quantified by their own in (“Cannabis Inflorescence & Leaf QC from The American individual calibration curves generated with analytical refer Herbal Pharmacopeia 2013). The in-house assays for cannab ence standards (Restek) and all use tricosane as the internal inoids included orthogonal methods of GC-FID and HPLC standard. The retention time of THCV was determined by for the highest level of accuracy. 35 analyzing THVO1 (vide infra) by GCMS, however since ana Samples were prepared by grinding ~5g of dried Cannabis lytical standards were not available it was “quantified by flower material in a coffee grinder. From this homogenized referencing the calibration curve for THC. material, 500+20 mg was placed in a bead beater vial with ~1 There was no need to consider chromatographic separation g of 2 mm beads and 5 mL of working Solution. Each sample of acidic forms of the cannabinoids due to their immediate was placed in the bead beater (BioSpec Products Inc.) and 40 conversion to neutral form in the heated injector of the instru homogenized on high for 3 minutes. The vials were centri ment, although a thorough study of the conversion efficiency fuged at 1350xg, decanted into 50 mL falcon tubes, and the of THCA was performed and is discussed in section iv. (or process was repeated with fresh working solution. After the thogonal analyses of all samples). second extraction the caps were removed, the vials were The instrumentation includes a Clarus 680 gas chromato decanted into the appropriate falcon tubes, and the vials were 45 graph (GC) equipped with an autosampler, an Elite-1 column rinsed into the falcon tubes with an additional 5 mL of work (PerkinElmer (Waltham, Mass.), 30 m length, 0.25 mm inter ing solution. For samples Suspected of having lower concen nal diameter, 0.25um thickness film diameter) and a flame trations of analytes (i.e. <10% THC or total terpene content ionization detector (FID). Instrument control and data acqui ~0.5%), 3 mL portions of working solution could be sition and analyses was accomplished by TotalChrom Soft employed. Approximately 2 mL of the extracts were placed in 50 ware version 1.2.3.4 (Perkin Elmer, Waltham, Mass.). 2 mL centrifuge tubes, and the vials were centrifuged at Calibration curves were generated by injecting each stan 9500x g for 5 minutes. The supernatant was placed in a GC dard in triplicate and the RSDs provided the measure of vial for terpene analysis without dilution. The supernatant precision while the absolute accuracy was determined by was also diluted with working solution for GC and HPLC comparing the concentrations of the standards predicted by analysis. A 1:40 dilution provided the appropriate concentra 55 the calibration curve to their “known values determined by tion for analysis of cannabinoids present at concentrations dilution ratios. AOAC International standards for accuracy above 1.5%, while a 1:3 dilution allowed for analysis of and precision were used as quality guidelines for every cali cannabinoids below this level. bration. Check standards were run at the start, middle, and i. Terpenoids by Gas Chromatography-Flame Ionization end of every analysis, and recalibration was performed when Detector (GC-FID) 60 they varied more than +/-5% of their initial average response. Terpenes were quantified by a method developed on a Levels that failed the acceptance criteria and analytes were GC-FID instrument from Perkin Elmer (Waltham, Mass.). not quantified at those levels until recalibration of the instru This method separates and quantifies 17 different terpenoids ment corrected the deficiency. Due to the very linear nature of commonly found in Cannabis plant tissue. The terpenoids are the FID detector, the GC-FID cannabinoid assay generally each quantified by their own individual calibration curves 65 provided satisfactory results over nearly two orders of mag generated with analytical reference standards (Sigma Ald nitude (up to 1.0 mg/mL), however in order to use the same rich) and all use n-nonane as the internal standard. calibration solutions and “validation' procedures for both GC US 9,370,164 B2 61 62 and HPLC the range was reduced to that of the HPLC method. GC should be used since conversion to these forms in the Based on the sample mass extracted (500mg) and a 3x3 mL injector of the GC is an inherent part of the analytical method. extraction (low oil samples), a 1:3 dilution provided quanti The second reason is also related to the difference between tation of cannabinoid levels from 0.09-1.35% and the 1:40 the acidic and neutral cannabinoids, but has to do with the dilution from 1.15-18% in the plant matrix. A 3x5 mL extrac- 5 availability of analytical standards to calibrate the instru tion (high oil samples, typical), a 1:3 dilution provided quan ments. While all of the neutral cannabinoids (THC, CBG, titation of cannabinoid levels from 0.14-2.25% and the 1:40 CBC, CBD, and CBN) are available as analytical standards, dilution from 1.9-30% in the plant matrix. THCA is the only acidic cannabinoid available as an analyti iii. Cannabinoids by High Performance Liquid Chromatog cal standard and the instruments were only calibrated for raphy Photo Diode Array Detector (HPLC-PDA) 10 quantification using actual analytical standards. Technically An HPLC-PDA (also known as HPLC-DAD, or simply the HPLC assay could characterize the naturally occurring HPLC) assay was developed as an orthogonal method to chemotypes, but the acidic analytes are not available as stan GC-FID for cannabinoid analyses. This method quantifies six dards, so this quantification is approximate and considered neutral cannabinoids (CBD, CBG, CBN, THC, A8-THC, and for information only. The acidic analytes are all quantified by CBC) as well as THCA based on calibration curves generated 15 reference to the calibration curve for THCA, and this is not an with analytical standards and an internal reference standard unreasonable assumption as many of them have approxi (ibuprofen). The only acidic cannabinoid that is readily avail mately the same spectral properties. The GC assay is cali able as an analytical standard in the United States is THCA, so brated with analytical standards, but these are the neutral levels of CBDA, CBGA, and THCVA are estimated by ref cannabinoids and their formation from the naturally occur erence to THCA calibration. 2O ring acidic cannabinoids in the GC injector is not quantitative, All HPLC analyses were performed using a PerkinElmer which complicates exact characterization of the naturally (Waltham, Mass.) HPLC system comprised of a Flexar FX-15 occurring chemotype. binary pump, a Flexar 5-CH solvent manager, an FX UHPLC The final reason is simply to have an internal crosscheck of autosampler, and a Peltier LC column oven. UV data was our results by using orthogonal testing methods. Each type of collected at 228 nm and 280 nm with a Flexar FX-PDA 25 assay (GC and HPLC) has its strengths and weaknesses, and UHPLC detector. Chromatography was performed on a by using both methods one can compare results and ensure Brownlee SPPC18 column (PKIN9308411, 2.7 um, 3.0x150 that both the identification and quantitation of the compo mm), protected by a Brownlee SPP C18 guard column (2.7 nents are accurate. A caveat to this, as mentioned above, is um, 2.1x5 mm). HPLC system control, data acquisition and that the conversion of the acidic forms to the neutral forms is analyses were performed with Chromera software version 30 not quantitative due to thermal degradation. Under the highly 34.15904. optimized conditions of a GC injector we have found conver Calibration was achieved by performing a five-point cali sion can vary between 75-85% (for analytical THCA stan bration curve (0.016-0.25 mg/mL for each analyte) followed dards), while Cannabis samples generally have a conversion by linear regression analysis. This analysis was performed of 70-80%. Similar conversion rates are also described in with Microsoft Excel (Redmond, Wash.) software. The cali- 35 literature for highly optimized analytical instruments (Dussy bration curves were generated by injecting each standard in et al. 2004). Because of this incomplete conversion our GC triplicate and the RSDs provided the measure of precision results are consistently 20-30% lower than the HPLC results while the absolute accuracy was determined by comparing for Cannabis samples. This same conversion efficiency can the concentrations of the standards predicted by the calibra be applied to estimate the maximum availability of THC tion curve to their “known values determined by dilution 40 based on THCA content when Smoking or vaporizing Can ratios. AOAC International standards for accuracy and preci nabis. sion were used as quality guidelines for every calibration. v. Method “Validation Check standards were run at the start, middle, and end of In order to demonstrate the performance of a method of every analysis, and recalibration was performed when they analysis, a systematic process known and method validation varied more than +/-5% of their initial average response. 45 can be carried out. This process demonstrates the method is fit iv. Orthogonal Analyses of all Samples for its intended purpose and is necessary for the confident use The cannabinoid content was quantified by both GC-FID of that method, providing assurance that the results that are and HPLC. The main difference between GC and HPLC is reported are precise, accurate, and reflective of the sample. that GC involves thermal stress and mainly resolves analytes Very few labs in the Cannabis industry attempt to validate by boiling points while HPLC does not involve heat and 50 their assays and this fact, combined with inappropriate sam mainly resolves analytes by polarity. There are several rea pling have resulted in erroneous data for several varieties. In Sons that this orthogonal approach to analyses is desirable for order to validate the analytical methods employed for this highly accurate and reproducible results in determining project, an abbreviated protocol similar to Single Laboratory chemotype. The first reason is related to the difference Validation (SLV) was carried out. Assay “validation was between the cannabinoids produced naturally by the plant 55 carried out by spiking blank matrix with the analytes at low, (the acidic cannabinoids) and those that are bioactive (the med, and high concentrations and carrying out the assay neutral cannabinoids). Cannabis biosynthesizes all the can procedure in replicate (n=5). While some analytes provided nabinoids in their relatively unstable acidic forms, and these better results than others the analyte RSDs, recoveries, and forms are generally not bioactive in the traditional sense. The precisions at these concentrations satisfied AOAC guidance application of heat (flame, vaporizer, oven, etc) causes a loss 60 (based on mg/mL). In general the RSDs for the terpenes at the of the carboxylic acid group and generates the neutral forms low, medium, and high concentrations (varied by terpene but of the cannabinoids, which are generally the bioactive forms generally 0.016, 0.125, and 1.0 mg/mL) were less than 5%, that are sought after, however this process is highly variable 4%, and 3% respectively. The absolute bias for these analytes and not quantitative. If one wants to know the native phy was generally less than 10%, 4%, and 2%. In general the tochemical profile of the plant then HPLC should be used 65 RSDs for the cannabinoids by both GC and HPLC at the low, since this assay does not involve heat. If one wants to know medium, and high concentrations (0.016, 0.61, and 0.250 the possible available amount of bioactive cannabinoids, then mg/mL) were less than 2%, 2%, and 1% respectively. The US 9,370,164 B2 63 64 absolute bias for these analytes was generally less than 10%, apical dominance can be disrupted easily with removal of 2%, and 2%. The assays all provided satisfactory S/N ratios at apical meristem. Main stems also exhibit purpling, but inflo the lowest level and this was initially taken as the LOO. After rescences are not purple. subsequent re-calibrations (n=3 at each level), the LOO was Onset of Flowering and Inflorescences. taken as the lowest level of the calibration curve that provided Leaves are 3 and 5 leaflet patterns with 3 being predomi acceptable accuracy (<10% error) determined by comparing nant and overall decreasing to 1 and to none in the presence of the known concentration levels (determined by dilution female flowers. ratios) to the predicted levels (obtained from the signal and Female flowers are spread out due to the large internode calibration curve). The error between the known and mea spacing. Upon flower set, buds and Supporting structures 10 (stems, leaves, etc.) are quickly covered with an extremely sured values establishes the accuracy of the method and veri dense field of trichome bodies. Again, this variety tends to be fies that real samples do not present any matrix effects that more densely covered with trichome bodies than its parent influence the resulting measurements. The precision, or and other gold class varieties. In fact, the inflorescences are closeness of individual measurements, of the method is also very dense and have large calyxes covered in highly resinous determined by carrying out all analyses in replicate (n=5). 15 glands that exhibit this variety’s distinct lemon Pine-Sol scent Guidance for acceptable values was taken from publications after only 7-10 days. As inflorescences mature, the density provided by the AOAC. compact sets give way to foxtailing and reaching by indi The in-house validation revealed that the above-described vidual calyxes, resulting in an overall increase in Surface area chemical analysis methods were accurate and reliable, and dedicated to trichome production. In particular, the oily char the use of orthogonal methods of analyses provided an inter acter of these flowers set this gold class apart from its parent nal check on the assays as well as an understanding of the use and other gold class varieties. Textures are extremely sticky of GC to analyze thermally unstable molecules. Using mul and fibrous. Stems do not break they tear, but remain tiple dilution ratios kept samples in the linear ranges of the attached via intense fiber strands. assays, and method validation verified that precise and accu Description of Finished Flower. rate results were obtained. 25 GO13 consistently produces among the highest THCA levels of Cannabis known in California and is often noted for Example 2 an intense and crushing physical effect combined with a Sublime and inspiring mental flight. Aromas of lemon peel, Proprietary Parental Variety Phenotypes fuel and Pine-Sol combine to produce a pure menthol exha 30 lation when smoked. Noted for excellent appetite and sexual More detailed descriptions of the development and char stimulation often accompanied by uninterrupted sleep. acteristics of representative Parental Classes of Cannabis Description of Planting, Harvesting and Processing of the Varieties of the present invention are provided below. In some Plants. embodiments, the THC parental varieties of the present This variety is asexually propagated via taking cuttings of invention were selected for their morphologies and desirable 35 shoots and putting them in rock wool cubes. These cubes were phenotypes. presoaked with pH adjusted water and kept warm (~80° C.). GOD13 Full trays were covered, left under 18 hours of light and Description of Breeding Stock. allowed to root (7-14 days). Upon root onset, the plantlets Inflorescences were obtained for a land race of Gold class were transplanted into rigid 1 gallon containers filled with a varieties and seeds from these inflorescences were isolated 40 proprietary soil mix A and remain in 18 hours of daylight for and put into conditions proper for their germination. The another 14-21 days. Once root bound, plants are transplanted seeds which germinated grew identically. However, upon into rigid 3 gallon containers filled with proprietary soil mix flower onset, the seedlings were selected for the strongest B. Immediately, the light cycle is altered to 12/12 and flower limonene/Pine-Sol fragrance and narrowed to two pheno initiating begins. The plants remain in 12/12 lighting until types. Of these, the individual phenotype with the best user 45 harvesting. They undergo a propriety nutrient regimen and experience based on testing was selected to create GO13, a grow as undisturbed as possible for 60-70 days depending on variety classified into the Gold Class. chemotype analysis. Hypothesized Genetics. All Sun leaves are removed and plant dismantled to result in Cannabis indica ssp. afghanica WLD “Purple Afghan'xC. approximately 12" branches covered in inflorescences and indica ssp. indica var. indochinensis NLD "Lemon Thaix.C. 50 trichomes. The goal in harvesting is to realize that we are indica ssp. Kafiristanica NLDA. actually harvesting trichome heads but not buds. Thus, great Propagation and Vegetative Growth. care is taken not to disturb the trichome heads and as much of Cuttings from GO13 are marked by 3-finger leaflet sets the plant remains intact as possible to promote even and slow with internode buds asymmetrically located on alternate sides drying. on main shoot. In particular, the internode space of this vari 55 Yield Data. ety tends to be greater than that of other gold class varieties Yield determined on a per plant basis and determined by and stems harden quicker. Roots nodes appear with 7-10 days specified cultivation techniques employed. In this case, and roots within 10-14 days. The GOD13 grows extremely indoor Controlled Environment Agriculture (CEA) technique tall and thin with extreme stretching and asymmetrical bud following the protocol described elsewhere herein. Flower and leaf sets. When root system is not limited or pruned, this 60 onset was initiated with 12/12 day/night at approximately 12" variety of gold class varieties exhibits unparalleled vigor and in vegetative height. Total biomass ~150 g, finished flowers stretch. Vegetative growth is marked by a deep blue-green ~50 g, and/or -50 g of seed per plant. (Munsell ID) hue with lime green thin stalks. Petioles are Potential Uses of this Line. marked by purple pointillism increasing on sides exposed to Potential uses of GO13 include but are not limited to medi light and the end closest to palm of the leaflet set. Root bodies 65 cal applications, as a source for extractions of plant constitu are typically full and bright white. Stalks radiate a pungent ents and chemicals, for commercial raw materials, fiber and Smell of body odor or urine. Canopy extremely sparse and pulp. US 9,370,164 B2 65 66 Patient Testimonials/Comments and Visual Observations. Onset of Flowering and Inflorescences. Patients rave about the flavor and oily composition by com Leaves are 9 and 7 leaflet patterns with 7 being predomi parison to other Gold class varieties. In fact, besides the nant and overall decreasing to 1 and to none in the presence of extremely high potency from its combined cannabinoid/ter female flowers. In particular, flower onset is very slow with penoid entourage effects', this line of gold class has been 5 this variety. Hairy flowers are not very dense. Female flow noted by patients for being particularly effective for sexual ers are spread apart due to the large internode spacing. and appetite stimulation. Upon flower set, buds and Supporting structures (stems, Palatable CBDA varieties with ideal CBDA:THCA ratio leaves, etc.) take longer than most to become covered with can be developed from GO13 to reduce side-effects associ trichome bodies. Everything about this plant takes longer. As ated with extant recreational Cannabis varieties related to 10 inflorescences mature, they become more hardened and GO13. Additionally reduced THCA varieties can be devel dense. In particular, the oily character of these flowers was the oped that are intended to reduce side-effects from extant driving force for selection. recreational Cannabis varieties related to GO13. Description of Finished Flower. Flavor when smoked includes distinct citrus and mentho 15 BRO5 defines heady, hazy medicine with highly functional lated notes. Significant analgesia accompanies its deep range mental effects. This variety has the structure and scent of the of effects, but with little sedation, but the “rising/falling BRO5 lines famous around the world. With aromas of spice physical sensations associated with gold class. Some patients and anise, the hashish flavor when Smoked is enlightening. have compared its flavor to bergamot orange. Patients also BRO5 is noted for mood elevation, inspiration and creativ remark on the “clarity of this variety's psychoactivity, with ity and is also likely to improve home hygiene. less sedation and disorientation, and with considerable Chemotype Description for Patient. euphoria. Relative potency: Strong. Headspace Terpenes: pinenes, Its aroma has been characterized as a tangy, sharp, naph limonene. Caryophyllene content: high. thalene aroma with orange notes and a Sweet undertone. Also Description of Planting, Harvesting and Processing of the the range of pharmacologically active terpenoids that this 25 Plants. variety produces provide a significant “entourage effect” that This variety is asexually propagated via taking cuttings of accompanies the effects of its THC content. While it stimu shoots and putting them in rock wool cubes. These cubes were lates appetite, it does not appear to encourage overeating. presoaked with pH adjusted water and kept warm (~80° C.). BRO5 Full trays were covered, left under 18 hours of light and Description of Breeding Stock. 30 allowed to root (7-14 days). Inflorescences were obtained for a landrace of Haze and Upon root onset, the plantlets were transplanted into rigid seeds from these inflorescences were isolated and put into 1 gallon containers filled with a proprietary soil mix A and conditions proper for their germination. remain in 18 hours of daylight for another 14-21 days. Once The seeds which germinated grew identically, being short root bound, plants are transplanted into rigid 3 gallon con and squatty with purple leaves and Sweet scent, with one 35 tainers filled with proprietary soil mix B. Immediately, the exception which was talland stretchy with a savory and musty light cycle is altered to 12/12 and flower initiating begins. The scent. There was absolutely no sweetness in the smell of plants remain in 12/12 lighting until harvesting. They BRO5. Testing proved that its effects were the most enjoyable undergo a propriety nutrient regimen and grow as undisturbed and virtually myrcene free. The lack of myrcene and presence as possible for 60-70 days depending on chemotype analysis. of pinene and limonene is quite rare and sets this variety apart 40 All Sun leaves are removed and plant dismantled to result in from most Cannabis varieties. approximately 12" branches covered in inflorescences and Upon flower onset, the seedlings were selected for being trichomes. The goal in harvesting is to realize that we are short and squatty with purple leaves and berry scent to create actually harvesting trichome heads but not buds. Thus, great BRO5, a variety classified into the Gold Class. care is taken not to disturb the trichome heads and as much of Hypothesized Genetics. 45 the plant remains intact as possible to promote even and slow “NL#5xHaZexinbred Thai drying. Slow drying followed by a one to two month curing Propagation and Vegetative Growth. process. Cuttings from BRO5 are marked by 9-finger very thin Yield Data. leaflet sets with internode buds asymmetrically located on Yield determined on a per plant basis and determined by alternate sides on main shoot. In particular, the internode 50 specified cultivation techniques employed. In this case, space of this variety tends to be extremely large. Stems are indoor CEA technique following the protocol described else tall, frail and stretchy. Cuttings roots appear within 10-14 where herein. Flower onset was initiated with 12/12 day/night days. The BRO5 grows tall and stretchy with flimsy stems. It at approximately 16" in vegetative height. Organic mix of soil possesses the classic narrow-leafleted morphology associated in fabric pots, a regimen of nutrients following standard NPK with 1970’s Haze cultivars that were inherited from HaZe’s 55 feeding schedules and addition of proprietary mixture. tropical drug Cannabis parents, including Colombian and Flower onset was initiated with 12/12 day/night when plant Thai varieties. reached approximately 16" in vegetative height. BRO5 grows with asymmetrical bud and leaf sets. Vegeta Potential Uses of this Line. tive growth is marked by a lightened green (Munsell ID) hue Potential uses of BRO5 include but are not limited to medi with lime green thin stalks. Leaflets are longer and narrower 60 cal applications, extractions, commercial raw material than most of drug Cannabis varieties. (chemical), fiber and pulp. BRO5 displays vigorous hybrid character. Patient Testimonials/Comments and Visual Observations. There is little or no purple on this plant until the final weeks Patients rave about the great experience of using BRO5. of flowering. Leaves turn deep purple with flowers silvering The effects are mind stimulating with some visual crisp up as time goes on. Stalks radiate a hazy or musty urine 65 ness. The patients often comment that this variety is good for scent. Canopy extremely sparse and topping near flowering is the new user because of its lower THC concentration and the encouraged for even growth. clarity of the experience. US 9,370,164 B2 67 68 SILO)4 presoaked with pH adjusted water and kept warm (~80° C.). Description of Breeding Stock. Full trays were covered, left under 18 hours of light and Inflorescences were obtained for a proprietary breeding allowed to root (7-14 days). program and seeds from these inflorescences were isolated Upon root onset, the plantlets were transplanted into rigid and put into conditions proper for their germination. The 1 gallon containers filled with a proprietary soil mix A and seeds which germinated grew identically. The resulting plants remain in 18 hours of daylight for another 14-21 days. Once were then crossed with GO13 plants and seeds were planted root bound, plants are transplanted into rigid 3 gallon con and germinated for selection based on oil content of the tainers filled with proprietary soil mix B. Immediately, the plants. Plants with higher oil content were selected to create light cycle is altered to 12/12 and flower initiating begins. The 10 plants remain in 12/12 lighting until harvesting. They SILO4. undergo a propriety nutrient regimen and grow as undisturbed Hypothesized Genetics. as possible for 60-70 days depending on chemotype analysis. “Cannabis indica ssp. afghanica WLD “SB Purple'xC. All Sun leaves are removed and plant dismantled to result in indica ssp. indica NLDxC. indica ssp. Kafiristanica NLDA' approximately 12" branches covered in inflorescences and Propagation and Vegetative Growth. 15 trichomes. The goal in harvesting is to realize that one is Cuttings from SIL04 are marked by 5-finger leaflet sets actually harvesting trichome heads but not buds. Thus, great with internode buds asymmetrically located on alternate sides care is taken not to disturb the trichome heads and as much of on main shoot. In particular, the internode space of this vari the plant remains intact as possible to promote even and slow ety tends to be longer and stalks thinner (~4-8" Veg, decreas drying. Slow drying followed by a one to two month curing ing flower onset). Plants are tall, stretchy and productive. process. Roots of the cuttings appear within 10-14 days. Yield Data. The SIL04 grows tall and stretchy and exhibits little or no Yield was determined on a perplant basis and determined apical dominance. SIL04 grows with asymmetrical bud and by specified cultivation techniques employed. In this case, leafsets. Vegetative growth is marked by a lavish green (Mun indoor CEA technique following the protocol described else sell ID) hue with green undersides and hard wood like stalks. 25 where herein. Flower onset was initiated with 12/12 day/night When healthy, fan leaves are extremely jagged and Serrations at approximately 16" in vegetative height. Total biomass are very pronounced. ~120g, finished flowers ~40 g, and/or -30g of seed perplant. The stems are strong and fibrous, but extremely thin. The Potential Uses of this Line. standout quality of SIL04 is the amount of trichomes and their Potential uses of SIL04 include but are not limited to medi density. The flower sets look frosty before most other vari 30 cal applications, extractions, commercial raw material (e.g., eties. chemical), fiber and pulp. Stalks are vanilla spice scent. Patient Testimonials/Comments and Visual Observations. Canopy is extremely sparse with clustered bud formation. Very interesting from an organoleptic standpoint (Sweet Topping extremely encouraged. Amsterdam flavor) and a caryophyllene content standpoint. Onset of Flowering and Inflorescences. 35 SIL04 produces a happy laughing high, with the classic com Leaves are 5 leaflet patterns with 5 being predominant and busted aroma of 1990's landrace varieties of the same Can overall decreasing to 1 and to none in the presence of female nabis class. flowers. In particular, flower onset is fast by comparison to WHIO4 most varieties. Description of Breeding Stock. Trichome density and smell are almost immediate. Female 40 Inflorescences were obtained for a landrace of WHIO4 and flowers are spear-shaped, dense and thick although relatively seeds from these inflorescences were isolated and put into large internode lengths. Again, this variety tends to be more conditions proper for their germination. The seeds which densely covered with trichome bodies than most other vari germinated grew fairly similarly. However, upon flower eties. onset, the seedlings were selected for trichome density, leaflet The flowers are compact and well-formed in the shape of 45 width and root vigor to create WHIO4. Small pinecones. As inflorescences mature, the density com Hypothesized Genetics. pact sets compound to form bright orange and silver flowers “Cannabis indica ssp. afghanica WLD Propagation and that give way to yellow and purple Sun leaves. Vegetative Growth. Plants are marked by unusually high oil mass content and Cuttings from WHIO4 are marked by 7-finger leaflet sets extremely dense Small resinous buds. 50 with internode buds asymmetrically located on alternate sides Apical inflorescences are often smaller than lowers. Inflo on main shoot. In particular, the internode space of this vari rescences particularly are resistant to fungal infestation due to ety tends to be greater than that of other Silver varieties and compact oil flowers. stems harden more slowly. In particular, the cutting roots Description of Finished Flower. more rapidly than other Silver varieties. In fact, the root SIL04 (a.k.a., internally known as Heiress’ or “Oily Heir 55 bodies of the plant are the most robust and vigorous of all ess) was bred from a dream team of Cannabis genetics: Cannabis plants tested in our laboratory. Root time varies Northern LightsxHaze, Santa Barbara Purps, a Midwest G-13 with nodes appearing within 7-10 days and roots within 10-14 and the aforementioned GO13. The chemotype of this variety days. is indicative of this diverse genetic heritage. The aroma con The WHIO4 grows medium in stature with stocky branches sists of vanilla, grapefruit, and even has petroleum notes, but 60 and stalks. Even growth throughout with asymmetrical bud a rich creamy vanilla flavor emerges when smoked. Noted for and leaf sets. Vegetative growth is marked by a deep blue its rare combination of clarity and profound potency, it deliv green (Munsell ID) hue with lime green thin stalks. Leaflets ers functional and long lasting inspiration and positivity. are fat and exhibit classic recreational indica look. These Description of Planting, Harvesting and Processing of the broad leaflets are indicative of this variety. Petioles are Plants. 65 marked by purple pointillism increasing on sides exposed to This variety is asexually propagated via taking cuttings of light and the end closest to palm of the leaflet set. Root bodies shoots and putting them in rock wool cubes. These cubes were are typically full and bright white. Stalks radiate a pungent US 9,370,164 B2 69 70 Smell of bubble gum coffee and green class. Canopy Flower onset was initiated with 12/12 day/night at approxi extremely sparse and apical dominance can be clearly mately 12" in vegetative height. Total biomass ~120 g, fin observed and removal of apical meristem often results in ished flowers ~30 g, and/or ~15 g of seed per plant. stunted growth. Main stems may also exhibit purpling, and Potential Uses of this Line. inflorescences sets are large, but spread out. 5 Potential uses of WHIO4 include but are not limited to Onset of Flowering and Inflorescences. medical applications, extractions, commercial raw material Leaves are 7 and 5 leaflet patterns with 3 being predomi (e.g., chemical), fiber and pulp. nant and overall decreasing to 1 and to none in the presence of Patient Testimonials/Comments and Visual Observations. female flowers. Female flowers are spread out due to the large Patients rave about the coffee flavor and oily and silver internode spacing. 10 composition of WHIO4. In fact, besides the mellow effects, Upon flower set, buds and Supporting structures (stems, WHIO4 is particularly noted for treating pain and inspiration. leaves, etc.)are quickly covered with an extremely dense field REDO8 of trichome bodies. Again, this variety tends to be more Description of Breeding Stock. densely covered with trichome bodies than its parent and Inflorescences were obtained from a DJ Short's Flo (a.k.a. other Silver varieties. In fact, the inflorescences are very 15 DJ’s Flo) pollinated by a hermaphroditic Hawaiian plant and dense and have large calyxes covered in highly resinous seeds from these inflorescences were isolated and put into glands that exhibit this variety’s distinct lemon Pine-Sol scent conditions proper for their germination. after only 7-10 days. The seeds which germinated grew very uniformly in As inflorescences mature, the dense and compact calyx appearance. However, the seedlings were selected for vigor clusters or flower sets give way to foxtailing and reaching by ous phenotype with highest trichome density and oily feel of individual calyxes, resulting in an overall increase in Surface resin glands to create RED08. area dedicated to trichome production. In particular, the oily Hypothesized Genetics. character of these flowers set WHIO4 apart from its parent and “1995 Hawaiian Bag Seed:XThai'. other Silver varieties. Textures are extremely sticky and Propagation and Vegetative Growth. fibrous. Stems do not break they tear, but remain attached 25 Cuttings from RED08 are marked by 7-finger leaflet sets via intense fiber strands. with internode buds asymmetrically located on alternate sides Description of Finished Flower. on main shoot. In particular, the internode space of this vari WHIO4 has descended from the great Afghan hashish Can ety tends to be medium-stretchy (~4" veg, decreasing flower nabis cultivars and is a nearly perfect choice for vaporization. onset). Plants are tall, robust and lanky. Cuttings root within The resin content delivers a range of tastes and effects with 30 10-14 days. each draw. The RED08 grows tall and stout with mixed apical domi The aroma consists of coffee, spice and exotic incense. aC. This variety is noted for its ability to mellow without sedation RED08 grows with asymmetrical bud and leaf sets. Veg or fatigue, excellent analgesic effects and deep introspection. etative growth is marked by a deeper off green (Munsell ID) Chemotype Description for Patient. 35 hue with deep purple strong hollow stalks. Relative potency: mild. Headspace Terpenes: pinenes, When healthy, Sun leaves are gigantic with magenta and myrcene, limonene, linalool. Caryophyllene content: purple under side coloring. Plants have Super vigorand hybrid medium character. RED08's stand-out quality feature is the high Description of Planting, Harvesting and Processing of the amount of trichomes and the high amount of oil. Stalks have Plants. 40 a pungent medical scent. Plant canopy is dense with large This variety is asexually propagated via taking cuttings of cola formation. Topping encouraged. shoots and putting them in rock wool cubes. These cubes were Onset of Flowering and Inflorescences. presoaked with pH adjusted water and kept warm (~80° C.). Leaves are 7 leaflet patterns with 7 being predominant and Full trays were covered, left under 18 hours of light and overall decreasing to 1 and to none in the presence of female allowed to root (7-14 days). 45 flowers. In particular, flower onset is medium-fast by com Upon root onset, the plantlets were transplanted into rigid parison to most varieties. Trichome density and Smell are 1 gallon containers filled with a proprietary soil mix A and almost immediate. Female flowers are clustered to do remain in 18 hours of daylight for another 14-21 days. Once decreased internode spacing. Again, this variety tends to be root bound, plants are transplanted into rigid 3 gallon con more densely covered with trichome bodies than its parents tainers filled with proprietary soil mix B. Immediately, the 50 and other varieties. In fact, the inflorescences are very dense light cycle is altered to 12/12 and flower initiating begins. The and have large calyxes covered in highly resinous glands that plants remain in 12/12 lighting until harvesting. They exhibit this variety’s distinct blueberry pine medicine/me undergo a propriety nutrient regimen and grow as undisturbed dicinal scent after only 7-10 days. as possible for 60-70 days depending on chemotype analysis. As inflorescences mature, the density compact sets com All Sun leaves are removed and plant dismantled to result in 55 pound to form bright green and extremely oily buds. In par approximately 12" branches covered in inflorescences and ticular, the oily character of these flowers set this its parent trichomes. The goal in harvesting is to realize that we are and phenotypes. actually harvesting trichome heads but not the buds. Thus, Description of Planting, Harvesting and Processing of the great care is taken not to disturb the trichome heads and as Plants. much of the plant remains intact as possible to promote even 60 This variety is asexually propagated via taking cuttings of and slow drying. Slow drying followed by a one to two month shoots and putting them in rock wool cubes. These cubes were curing process. presoaked with pH adjusted water and kept warm (~80° C.). Yield Data. Full trays were covered, left under 18 hours of light and Yield was determined on a perplant basis and determined allowed to root (7-14 days). by specified cultivation techniques employed. In this case, 65 Upon root onset, the plantlets were transplanted into rigid indoor CEA technique following the protocol described else 1 gallon containers filled with a proprietary soil mix A and where herein. remain in 18 hours of daylight for another 14-21 days. Once US 9,370,164 B2 71 72 root bound, plants are transplanted into rigid 3 gallon con Description of Finished Flower. tainers filled with proprietary soil mix B. Immediately, the SILO3 combines a beautifully sweet cherry WLD Afghan light cycle is altered to 12/12 and flower initiating begins. with a NLD to deliver a strong, cheerful, dreamy psychoac The plants remain in 12/12 lighting until harvesting. They tivity. This variety produces a pleasant silliness and a undergo a propriety nutrient regimen and grow as undisturbed where'd I put my keys!? memory effect and obliterates most as possible for 60-70 days depending on chemotype analysis. patient troubles. All Sun leaves are removed and plant dismantled to result in Aroma consists of cherry cough drops, fresh Strawberries approximately 12" branches covered in inflorescences and and just a hint of spice. SIL03 is often noted for long-lasting trichomes. The goal in harvesting is to realize that we are effects and positive mood impact. 10 Description of Planting, Harvesting and Processing of the actually harvesting trichome heads but not buds. Thus, great Plants. care is taken not to disturb the trichome heads and as much of This variety is asexually propagated via taking cuttings of the plant remains intact as possible to promote even and slow shoots and putting them in rock wool cubes. These cubes were drying. Slow drying followed by a one to two month curing presoaked with pH adjusted water and kept warm (~80° C.). process. 15 Full trays were covered, left under 18 hours of light and Potential Uses of this Line. allowed to root (7-14 days). Potential uses of RED08 include but are not limited to Upon root onset, the plantlets were transplanted into rigid medical applications, extractions, commercial raw material 1 gallon containers filled with a proprietary soil mix A and (e.g., chemical), fiber and pulp. remain in 18 hours of daylight for another 14-21 days. Once Patient Testimonials/Comments and Visual Observations. root bound, plants are transplanted into rigid 3 gallon con RED08 is very interesting from an organoleptic standpoint tainers filled with proprietary soil mix B. Immediately, the and it is unique in almost all visual categories. light cycle is altered to 12/12 and flower initiating begins. The SILO3 plants remain in 12/12 lighting until harvesting. They Description of Breeding Stock. undergo a propriety nutrient regimen and grow as undisturbed Inflorescences were obtained froma, and selfed seeds from 25 as possible for 60-70 days depending on chemotype analysis. these plants were germinated. The seeds which germinated All Sun leaves are removed and plant dismantled to result in grew very similarly. The resulting seedlings were selected for approximately 12" branches covered in inflorescences and Vigor. trichomes. The goal in harvesting is to realize that we are Hypothesized Genetics. actually harvesting trichome heads but not buds. Thus, great “Cannabis indica ssp. afghanica WLD “Cherry Afghan'x 30 care is taken not to disturb the trichome heads and as much of the plant remains intact as possible to promote even and slow C. indica ssp. indica NLD hybrid drying. Slow drying followed by a one to two month curing Propagation and Vegetative Growth. process. Cuttings from SIL03 are marked by 7-finger leaflet sets Yield Data. with internode buds asymmetrically located on alternate sides 35 Yield was determined on a perplant basis and determined on main shoot. In particular, the internode space of this vari by specified cultivation techniques employed. In this case, ety tends to be medium-stretchy (~4" veg, decreasing flower indoor CEA technique following the protocol described else onset). The plants are tall, robust and lanky. Cuttings root where herein. Flower onset was initiated with 12/12 day/night within 10-14 days. at approximately 16" in vegetative height. Total biomass The SIL03 grows tall and strong with little apical domi 40 ~180 g, finished flowers ~60 g, and/or ~50g of seed perplant. nance. SIL03 grows with asymmetrical bud and leaf sets. Potential Uses of this Line. Vegetative growth is marked by a lighter shade of green Potential uses of SIL03 include but are not limited to medi (Munsell ID) hue with deep purple strong hollow stalks. cal applications, extractions, commercial raw material (e.g., When healthy, sun leaves are point upward toward light chemical), fiber and pulp. SOUC. 45 Patient Testimonials/Comments and Visual Observations. The stems are strong and fibrous. The plants are Super Noted as being very interesting from an organoleptic stand vigorous and hybrid in character. The stand-out quality is the point. SIL03 is unique in almost all visual categories. high amount of trichomes and the high amount of oil. Stalks GREO1 have a Sweet scent. Canopy is dense with large cola forma Description of Breeding Stock. tion. Topping encouraged. 50 Inflorescences were obtained and isolated and put into Onset of Flowering and Inflorescences. conditions proper for their germination. Leaves are 7 leaflet patterns with 7 being predominant and The seeds which germinated grew identically. However, overall decreasing to 1 and to none in the presence of female the seedlings were selected for the phenotype that is more flowers. In particular, flower onset is medium-fast by com densely covered in trichomes, where the oil content of the parison to most varieties. 55 gland heads was higher than other phenotypes of this variety. Trichome density and smell are almost immediate. Female Hypothesized Genetics. flowers are clustered to do decreased internode spacing. “Cannabis indica ssp. afghanica WLD Afghan #1'xC. Again, this variety tends to be more densely covered with indica ssp. indica NLD hybrid trichome bodies than its parents and other varieties. In fact, Propagation and Vegetative Growth. the inflorescences are very dense and have large calyxes 60 Cuttings from GRE01 are marked by 9-finger very thin covered in highly resinous glands that exhibit this variety's leaflet sets with internode buds asymmetrically located on distinct blueberry pine medicine/medicinal scent after only alternate sides on main shoot. In particular, the internode 7-10 days. space of this variety tends to be extremely large. Plants are As inflorescences mature, the dense and compact calyx tall, frail and stretchy. Cuttings root appears within 10-14 clusters or flower sets form bright green and extremely oily 65 days. buds. In particular, the oily character of these flowers set this GRE01 grows tall and stretchy with flimsy stems and variety apart from its parent and phenotypes. embodies what it means to be a true hybrid. US 9,370,164 B2 73 74 GRE01 grows with asymmetrical bud and leaf sets. Veg Flower onset was initiated with 12/12 day/night at approxi etative growth is marked by a lush green (Munsell ID) hue mately 16" in vegetative height. Total biomass ~160 g, fin with lime green thin stalks. ished flowers ~50 g, and/or ~50 g of seed per plant. Leaflets are longer and thinner than varieties. Plants have a Potential Uses of this Line. vigorous hybrid character. GRE01 has little or no purple color Potential uses of GRE01 include but are not limited to on the plant. The stand-out quality is the high amount of medical applications, extractions, commercial raw material trichomes and the high amount of oil. Plant stalks have a (e.g., chemical), fiber and pulp. Sweet citrus creamsicle scent. Patient Testimonials/Comments and Visual Observations. Plant canopy is dense and even topping near flowering is Patients rave about the great experience of using this plant. encouraged for even growth. 10 The flowers of GRE01 consistently produce approximately Onset of Flowering and Inflorescences. 2.0% CBGA in finished flowers. Its wonderful smell/taste is Leaves are 9 and 7 leaflet patterns with 7 being predomi patient's major reason for appeal. nant and overall decreasing to 1 and to none in the presence of PURO3 female flowers. In particular, flower onset is fast by compari 15 Description of Breeding Stock. son to most varieties. Inflorescences were obtained for a landrace of purple class Trichome density and smell are almost immediate. Female pollinated with a hermaphroditic purple class variety and flowers are clustered to do decreased internode spacing. seeds from these inflorescences were isolated and put into Again, this variety tends to be more densely covered with conditions proper for their germination. trichome bodies than its parent and other green class varieties. The seeds which germinated grew very uniformly in In fact, the inflorescences are very dense and have large appearance. However, upon flower onset, the seedlings were calyxes covered in highly resinous glands that exhibit this selected for the two phenotypes that most smelled like grape variety’s distinct green class creamsicle scent after only 7-10 and dank, and producing flowers with the highest trichome days. As inflorescences mature, the density compact sets density and robust examples of these two phenotypes were compound to form orange and bright green extremely oily 25 subsequently crossed to create PUR03. buds. In particular, the oily character of these flowers set this Hypothesized Genetics. green class apart from its parent and other green class variet “2007 SB PUP1 X2009 PPS7. 1CS Propagation and Vegetative Growth. Description of Finished Flower. Cuttings from PUR03 are marked by 7-finger leaflet sets 30 with internode buds asymmetrically located on alternate sides GRE01 defines sweet, delicious medicine/medicinal with on main shoot. In particular, the internode space of this vari functional mental effects. This variety has resin production ety tends to be decreased. Short, squatty and bushy. Cuttings akin to Afghan and psychoactivity reminiscent of original root within 10-14 days. Green class. The PURO3 grows stout in the traditional Christmas tree GRE01 has aromas of citrus, brown Sugar, and banana nut 35 shape. PUR03 grows with asymmetrical bud and leaf sets. bread combine to produce a fantastic fruity hashish flavor Vegetative growth is marked by a deeper off green (Munsell when smoked. It is noted for mood elevation and daytime ID) hue with lime green thin stalks. Leaflets are longer and bursts of energy that provide for short-term pain relief. thinner than varieties. When healthy, Sun leaves are gigantic. Description of Planting, Harvesting and Processing of the It has vigorous hybrid character. The stand-out quality is the Plants. 40 high amount of trichomes and the high amount of oil. There is This variety is asexually propagated via taking cuttings of an extremely high cannabinoid content in PUR03. Stalks shoots and putting them in rock wool cubes. These cubes were have a Sweet dank scent. Canopy dense and do not need to presoaked with pH adjusted water and kept warm (~80° C.). top. Full trays were covered, left under 18 hours of light and Onset of Flowering and Inflorescences. allowed to root (7-14 days). 45 Leaves are 7 leaflet patterns with 7 being predominant and Upon root onset, the plantlets were transplanted into rigid overall decreasing to 1 and to none in the presence of female 1 gallon containers filled with a proprietary soil mix A and flowers. In particular, flower onset is fast by comparison to remain in 18 hours of daylight for another 14-21 days. Once most varieties. root bound, plants are transplanted into rigid 3 gallon con Trichome density and smell are almost immediate. Female tainers filled with proprietary soil mix B. Immediately, the 50 flowers are clustered to do decreased internode spacing. light cycle is altered to 12/12 and flower initiating begins. The Again, this variety tends to be more densely covered with plants remain in 12/12 lighting until harvesting. They trichome bodies than its parents and other purple varieties. In undergo a propriety nutrient regimen and grow as undisturbed fact, the inflorescences are very dense and have large calyxes as possible for 60-70 days depending on chemotype analysis. covered in highly resinous glands that exhibit this variety's All Sun leaves are removed and plant dismantled to result in 55 distinct grape lollipop scent after only 7-10 days. As inflores approximately 12" branches covered in inflorescences and cences mature, the density compact sets compound to form trichomes. The goal in harvesting is to realize that we are deep purple and darkgreen extremely oily buds. In particular, actually harvesting trichome heads but not buds. Thus, great the oily character of these flowers set this purple apart from its care is taken not to disturb the trichome heads and as much of parent and other green class varieties. the plant remains intact as possible to promote even and slow 60 Description of Finished Flower. drying. Slow drying followed by a one to two month curing PUR03 defines sweet, delicious grape flavored medicine process. with functional mental effects and pain relief. This variety has Yield Data. resin production akin to Afghan and psychoactivity reminis Yield was determined on a perplant basis and determined cent of the PUR03. PUR03 has aromas of grape, Sweet sugar, by specified cultivation techniques employed. In this case, 65 and dank which all combine to produce a fantastic grape indoor CEA technique following the protocol described else flavor when smoked. It is noted for mood elevation, short where herein. term pain relief and hunger stimulation. US 9,370,164 B2 75 76 Description of Planting, Harvesting and Processing of the and the high amount of oil. YEL03 has stalks with a sweet Plants. scent. Plant canopy is sparse with scattered bud formation. This variety is asexually propagated via taking cuttings of Topping encouraged. shoots and putting them in rock wool cubes. These cubes were Onset of Flowering and Inflorescences. presoaked with pH adjusted water and kept warm (~80° C.). Leaves are 9 leaflet patterns with 9 being predominant and Full trays were covered, left under 18 hours of light and overall decreasing to 1 and to none in the presence of female allowed to root (7-14 days). flowers. In particular, flower onset is fast by comparison to Upon root onset, the plantlets were transplanted into rigid most varieties. 1 gallon containers filled with a proprietary soil mix A and Trichome density and smell are almost immediate. Female remain in 18 hours of daylight for another 14-21 days. Once 10 root bound, plants are transplanted into rigid 3 gallon con flowers are sparse due to large internode spacing. Again, this tainers filled with proprietary soil mix B. Immediately, the variety tends to be more densely covered with trichome bod light cycle is altered to 12/12 and flower initiating begins. The ies than its parents and other varieties. The flowers are not plants remain in 12/12 lighting until harvesting. They compact or well-formed. Inflorescences are spirals of indi undergo a propriety nutrient regimen and grow as undisturbed 15 vidual foxtails that form a coral looking structure. Although as possible for 60-70 days depending on chemotype analysis. buds are made of individual spirals, the inflorescences are All Sun leaves are removed and plant dismantled to result in dense and tightly packed. As inflorescences mature, the den approximately 12" branches covered in inflorescences and sity compact sets compound to form bright green and trichomes. The goal in harvesting is to realize that we are extremely oily buds. actually harvesting trichome heads but not buds. Thus, great Description of Finished Flower. care is taken not to disturb the trichome heads and as much of YEL03 has descended from the great Oaxacan and Thai the plant remains intact as possible to promote even and slow Cannabis landrace plants of the 1970s. This variety delivers drying. Slow drying followed by a one to two month curing an intense 'up' Stimulating effect that can be great for coun process. tering the debilitating aspects of many medical conditions. A Yield Data. 25 complex aroma of spicy spruce and lemon peel release a Yield was determined on a perplant basis and determined cornucopia of Sweet and spicy piney flavors when Smoked. It by specified cultivation techniques employed. In this case, is often characterized by a clear head, accompanied by mood indoor CEA technique following the protocol described else elevation. where herein. Description of Planting, Harvesting and Processing of the Flower onset was initiated with 12/12 day/night at approxi 30 Plants. mately 16" in vegetative height. Total biomass ~160 g, fin This variety is asexually propagated via taking cuttings of ished flowers ~50 g, and/or ~50 g of seed per plant. Potential Uses of this Line. shoots and putting them in rock wool cubes. These cubes were Potential uses of PURO3 include but are not limited to presoaked with pH adjusted water and kept warm (~80° C.). medical applications, extractions, commercial raw material 35 Full trays were covered, left under 18 hours of light and (e.g., chemical), fiber and pulp. allowed to root (7-14 days). Upon root onset, the plantlets Patient Testimonials/Comments and Visual Observations. were transplanted into rigid 1 gallon containers filled with a Patients rave about the great experience. This flower con proprietary soil mix A and remain in 18 hours of daylight for sistently produces approximately 2.0% CBGA in finished another 14-21 days. Once root bound, plants are transplanted flowers. Its wonderful smell/taste is patients major reason for 40 into rigid 3 gallon containers filled with proprietary soil mix appeal. B. Immediately, the light cycle is altered to 12/12 and flower YELO3 initiating begins. The plants remain in 12/12 lighting until Description of Breeding Stock. harvesting. They undergo a propriety nutrient regimen and Inflorescences were obtained from an unknown landrace. grow as undisturbed as possible for 60-70 days depending on Seeds from these inflorescences were isolated and put into 45 chemotype analysis. conditions proper for their germination. The seeds which All Sun leaves are removed and plant dismantled to result in germinated grew uniformly in appearance. However, the approximately 12" branches covered in inflorescences and seedlings were selected for their narrow-leafleted tropical trichomes. The goal in harvesting is to realize that we are Cannabis morphology and pinene production to create actually harvesting trichome heads but not buds. Thus, great YELO3. 50 care is taken not to disturb the trichome heads and as much of Hypothesized Genetics. the plant remains intact as possible to promote even and slow “Cannabis indica ssp. indica NLD “Thai'xC. indica ssp. drying. Slow drying followed by a one to two month curing indica NLD “Highland Mexican.” process. Propagation and Vegetative Growth. Yield Data. Cuttings from YEL03 are marked by 9-finger leaflet sets 55 Yield was determined on a perplant basis using the speci with internode buds asymmetrically located on alternate sides fied cultivation techniques employed. In this case, indoor on main shoot. In particular, the internode space of this vari CEA technique following the protocol described elsewhere ety tends to be lengthy and stretchy (~4" veg, decreasing herein. Flower onset was initiated with 12/12 day/night at flower onset). The plants are tall, robust and lanky. Cuttings approximately 16" in vegetative height. Total biomass ~120 root within 10-14 days. 60 g, finished flowers ~40 g, and/or ~30g of seed per plant. The YELO3 grows tall and strong with pronounced apical Potential Uses of this Line. dominance. YEL03 grows with asymmetrical bud and leaf Potential uses of YEL03 include but are not limited to sets. Vegetative growth is marked by a lighter dark green medical applications, extractions, commercial raw material (Munsell ID) hue with purple undersides and strong hollow (e.g., chemical), fiber and pulp. stalks. When healthy, sun leaves are point upward toward 65 Patient Testimonials/Comments and Visual Observations. light source at twisted angles. The stems are strong and Plants have a very interesting from an organoleptic stand fibrous. The stand-out quality is the high amount of trichomes point and are unique in almost all visual categories. US 9,370,164 B2 77 78 PUR 12 remain in 18 hours of daylight for another 14-21 days. Once Description of Breeding Stock. root bound, plants are transplanted into rigid 3 gallon con Inflorescences were obtained for an unknown landrace. tainers filled with proprietary soil mix B. Immediately, the Seeds from these inflorescences were isolated and put into light cycle is altered to 12/12 and flower initiating begins. The conditions proper for their germination. The seeds which 5 plants remain in 12/12 lighting until harvesting. They germinated grew uniformly in appearance. However, the undergo a propriety nutrient regimen and grow as undisturbed seedlings were selected for trichome density and hybrid leaf as possible for 60-70 days depending on chemotype analysis. morphology to create PUR12. All Sun leaves are removed and plant dismantled to result in Hypothesized Genetics. approximately 12" branches covered in inflorescences and “Cannabis indica ssp. afghanica WLDAfghan #1 ''xCan 10 trichomes. The goal in harvesting is to realize that we are nabis indica ssp. indica NLD “Brazilian'x.C. indica ssp. actually harvesting trichome heads but not buds. Thus, great indica NLD “Indian. care is taken not to disturb the trichome heads and as much of Propagation and Vegetative Growth. the plant remains intact as possible to promote even and slow Cuttings from PUR12 are marked by 5-finger leaflet sets drying. Slow drying followed by a one to two month curing with internode buds asymmetrically located on alternate sides 15 process. on main shoot. In particular, the internode space of this vari Yield Data. ety tends to be shorter and stout (-2-4" veg, decreasing flower Yield was determined on a perplant basis and determined onset). The plants are short, robust and bushy. Cuttings root by specified cultivation techniques employed. In this case, within 10-14 days. indoor CEA technique following the protocol described else The PUR12 grows short and bushy with classic Christmas where herein. Flower onset was initiated with 12/12 day/night tree apical dominance. PUR12 grows with asymmetrical bud at approximately 16" in vegetative height. Total biomass and leaf sets. Vegetative growth is marked by a dark green ~140g, finished flowers ~50 g, and/or ~50g of seed perplant. (Munsell ID) hue with green undersides and hard wood like Potential Uses of this Line. stalks. When healthy, sun leaves are point upward toward Potential uses of PUR12 include but are not limited to light source and reach. The stems are strong and fibrous. 25 medical applications, extractions, commercial raw material The stand out quality is the high amount of trichomes and the (e.g., chemical), fiber and pulp. high amount of oil. The flower sets look white before most Patient Testimonials/Comments and Visual Observations. other varieties. Stalks are sweet scent. Plant canopy is dense Very interesting from an organoleptic standpoint (Sweet with clustered bud formation. Topping discouraged. Amsterdam flavor) and a caryophyllene content standpoint. Onset of Flowering and Inflorescences. 30 Happy laughing high. PUR12 has the burnt scent of 1990s Leaves are 5 leaflet patterns with 5 being predominant and landraces. overall decreasing to 1 and to none in the presence of female Parental Plant Phenotypes flowers. In particular, flower onset is fast by comparison to In order to better describe the morphologies of parental most varieties. Cannabis lines, plants were grown indoor to maturity at 120 Trichome density and smell are almost immediate. Female 35 days post transfer under standard production and pruning flowers are dense and thick due to relatively small internode methods. These plants were assayed for several phenotypes lengths. Again, this variety tends to be more densely covered important for Cannabis production. These phenotypes and with trichome bodies than other varieties in the white class. their descriptions are listed below, and their measurements The flowers are compact and well-formed in the shape of for parental varieties summarized in Table 3. pinecones. Pistils are fat and of high density. As inflores 40 Plant Sex-In order to properly assess the progeny mor cences mature, the density compact sets compound to form phology of the female inflorescence important for Cannabis bright neon-green flowers that give way to red-orange hair. It production, only pistillate plants were allowed to fully is marked by unusually high sesquiterpene content and develop. Breeding of pistillate flowers was carried out by extremely resinous buds. Inflorescences are subject to fungal reversing the sex of a branch of female flowers through the infestation due to large size and extreme density. 45 application silver thiosulfate. Sex determination was made Description of Finished Flower. during vegetative growth through the identification of the PUR12 produces prodigious amounts of psychoactive earliest pre-flowers (see Cervantes 2006 “Marijuana Horti resin. This variety was derived from Brazilian, Indian, and culture The indoor/outdoor medical grower's bible editors Afghan gene pools. Its aroma of green classy, balsamic, pine Linda Meyer and Estella Cervantes). Male plants were not apple gazpacho delivers a Sweet, hashy flavor when Smoked. 50 allowed to develop in order to avoid accidental pollination of It is noted for fast-onset psychoactivity reminiscent of tradi female plants. tional Cannabis experiences that will leave you right where it Plant Height measured in centimeters from the base of found you. Happiness induced pain relief and considerable the plant to the top of the apical meristem. Plants were mea relaxation. sured 120 days post transfer. Chemotype Description for Patient. 55 Plant Diameter measured in centimeters as width of the Relative potency: very strong. Headspace Terpenes: plant at its widest diameter. Plants were measured 120 days pinenes, myrcene, limonene, humulene, and naphthalene. post transfer. Caryophyllene content: Very high Number of Leafletts—Leafletts on leaves were counted. Description of Planting, Harvesting and Processing of the The leaf with the most leaflets was recorded at 120 days post Plants. 60 transfer. This variety is asexually propagated via taking cuttings of Leaf Type Leaves were visually inspected for broad or shoots and putting them in rock wool cubes. These cubes were narrow leaf morphologies. Narrow leaf morphologies pro presoaked with pH adjusted water and kept warm (~80° C.). duce leaflets less than 1 cm wide (N). This type of leaf Full trays were covered, left under 18 hours of light and morphology is most closely associated with Cannabis sativa allowed to root (7-14 days). 65 varieties. Broad leaf morphologies produce leaflets wider Upon root onset, the plantlets were transplanted into rigid than 2 cm (B). This type of leaf morphology is most closely 1 gallon containers filled with a proprietary soil mix A and associated with Cannabis indica. Leaves were designated as US 9,370,164 B2 79 80 medium (M) morphologies if they fell in between broad and cm and 300 cm were designated “medium”. Inflorescences narrow leaf values, indicating the progeny included genetics with volumes greater than 300 cm were designated “large”. from both C. sativa and C. indica. Average Apical Inflorescence Size—Inflorescence size Average Internodes—Internodes were counted at plant was measured by Volume by measuring the height and radius maturity at 120 days. Number of internodes is highly corre of apical inflorescences at plant maturity at 120 days post lated to plant branching and number of inflorescences. Inter transfer. These measurements were used to determine volume nodes are defined as the sections of stem between nodes. of the inflorescence using a cylinder shape approximation Node Branching Node branching was visually deter (formula Pixradius'xheight). Values for apical inflorescences mined by inspecting nodes and determining the amount of of multiple plants were averaged. Apical inflorescences with branching at plant maturity at 120 days post transfer. Higher 10 average volumes of less than 400 cm were designated branching can increase total flower yield, but can also pro “small'. Apical inflorescences with average volumes duce plants that cannot be grown closely for indoor produc between 400 cm and 600 cm were designated “medium”. tion. Inflorescences with volumes greater than 600 cm were des Leaf Color Representative leaves from each plant were ignated "large'. harvested and pictures were taken. Colors will be analyzed 15 Floral Cluster Density—Floral cluster density is a measure and will be provided using Royal Horticultural Society color of how tightly packed floral buds are in a plant inflorescence. chart values. This measure is correlated with total yield and is also associ Average Number of Inflorescences at Maturity—Inflores ated with the amount of labor necessary for trimming the cences were visually inspected and counted at plant maturity inflorescence post harvest. For parental varieties of this at 120 days post transfer. Plants were designated as having example, floral cluster density was approximated by measur “low” number of inflorescences if they produced less than 10 ing the time it took for the inflorescences to dry (reach ~10% inflorescences per plant. Plants were designated as having relative humidity). Faster drying time were associated with “medium number of inflorescences if they produced “low” floral cluster density. Slower drying times were asso between 10 and 15 inflorescences per plant. Plants were des ciated with “high floral cluster densities. Low density floral ignated as having “high number of inflorescences if they 25 clusters dry in 4-5 days, medium density floral clusters take produced more than 15 infloresences per plant. In general, 6-7 days, and dense floral clusters take 8-9 days. higher number of inflorescences are associated with higher Trichome Density Trichomes on the inflorescences of Cannabis flower yield. mature plants at 120 days post transfer were visually Average Non-Apical Inflorescence Size Inflorescence inspected for trichome density and assigned a score of 1-10 size was measured by Volume by measuring the height and 30 based on past experiences of the grower. Lower scores indi radius of each non-apical inflorescence at plant maturity at cated lower trichome densities, whereas higher scores indi 120 days post transfer. These measurements were used to cated higher trichome densities. Trichome density is also determine Volume of the inflorescence using a cylinder shape commonly referred to as “frostiness”. Inflorescences with approximation (formula Pixradius'xheight). Values for all scores higher than 7 appear to be completely covered in white non-apical inflorescences were averaged. Inflorescences with 35 trichomes giving a "frost' like appearance. Density Scores of average volumes of less than 100 cm were designated 8-10 were equivalent to what could be expected of an OG “small'. Inflorescences with average volumes between 100 Kush Strain. TABLE 3 Phenotype table of parental varieties.
Plant Plant Avg Avg i height dia Number Oil- Avg Tri- Num at leter at Inflores- apical apical (a) chome ber Variety matur- matur- Avg # CeCe:S inflores- inflores- Floral density of (W ity ity Leaf inter at CeCe. CeCe. cluster (1-10 Leaf l8le (cm) (cm) type nodes Branching a each node maturity size size (cm) density Scale) letts
PUR13 S4 63.5 B 23 every node: ear, 1 branch Low Large High High 9 5 SILO)4 45 65.2 B 27.5 every node: eaf, 1 branch High Large High LOW 5 5 REO1 95 45.7 M 29.3 every node: eaf, 1 branch Medium Mediumium High Medium 7 5 SILO3 33 49.8 B 26.5 every node: eaf, 1 branch Medium Large High Medium 7 7 PURO3 71 47.4 B 23 every node: ear, 1 branch Low Medium Medium High 5 7 SILO1 78 46.5 B 15.7 every node: ear, 1 branch Low Medium LOW High 7 5 SILO6 83 22.9 B 21 every node: ear, 1 branch LOW Medium LOW LOW 9 7 YELO3 O4 67.6 B 20.8 every node: eaf, 1 branch Medium Large High LOW 7 9 WHIO7 12 SO.2 B 29.5 every node: eaf, 1 branch Medium Medium High Medium 9 7 GOD13 21 45.7 B 22 every node: eaf, 1 branch Medium Small Medium LOW 7 5 ORAO2 35 43.2 B 32.3 every node: eaf, 1 branch High Small High LOW 9 5 WHIO4 O3 36.2 B 19.8 every node: ear, 1 branch LOW Medium Medium Medium 7 7 PURO1 25 39.4 M 28.8 every node: eaf, 1 branch Medium Medium High Medium 9 7 CBDO3 89 41.5 M 26.5 every node: eaf, 1 branch High Small LOW High 9 7 SILO2 94 43.8 M 28.5 every node: eaf, 1 branch High Medium Medium LOW 7 5 BROO1 18 43.2 M 27 every node: eaf, 1 branch Medium Medium High LOW 9 7 PUR12 O3 45.1 M 31.7 every node: eaf, 1 branch High Medium Medium High 5 5 CBDO1 25 44.9 M 38.3 every node: eaf, 1 branch High Small LOW LOW 2 5 PURO6 94 445 B 27.3 every node: eaf, 1 branch Medium Medium LOW Medium 7 7 CBDS 32 48.3 B 37 every node: eaf, 1 branch High Low Medium Medium 7 5 US 9,370,164 B2 81 82 Example 3 The analysis of Cannabis class varieties led to the slightly different abbreviations for color classes and also to the Analysis of Proprietary THC Parental Varieties renaming of varieties disclosed in the original filing. Name changes in this application from priority documents (U.S. One objective of the present invention was to develop 61/801,528 and U.S. 61/897,074) are summarized in Table 5. Cannabis varieties with high terpene oil contents and differ New class categories violet and pink are included in the ent terpene profiles to meet various aroma/flavor and medici following table. nal needs. The parental varieties developed in the present invention, underwent chemical analyses as described in TABLE 5 Example 1. The resulting cannabinoid and terpene profiles 10 were further subjected to agglomerative hierarchal clustering Changes in variety color classification and naming. (AHC) using XLStatto classify varieties into "classes'. Vari eties in a given class of Cannabis share certain common Old Name New Name physiological, chemical and/or morphological characteris BLU4 BLU04 tics. Thus, according to the present invention, Cannabis 15 BLU8 REDO8 BLU9 GRE09 plants are grouped into named classes according to their GRE GREO1 primary/dominant flavor(s) in order to establish standard VLT Cannabis classes of plants herein referred to collectively as GO13 GOD13 Classes of Cannabis Varieties. GOD3 GODO3 GOD2 GODO2 As explained in greater detail below, individual Cannabis GOD11 GOD11 plants of the proprietary Cannabis varieties were identified, GOD10 GOD10 tested and grouped to form class categories of similar variet BLUS SILO6 ies. According to the present invention, more than one variety GOD12 SIL12 GOD8 SILO8 of Cannabis may have been established within a single Can RED2 SILO3 nabis class. Selected candidate Cannabis plants for a specific 25 RED1 SILO2 variety may have been subjected to further breeding and SIL SILO1 WHI2 SILO)4 selection before being chosen as a Cannabis variety for a GODS WHIO2 particular class. The final selected varieties were designated WHIT WHIO7 as Classes of Cannabis Varieties. Therefore, as used herein, GOD6 WHIO6 Classes of Cannabis Varieties or variety classes or the like 30 GO14 WHI14 SIL4 WHIO4 each refer to certain Cannabis varieties originating from pro CHM1 WHIO1 prietary varieties, wherein they were selected based on cer SILOS WHIO5 tain desirable phenotypical characteristics and morphologi GOD4 WHIO9 cal characteristics for a particular class of Cannabis. Color THCO1 BLKO1 class parental Cannabis tended to be chemotype I plants. 35 THCO2 BLKO2 THCO3 BLKO3 Table 4 summarizes the classes of the Cannabis varieties THCO)4 BLKO4 provided by the present invention and the Class color, Class YEL3 YELO3 name, Class abbreviations (ABRV), flavor associated with YELO5 YELO5 each class, and the major terpenes measured in each class. TABLE 4 Color class characteristics of THC parental varieties. Color Abbrev Class Terpene Characteristics Flavor Azure AZR myrcene >> limonene > caryophyllene Woody, fruity Black BLK caryophyllene > limonene > myrcene Camphoreous, baked Sweets Blue BLU pinenes > myrcene > caryophyllene Berry, terpy, solvent Bronze BRZ. limonenes myrcene > caryophylene Sweet, lemons Brown BRO myrcene >> Ocimene > pinene Musky, Sweet, apple cider Fuscia FSC myrcene, caryophyllene Fuel, grass, baked lemon Gold GOD limonene = caryophylene = myrcene Lemon, pine-Sol, fuel Green GRE myrcene > limonene socimene Sweet, cream, citrus, skunk Grey GRA myrcene, pinene, limonene Woody, green, Sweet, bubblegum, pine Jade JAD terpinolene, myrcene Sweet, pepper, spice Lemon LMN limonene > myrcene socimenes caryophyllene Citrus, Sweet, spice Magenta MAG myrcene >> Ocimene > limonenes caryophyllene Sweet, orange peel, spice Navy NVY myrcene > pinene > limonene socimene Sweet, pine, citrus Olive OLV myrcene >> Ocimenes limonene Sweet, orange, lemon Orange ORA myrcene, terpinolene, Ocimene, pinene Intense orange peel, Sweet Pink PNK myrcene socimene spinene Sweet, orange, pine Purple PUR myrcene, pinene, caryophyllene Grapes, pine, Sweet, pineapple, berry, floral, acrid, menthol Red RED ocimenes limonene spinene Floral, vanilla, Skunk Sea SEA limonenes caryophylenes myrcene > Ocimene Lemons, pepper, Sweet Silver SIL limonene, > caryophyllene, myrcene Lime, pomegranate, creamy, blueberry, spice, menthol Tan TAN myrcene > Ocimene > limonene > pinene Sweet, citrus, pine Violet VLT myrcene socimene Sweet, oranges White WHI limonene = caryophylene, > myrcene Berry, lime, skunk, fuel, incense, citrus, pine Yellow YEL terpinolene, Ocimene, myrcene Lemon, pine, skunk US 9,370,164 B2 83 84 TABLE 5-continued TABLE 5-continued Changes in variety color classification and naming. Changes in variety color classification and naming.
Old Name New Name 5 Old Name New Name
PUR2 YELO2 GOD7 GRAO7 K11 AD11 GRE31 GRA31 K12 AD12 WHI4 GRAO4 CK4 ADO)4 WHIS GRAOS ORA3 ORAO3 ORA2 ORAO2 10 PUR1 PURO1 The cannabinoid and terpene profiles of eachTHC parental PUR3 PURO3 variety were determined using both GC-FID and HPLC as GRA3PURS PURO5PUR13 described in Example 1. The resulting measurements are BLU6 PURO6 summarized in Tables 6, 7, 8, and 9. The GC-FID cannabinoid So I 15 analysis of Table 6 also included measurements for THCV. WHI3 PUR12 CBDV, CBGV, CBN, and delta 8 THC, all of which were GRE FSCO)4 measured to be less than 0.05% and were therefore not CHM3 FSC03 included in the table. Similarly, the HPLC cannabinoid analy SS iOS, sis of Table 7 included measurements for THCV, THCVA, ORA4 BROO2 20 CBDV, CBDVa, CBGV, CBGVA, CBC, CBCA, CBD, and PNK CBN, all of which were measured to be less than 0.01%, and were therefore not included in the table. TABLE 6 Cannabinoid measurement by GC-FID for THC color class parental varieties. Blank values indicate undetectable levels or 0. GC-FID THC Color Class Parental Lines THC CBD CBG CBC Cannabs by GC THC:CBD by GC Cannabs/Terps (GC Sample Wt 9%. 95% CI Wt 9%. 95% CI Wt 9%. 95% CI Wto 95% CI Wt 9/o 95% CI Wt 9/o 95% CI Wt 9% 95% CI BLKO1 18.82% 0.51% 0.23% 19.56% 13.77 BLKO2 20.23% 1.37% O.37% 21.97% 18.78 BLKO3 16.54% O.71% 0.43% 17.67% 12.06 BLKO4 20.70% O.S8% O.19% 21.90% 12.37 BLUO4 7.52% O.16% O.16% O.10% 7.98% 47.26 5.94 BROO1 12.23%. 2.32% O.S2% 0.01% O.26% O.07%. 13.08% 2.27% 9.01 3.27 BROO2 13.47% 0.97% 0.02% 0.02% 0.74% 0.06% O.16% O.02%. 14.44% O.90%. 1036.07 1147.94 12.66 2.88. FSCO)4 12.49% O.34% 0.42%. 13.29% 6.62 FSCO3 16.20% 1.84% O.29% 0.14% O.16% O.03%. 16.71% 1.91% 10.08 1.71 FSCO2 17.57% O.S190 O.19%. 18.39% 7.89 GOD13 19.79% 2.10% O.S3% 0.07% O.18% O.02%. 20.55% 2.17% 7.30 O.S6 GODO3 21.12% O.82% O.17%. 22.19% 10.13 GODO2 19.36% 1.70% O.64%, 1.08% O.16% O.01%. 20.22% 2.75% 8.72 3.85 GOD11 20.45% 1.00% O.15%. 21.65% 10.38 GOD10 21.37% O.97% O.18%. 22.58% 9.00 GRAO7 16.07%. 1.25% O.43% 0.03% O.18% O.07%. 16.74%. 1.22% 7.71 O.83 GRA31 11.43%. 2.35% O.13% 0.09% O.21% O.09%. 11.81% 2.54% 9.19 120 GRAO4 8.30% 1.56% O.O1% 0.00% O.06% 0.01% O.15% O.01% 8.54%. 1.56%. 1193.88 110.99 8.77 1.65 GRAOS 11.52%. 1.45% 0.01% 0.00% O.14% 0.05% O.32% O.03%. 12.02%. 1.50% 850.84 193.14 7.01 1...SO GREO9 7.97% O.31% O.16% 8.48% 11.30 GREO1 16.43% 0.83% 1.31% 0.08% O.31% O.04%. 18.10% 0.90% 9.59 1.57 JAD11 12.45% O.S.4% O.25% 3.28% 5.41 JAD12 8.32% O.47% O.19% 9.03% 5.23 JADO4 10.29% O.67% 0.18%. 11.20% S.80 ORAO2 11.83% 0.79% O.S.0% 0.04% 0.13% O.01%. 12.50% 0.83% 9.32 1.53 ORAO3 11.60% 1.23% 0.03% 0.04% 0.20% 0.04% O.18% O.01%. 12.03%. 1.25% 818.90 1083.82 7.33 2.92 PURO3 15.52%. 1.12% O.32% 0.07% O.30% O.03%. 16.19%. 1.17% 9.28 O.64 PURO1 11.45%. 1.01% O.25% O.06% O.16% O.03%. 11.91% 0.98% 7.06 O.81 PUR13 16.13%. 2.35% O.75% 0.06% O.19% O.02%. 17.14% 2.38% 9.45 1.30 PURO6 14.08% 2.66% O.16% 0.03% O.17% O.03%. 14.48% 2.70% 9.56 2.93 PUROS 18.96% 0.92% O.39% 0.01% O.32% O.01%. 19.70% O.91% 6.45 O.30 PUR11 13.49% O.41% O.15% 4.09% 9.93 PUR13 9.89% O.16% O.18% O.26% 6.55 PUR12 13.89% 2.42% O.31% 0.10% O.15% O.03%. 14.39% 2.43% 12.44 2.26 REDO8 8.42% 0.88% 1.18% 0.67% O.22% O.11% 9.86%. 1.38% 1427 2.15 SILO4 15.27%. 1.28% O.41% 0.06% O.19% O.03%. 15.92%. 1.36% 7.02 O.68 SILO6 1.25%. 1.09% O.39% 0.03% O.40% O.07%. 12.09%. 1.14% 14.25 1.07 SILO8 17.15%. 1.43% O.23% 0.11% O.15% O.02%. 17.59%. 1.39% 10.78 2.17 SILO3 3.37% 0.90% O.19% 0.02% O.16% O.04%. 13.74% 0.96% 11.07 18O SILO2 15.00% 2.38% O.12% 0.00% O.17% O.02%. 15.30% 2.39% 9.03 O.10 SILO1 4.23%. 2.05% O.40% 0.03% O.16% O.01%. 14.88%. 2.07% 10.89 1.36 WHIO7 15.44%. 1.75% O.24% 0.03% O.17% O.01% 15.92%. 1.78% 8.73 1.OS WHIO4 15.97%. 1.40% O.S.0% 0.20% O.16% O.02%. 16.76%, 1.65% 12.93 S.49
US 9,370,164 B2 87 88 TABLE 7-continued Cannabinoid measurement by HPLC for THC color class parental varieties. Blank values indicate undetectable levels, or 0. Cannabinoids (UHPLC Cannabs/Terps
THCA CBDA CBGA THC CBG Cannabs by HPLC HPLC Sample Wt 9%. 95% CI Wt 9%. 95% CI Wt 9%. 95% CI Wt 9%. 95% CI Wt 9% 95% CI Wt 9% 95% CI Ratio 95% CI WHIO2 22.19% 0.61% O.15% 23.09% 16.34 YELO3 16.71% 0.17% O.74% 0.03% 0.33%. O.10% 17.99% O.35% 12.66 1.99 YELOS 19.65% 3.39% 1.93% 0.41% 0.85%. O.11% 22.52% 3.81% 9.41 2.03 YELO2 13.97%. 1.29% 1.19% 0.33% 0.20% O.14% 12.62% 5.779, 6.65 3.11
*LOQ for all cannabinoids was 0.14%.
US 9,370,164 B2 95 96 TABLE 9 Relative terpene levels as measured by GC-FID for THC color class parental varieties. Blank values indicate undetectable levels, or 0. Terpenes ter- alpha beta gamma alpha beta alpha alpha beta pino- phellan- oci- limo- ter- alpha ter pin- fen- camp- ter- humu- caryo- lina- cary myr Sample lene drene mene carene nene pinene pinene pinene ene chol hene pineol lene phyllene lool oxide cene
4%. 3% 0%. 3% 12% 32% 10% 0%. 12% 4%. 3% 0%. 3% 12% 35% 10% 0%. 12% 3%. 2% 0%. 3% 15% 41% 10% 0% 8% 29, 290 0%. 3% 13% 39% 9% 0% 9% 18% 19 O%. 3% 3% 9% 3% 0% 15% 290 0% 0% O% 190 3% 4% 0% 73% 290 0% 0% O% 2% 79% 4% 0% 54% 190 0% 0% O% 8% 23% 3%. 490 S190 29, 190 0%. 2% 10% 26% 4% 0%. 39% 29, 190 0%. 2% 10% 25% 3% 0%. 43% 5%. 3% 0%. 3% 59% 1796 6%. 190 33% 5%. 2% 0%. 2% 6% 20% 5% 0%. 23% 4%. 2% 19 3% 79% 25% 6% 19 25% 4%. 2% 19 2% 6% 22% 6% 19 29% 4%. 2% 0%. 2% 6% 22% 6% 0%. 27% 3%. 2% 0%. 2% 2% 8% 9% 0% S6% 6% 19 O%. 3% 190 4% 5% 0%. 60% 5%. 190 0%. 2% 190 4% 3% 0%. 66% 7%. 190 0% 190 3% 79% 290 0%. 59% 5%. 2% 0%. 3% 2% 79% 4% 0%. 33% 4%. 2% 0%. 2% 4% 296 5% 0%. 33% 4% 0% 0%. 2% 2% 59 O%. 49/o 15% 5% 0% 0%. 2% 2% 79% O% 6%. 10% 5% 0% 0%. 2% 3% 296 190 3%. 11% 4% 19 O%. 2% 59% % 290 0%. 26% 6% 0% 0%. 2% 190 4% 29, 290 40% 4% 0% 0% 0% 5% 9% 6% O% 40% 7%. 3% 0%. 3% 3% 4% 4% 0%. 35% 8% 0% 0% O% 4% % 290 0%. 39% 11% 19 O% 190 4% % 4% 19 42% 3% 0% 0% O% 4% 4% 6% 0% 50% 7% 0% 0% O% 3% 9% 290 0%. 44% 11% 0% 0% O% 59% 296 O% 0%. 38% 8% 290 0% O% 59% % 290 0%. 44% 7%. 490 0%. 490 3% % 3% 0% 3% SILO4 O% 0%. 5% 0%. 31% 0% 3% 0% 5%. 490 19 496 6% 21% 9% 0% 9% SILO6 O% 0%. 12% 0%. 35% 0% 6% 0% 9% 6% 0% 6% 2% 9% 3% 0% O% SILOS 0% 0% O% 0%. 34% 0% 3% 0% 6%. 490 0%. 490 3% % 16% 0% 79% SILO3 O% 0% O% 0%. 31% 0% 3% 0% 5%. 3% 0%. 3% 4% 4% 14% 0%. 20% SILO2 O% 0% O% 0%. 35% 0% 3% 0% 6%. 490 0%. 490 6% 22% 13% 0% 6% SILO1 O% 0% O% 0%. 30% O% 3% 0% 5%. 490 0%. 490 59% 20% 14% 0% 4% WHIO7 O% 0%. 3% 0%. 31%. 3% 496 O% 5%. 3% 0%. 490 8% 31% 3% 0% 6% WHIO4 O% 0% O% 0%. 33% 0% 2% O% 5%. 3% 0%. 3% 79% 28% 8% 0% O% WHIO9 O% 0% O% 0%. 29% 0% 2% O% 5%. 3% 0% O% 12% 33% 3% 0% O% WHIO1 O% 0% O% 0%. 27% 0% 2% O% 4%. 3% 0%. 3% 11% 31% O% 0% 79% WHI14 O% 0% O% 0%. 28% 0% 2% O% 4%. 3% 0%. 490 10% 28% 10%. 190 190 WHIOS O% 0% O% 0%. 29% 0% 3% 0% 5%. 3% 0%. 2% 10% 37% 29, 190 9% WHIO6 190 0% O% 0%. 33% 0% 2% O% 5%. 3%. 196 190 10% 29% 3%. 190 190 WHIO2 O% 0% O% 0%. 28% 0% 3% 0% 5%. 2% 0% O% 13% 34% 3% 0% O% YELO3 35% 2%. 11%. 2%. 10% 0% 3% 196 5%. 5% 0%. 2% 2% 8% 29 O% 190 YELOS 43%. 2%. 12%. 2% 6%. 196 3%. 2% 5%. 190 0%. 2% 4% 11% O%. 190 8% YELO2 35% 2%. 15%. 196 9%. 196 3% 196 5%. 190 0%. 2% 2% 79% 3% 0% 190
Example 4 Example 1. The cannabinoid and terpene profiles of each CBS parental variety was determined using both GC-FID and Analysis of CBS Parental Varieties 60 HPLC as described in Example 1. The resulting measure ments are summarized in Tables 10-22 as average values and A. Proprietary CBs Parental Varieties 95% confidence interval ranges based on five replicate mea One objective of the present invention was to develop surements. The GC-FID cannabinoid analysis of the CBD Cannabis varieties producing non-THC cannabinoids (CBs) parental varieties in Table 10 also included measurements for with high terpene oil contents and different terpene profiles to 65 THCV, CBDV, CBGV, CBN, and Delta 8 THC, all of which meet various aroma/flavor and medicinal needs. Chemical were measured to be less than 0.01%, and were therefore not analysis of these CBs varieties was conducted as described in included in the table. Similarly, the HPLC cannabinoid analy US 9,370,164 B2 97 98 sis of the CBD parental varieties in Table 11 included mea other embodiments of the present invention, the THVO1 surements for CBCA, THCVA, CBDVA, CBGVA, CBC, parental line can be crossed with chemotype II varieties to THCV, CBDV, CBGV, and CBN, all of which were measured produce THCV and CBDV cannabinoids. In yet other to be less than 0.01%, and were therefore not included in the embodiments, the THVO1 parental line can be crossed with table. CBG accumulating varieties described to produce CBGV As can be seen in Tables 10 and 11, CBD01, 24, 11, and 13 accumulating plants. are chemotype III varieties, with a B/B genotype respon The present invention also teaches the use of two sources of sible for producing CBD, or CBDA (as measured by HPLC). CBG genetics. The first set of CBG-producing parental lines The other parental CBD lines (CBD02-05), have beenbred to are plants BLKO2, GOD11, GRE01, RED08, and YEL05 of be chemotype II plants with B/B genotypes producing both 10 the THC parentals in Table 6. While not wishing to be bound THC and CBD. These proprietary lines were bred for more by any one theory, the inventors of the present invention desirable terpene profiles through multiple rounds of crosses believe that the CBG produced by these plants is due to the with THC class varieties and selfing to obtain desired genet incomplete processing of CBG by the THC and CBD syn ics. CBD05 exhibits several desirable features such as the 15 thase enzymes. This may be caused by an over production of production of both THC and CBD, as well as a terpene profile CBG, or the inefficient processing of the THC synthase that is not dominated by myrcene (Table 13). enzymes of the plant. Progeny of these parental lines are THCV-producing parental line THVO1 was also bred for expected to produce low levels of CBG in combination with its ability to produce propyl CBGV. THVO1 does not accu other cannabinoids and desirable terpene profiles. mulate CBGV due to its conversion to THCV by THC syn- * B. Additional CBs Parental Varieties thase. The GC-FID cannabinoid analysis of the THVO1 Another source of CBG-producing parental lines is CBG parental line in Table 14 also included measurements for variety CBG02 in Table 18. While not wishing to be boundby CBDV, CBGV, CBN, and Delta 8 THC, all of which were any one theory, the inventors of the present invention believe measured to be less than 0.01%, and were therefore not 25 that the CBG accumulation in this variety is due to the pres included in the table. Similarly, the HPLC cannabinoid analy- ence of a null allele (B). Progeny of these parental varieties sis of the THVO1 parental line in Table 15 included measure- are expected to produce higher levels of CBG, alone, or in ments for CBDA, CBCA, CBDVA, CBGVA, CBD, CBC, combination with other cannabinoids and desirable terpene THCV, CBDV, CBGV, CBN, and delta 8 THC, all of which profiles. The HPLC cannabinoid analysis of the CBG02 were measured to be 0, and were therefore not included in the 30 parental line in Table 19 included measurements for CBDA, table. CBGV is produced by combining divarinic acid and CBCA, THCVA, CBDVA, CBGVA CBD, CBC, THCV, geranylpyrophosphate. This is regulated by locus A which CBDV, CBGV, CBN, and delta 8 THC, all of which were can encode for enzymes to generate pentyl CBG (A) or measured to be less than 0.09, and were therefore not included propyl CBGV (A.) (De Meijer et al. 2009 Euphytica, 165: 3s in the table. 293-31). Thus if crossed with a CBD producing chemotype II A CBC parental variety will be obtained by screening plant, the THCV locus A is expected to produce both THCV plants for CBC accumulation in mature tissue. While it is and CBDV. As can be seen in Tables 14 and 15, the parental believed that CBC biosynthesis is a feature of juvenile tissue, THVO1 line contains at least one allele encoding for propyl several reports have published reports suggesting the exist cannabinoids with THC and THCV cannabinoids accumulat- " ence of Cannabis varieties accumulating CBC in older tissue ing at roughly equal amounts. The alleles of locus B appear to (De Meijer et al., 2009 “The inheritance of chemical pheno be B/B, with no significant accumulation of CBD. Further, type in Cannabis sativa L. (III): variation in cannab these THC synthase genes appear to be functioning efficiently ichromene proportion'). Table 22 outlines some of the pub converting nearly all CBG and CBGV into THC and THCV as lications describing varieties with CBC accumulation that respectively. In some embodiments, the THVO1 parental line will be analyzed for high CBC accumulation. The best vari may be crossed with class varieties to produce THCV pro- eties identified through chemical and phenotypical analysis ducing specialty Cannabis with desirable terpene profiles. In will be designated as CBC01-CBCO5. TABLE 10 Cannabinoid measurement by GC-FID for CBD parental varieties. Blank values indicate undetectable levels or 0. Cannabinoids (GC-FID THC CBD CBG CBC Cannabs by GC THC:CBD by GC Cannabs/Tarps (GC Sample Wt 9%. 95% CI Wt 9%. 95% CI Wt 9%. 95% CI Wt 9%. 95% CI Wt 9%. 95% CI Wt 9% 95% CI Ratio 95% CI
CBDO1 O.42% 0.08%. 11.13% 0.01% 0.41% 0.10% 0.60% 0.05%. 12.56% O.23% O.04 O.O1 5.33 O.36 CBDO3 3.48% 0.73% 6.77%. 1.37% 0.27% 0.06% 0.46% O.06%. 10.99%. 2.19% O.S1 O.04 11.90 2.19 CBDO2 1.96%. 1.78% 4.53% 3.98% O.35% 0.28% 7.08% 6.36% O.43 O.O2 3.81 3.37 CBDOS 4.13% 0.36% 7.99% O.75% 0.30% O.08% 0.55% 0.05%. 12.96% 0.56% O.S2 O.09 8.06 1.21 CBDO4 S.24% 5.74% O.16% O.44% 11.65% O.91 13.19 CBD24 O.19% 8.03% O.19% O.47% 8.87% O.O2 9.01 CBDO11 0.18% 6.03% O.10% O.47% 6.78% O.O3 7.76 CBD13 O.25% 8.20% O.14% O.S9% 9.18% O.O3 4.03
*LOQ for all cannabinoids was 0.14%.
US 9,370,164 B2 101 102 TABLE 12-continued Absolute terpene measurements by GC-FID for CBD parental varieties. Blank values indicate undetectable levels or 0. Terpenes (GC-FID) Total alpha beta cary identified humulene caryophyllene linalool oxide myrcene oil (wt %)
Wt 95% Wt 95% Wt 95% Wt 95% Wt 95% Wt 95% Sample % CI % CI % CI % CI % CI % CI
CBDO1 O.269% O.OO3% O.O29% O.13.1% O.OO19 1122% 0.01.2% 2.360% 0.2O2% CBDO3 O.O27% O.OO8% 0.077% O.O22% O.O.34% 0.005% O.O.30% O.005% 0.4.19% 0.097% 0.949% 0.204% CBDO2 O.O46% OO15% 0.146% O.O2.0% O.O49% O.OS4% O.877% 0.208%. 1841% 0.139% CBDOS O.O90% OO15% 0.234% 0.048%. O.O.92% 0.049%. O.O14% O.358% 0.1.52%. 1635% 0.331% CBD04 O.O68% O.21.6% O.065% O.23.0% O.883% CBD24 O.O42% O.13.1% 0.0579. OSO3% O.984% CBD11 O.O16% O.O60% O.O.28% O.S26% O.874% CBD13 O.O62% O.204% O.O.22% 1.450% 2.28.0% *LOQ for all terpenes was 0.02% except for alpha-pinene, linalool, and alpha-terpineol which were 0.04%.
TABLE 13 Relative terpene levels as measured by GC-FID for CBD parental varieties. Blank values indicate undetectable levels or 0. Terpenes ter- alpha beta gamma alpha alpha alpha beta pino- phellan- oci- car- limo- ter- alpha ter- beta fen- camp- ter- humu- caryo- lina- cary myr Sample lene drene mene ene nene pinene pinene pinene pinene chol hene pineol lene phyllene lool oxide cene CBD1 4% 19% 8% 196 190 11% 190 6%. 4.8% CBD3 6% 22% 9% 296 2% 3% 8% 4% 3%. 44% CBD2 6% 23% 10% 1% 2% 8% 3% 3%, 4.8% CBDS 79% 4% 28% 79% 14% 59% 14% 6% 19 22% CBD4 21% 3% 3% 6% 8% 24% 79% 26% CBD24 1796 190 3% 296 2% 4% 13% 6% 51% CBD11 18% 190 2% 196 59% 2% 79% 3% 60% CBD13 8% 8% 4% 196 3% 3% 9% 190 64%
TABLE 1.4 Cannabinoid measurement by GC-FID for THCV parental varieties. Blank values indicate undetectable levels. Cannabinoids (GC-FID
THC: Cannabs THCV Cannabs THC CBD CBG CBC THCV by GC by GC Terps (GC
95% 95% 95% 959, 95% 95% 95% 95% Sample Wt 9% CI W9% CI W9% CI Wt 96 CI Wt 9% CI Wt 9% CI Wt 9% CI Wt 9%. CI
THVO1 4.52%. 3.22% 0.01% O.S6% 0.39% 0.05% 0.03% 3.27%. 1.81% 8.40% S.46%. 135 0.24 S36 2.33
*LOQ for all cannabinoids was 0.14%.
TABLE 1.5 Cannabinoid measurement by HPLC for THCV parental varieties. Blank values indicate undetectable levels, or 0. Cannabinoids (UHPLC
THCA: Cannabs THCVA Cannabs/Terps THCA CBGA THCVA THC CBG by HPLC by HPLC HPLC
95% 95% 95% 9596 95% 95% 95% 95% Sample Wt 9% CI Wt 9% CI Wt 96 CI W9% CI Wt 9% CI Wt 96 CI Wt 9% CI Wt 9% CI
THVO1 4.05% O.S8% 3.85% O.22% O.06% 4.38% 8.58%. 1.05 6.50
*LOQ for all cannabinoids was 0.14%. US 9,370,164 B2 103 104 TABLE 16 Absolute terpene measurements by GC-FID for THCV parental varieties. Blank values indicate undetectable levels or 0. Terpenes (GC-FID) alpha beta gamma terpinolene phellandrene ocimene C8tle limonene terpinene
95% 95% 95% 95% 95% Wt 95% Sample Wt 9% CI W9% CI W9% CI Wt 96 CI Wt 9% CI % CI
THVO1 O.297% 0.008% O.192% O.O.31% Terpenes (GC-FID) alpha alpha alpha pinene terpinene beta pinene fenchol camphene terpineol
95% 95% 95% 95% 95% 95% Sample Wt 9% CI W9% CI W9% CI Wt 96 CI Wt 9% CI Wt 9% CI
THVO1 O.O63% 0.028% O.O39% 0.013% 0.023% 0.009% O.O29% O.O.12% Terpenes (GC-FID)
Total alpha beta identified humulene caryophyllene linalool cary oxide myrcene oil (wt %)
95% 95% 95% 95% 95% 95% Sample Wt 9% CI W9% CI W9% CI Wt 96 CI Wt 9% CI Wt 9% CI
THVO1 O.O86% O.O.28% 0.175% 0.098% O.O29% 0.01.2% O.S.97% 0.1.15%. 1528% 0.354%
TABLE 17 Relative terpene levels as measured by GC-FID for THCV parental varieties. Blank values indicate undetectable levels or 0. Terpenes alpha beta gamma alpha alpha beta Sample terpinolene phelland rene ocimene C8tle limonene terpinene pinene terpinene pinene
THVO1 1996 13% 4% 3% Terpenes alpha alpha beta Sample fenchol camphene terpineol humulene caryophyllene linalool cary oxide myrcene
THVO1 196 2% 6% 11% 2% 39%
TABLE 1.8 Cannabinoid measurement by GC-FID for CBG parental varieties. Blank values indicate undetectable levels or 0. Cannabinoids(GC-FID) THC CBD CBG CBC THCV CBDV
95% 95% 95% 9596 95% 95% Sample Wt 9% CI Wt 96 CI Wt 96 CI W9% CI Wt 9% CI Wt 96 CI
CBGO2 8.41% O.02% 2.66% O.17% Cannabinoids(GC-FID) Cannabs by THC: CBG Cannabs CBGV CBN D8-THC GC by GC Terps (GC)
95% 95% 95% 9596 95% 95% Sample Wt 9% CI Wt 96 CI Wt 96 CI W9% CI Wt 9% CI Wt 96 CI
CBGO2 O.15% 11.40% 3.16 13.13 US 9,370,164 B2 105 106 TABLE 19
Cannabinoid measurement by HPLC for CBG parental varieties. Blank values indicate undetectable levels or 0.
Cannabinoids (UHPLC)
THCA: Cannabs CBGA Cannabs THCA CBGA THC CBG by HPLC by HPLC Terps (HPLC)
95% 95% 95% 95% 95% 95% 95% Sample Wt 9% CI W9% CI Wt 96 CI Wt 9% CI Wt 9% CI Wt 90 CI Wt 9% CI
CBGO2 8.84% 3.89% 2.59% O.35% 15.75% 2.27 18.15
*LOQ for all cannabinoids was 0.14%.
TABLE 20 Absolute terpene measurements by GC-FID for CBG parental varieties. Blank values indicate undetectable levels or 0. Terpenes (GC-FID) alpha beta gamma terpinolene phellandrene ocimene C8tle limonene terpinene
Wt 95% 95% 95% W 95% 95% Wt 95% Sample % CI Wt 96 CI W9% CI 90 CI Wt 9-0 CI % CI
CBGO2 O.267% O.O.30% Terpenes (GC-FID) alpha alpha beta alpha pinene terpinene pinene fenchol camphene terpineol
95% Wt 95% 95% W 95% Wt 95% Wt 95% Sample Wt 9% CI % CI W9% CI 90 CI % CI % CI
CBGO2 0.066% O.O.26% Terpenes (GC-FID) Total alpha beta cary identified on humulene caryophyllene linalool oxide myrcene (wt %)
95% 95% 95% W 95% 95% 95% Sample Wt 9% CI W9% CI Wt 96 CI 90 CI Wt 96 CI Wt 96 CI
CBGO2 O.O76% O.2009 O.O.59% O.144% O.868%
*LOQ for all terpenes was 0.02% except for alpha-pinene, linalool, and alpha-terpineol which were 0.04%.
TABLE 21 Relative terpene levels as measured by GC-FID for CBG parental varieties. Blank values indicate undetectable levels or 0. Terpenes alpha beta gamma alpha alpha beta Sample terpinolene phelland rene ocimene C8tle limonene terpinene pinene terpinene pinene
CBGO2 31% 3% 8% 3% Terpenes alpha alpha beta Sample fenchol camphene terpineol humulene caryophyllene linalool cary oxide myrcene
CBGO2 9% 23% 79% 1796 US 9,370,164 B2 107 108 TABLE 22 CBD do not produce the pleasing organoleptic attributes of contemporary high-THC marijuana cultivars. Indeed, all Sources of CBC parental varieties. known chemotype II or chemotype III plants produce Reference for Cannabis Varieties Accumulating CBC myrcene dominant terpene profiles which do not have pleas Baker, PB et al., (1983) “The Physical and chemical features of Cannabis ing aroma/flavor, and do not have the entourage effects plants grown in the United Kingdom of Great Britain and Northern Ireland from Seeds of known origin- Part II: Second generation brought on by higher levels of non-myrcene terpenes. Thus, studies. Bull Narc 35:51-62. an objective of the present invention is to combine THC with Beutler JA, and Der Marderosian AH (1978) “Chemotaxonomy of higher CBS and diverse terpene profiles so as to produce Cananbis I. Crossbreeding between Cannabis sativa and C. ruderalis, with specialty Cannabis varieties with these pleasing aromas and analysis of cannabinoid content. Econ Bot 32:387-394. 10 Yotoriyama, Met al., (1980) “Plant breeding of Cannabis. Determination flavors that were unavailable until the present invention. of cannabinoids by high-pressure liquid chromatography. Yakugaku In other embodiments, the breeding programs of the Zasshi 100: 611-614. present invention were designed to produce THC:CBs Holley et al., (1975) “Contituents of Cannabis sativa L. XI: cannabidiol and cannabichromene in samples of known geographical origin. J expressing plants with higher terpene oil content. In some Pharm Sci 64: 892-894. 15 embodiments, the higher oil contents of the specialty Can nabis of the present invention produce pleasing aromas/fla vors. In other embodiments the higher oil levels of the spe Example 5 cialty Cannabis of the present invention allows the terpenes reach high enough levels to reduce THC side effects. In some Breeding Scheme for New Specialty Cannabis embodiments, the higher terpene oil contents of the specialty Varieties Cannabis of the present invention increase the amount of entourage effects of the terpenes in the terpene profile. In In another objective of the present invention, the Cannabis Some embodiments, the specialty Cannabis plants of the varieties of Examples 2-4 are used in Cannabis breeding present invention produce myrcene dominant plants with programs to develop specialty Cannabis plants and varieties. 25 improved aroma/flavor and entourage effects by increasing Furthermore, the specialty Cannabis varieties developed the terpene oil content. according to the present invention have specific aromas, fla One embodiment of the present invention is to produce Vor(s), and entourage effects in accordance with one of the specialty Cannabis varieties with highessential oil content, in classes of Cannabis varieties as discussed above. particularly, mono- and sesquiterpenes. The breeding objec This approach was designed in part, as a response to the 30 tives of the present invention are opposite to the face of fact that currently available Cannabis varieties have been skewed towards higher THC production, which has increased modern recreational marijuana breeding strategies which the likelihood of adverse effects from the elevated levels of have focused almost solely on breeding for higher levels of psychoactivity that these conventional high-THC varieties THC content alone. produce. 35 According to one embodiment of the present invention a Contemporary “recreational marijuana cultivars have THC class variety is crossed to a CBS producing line to been exclusively bred and selected primarily for their THC produce F1 seed which were grown to produce F1 progeny. acid content, secondarily (if at all) for their terpenoid aroma The resultant F1 progeny can be fixed through asexual repro and flavor chemistry, and rarely for their production of the duction and/or used in further breeding schemes. Five CBD other cannabinoid acids, such as CBDA. 40 lines were chosen to use in the initial breeding program: Cannabidiol (CBD), a cannabinoid that is rare in contem CBD1, CBD2, CBD3, CBD4 and CBD5 (see Example 3). porary Cannabis varieties, has been shown to reduce and Similarly, THC class varieties can be crossed to the THVO1, modulate the psychoactivity of THC and also reduce some of CBC01, and CBG01 parental varieties of the present inven THC's other adverse effects including tachycardia, anxiety, tion. According to one embodiment of the the present inven memory effects, etc. There is some evidence that CBD may 45 tion, each of these CBD, THCV, and CBG lines is crossed to reduce the buildup of tolerance to the effects of THC and also one or more Cannabis varieties which are described above reduce the likelihood of Cannabis dependency. Other can and Summarized in Example 3. In another embodiment, the nabinoids (CBs) such as CBDv, THCV, CBG, CBN, etc have present invention teaches crosses of any of the parental vari also recently been demonstrated to have a variety of medical eties with each other. Thus, for example, one or more GOLD and recreational uses. 50 Class varieties are crossed to each of CBD1, CBD2, CBD3, In some embodiments, the breeding programs of the CBD4, CBD5,THV01, CBC01, or CBG plants to produce F1 present invention were designed to combine THC with non populations to create (GOLD ClassXCBD: GOLD Classx THC CBs. Furthermore, the specialty Cannabis varieties of THV01; or GOLD ClassxCBG) combinations. In some the present invention were additionally selected for their abil embodiments, CBS producing parental varieties may also be ity to produce terpenes that are appealing to patients and that 55 crossed among themselves (e.g., CBD05 selfed, or CBD05x may also provide a pharmacological activity that modifies, THVO1) Following is a list of the iterations for each of the enhances or ameliorates the effects of THC. In contrast, pub ClassxCBD. ClassXTHV, and ClassxCBG crosses (Tables 23 licly-available contemporary hemp varieties that are high in and 24). TABLE 23 Example crosses between Color Class Cannabis varieties and CBD parental lines. CBDO1 Crosses CBDO2 Crosses CBDO3 Crosses CBD04 Crosses CBDOS Crosses
AZUREx CBDO1 AZUREX CBDO2 AZUREX CBDO3 AZUREx CBD04 AZUREX CBDOS BLACKx CBDO1 BLACKx CBDO2 BLACKx CBDO3 BLACKx CBD04 BLACKx CBDOS BLUEx CBDO1 BLUEx CBDO2 BLUEx CBDO3 BLUEx CBD04 BLUEx CBDOS US 9,370,164 B2 109 110 TABLE 23-continued Example crosses between Color Class Cannabis varieties and CBD parental lines. CBDO1 Crosses CBDO2 Crosses CBDO3 Crosses CBD04 Crosses CBDOS Crosses
BRONZEx CBDO1 BRONZEx CBDO2 BRONZEx CBDO3 BRONZEx CBD04 BRONZEx CBDOS BROWN X CBDO1 BROWN X CBDO2 BROWN X CBDO3 BROWN X CBD04 BROWN X CBDOS FUSCLAX CBDO1 FUSCLAX CBDO2 FUSCLAX CBDO3 FUSCIA X CBD04 FUSCLAX CBDOS GOLD X CBDO1 GOLD X CBDO2 GOLD X CBDO3 GOLD X CBD04 GOLD X CBDOS GREEN X CBDO1 GREEN X CBDO2 GREEN X CBDO3 GREEN X CBD04 GREEN X CBDOS GREY X CBDO1 GREY X CBDO2 GREY X CBDO3 GREY X CBD04 GREY X CBDOS JADEx CBDO1 JADEx CBDO2 JADEX CBDO3 JADEx CBD04 JADEX CBDOS LEMON X CBDO1 LEMON X CBDO2 LEMON X CBDO3 LEMON X CBD04 LEMON X CBDOS MAGENTAX CBDO1 MAGENTAX CBDO2 MAGENTAX CBDO3 MAGENTAX CBD04 MAGENTAX CBDOS NAVY X CBDO1 NAVY X CBDO2 NAVY X CBDO3 NAVY X CBD04 NAVY X CBDOS OLIVE X CBDO1 OLIVE X CBDO2 OLIVE X CBDO3 O LIVE X CBD04 OLIVE X CBDOS ORANGEx CBDO1 ORANGEx CBDO2 ORANGEx CBDO3 O RANGEx CBD04 ORANGEx CBDOS PINKX CBDO1 PINKX CBDO2 PINKX CBDO3 PINKx CBDO4 PINKX CBDOS PURPLE X CBDO1 PURPLE X CBDO2 PURPLE X CBDO3 PURPLE X CBD04 PURPLE X CBDOS RED X CBDO1 RED X CBDO2 RED X CBDO3 RED X CBD04 RED X CBDOS SEAx CBDO1 SEAx CBDO2 SEA X CBDO3 SEAx CBD04 SEA X CBDOS SILVERX CBDO1 SILVERX CBDO2 SILVERX CBDO3 SILVERX CBD04 SILVERX CBDOS TANX CBDO1 TANX CBDO2 TAN X CBDO3 TAN X CBD04 TAN X CBDOS VIOLET X CBDO1 VIOLET X CBDO2 VIOLET X CBDO3 VIOLET X CBD04 VIOLET X CBDOS WHITEx CBDO1 WHITEx CBDO2 WHITEx CBDO3 WHITEx CBDO4 WHITEx CBDOS YELLOW X CBDO1 YELLOW X CBDO2 YELLOW X CBDO3 Y ELLOW X CBD04 YELLOW X CBDOS
TABLE 24 25 crossed with a CBD variety in order to produce THV, CBDV progeny. Regardless of the exact crossing/selection proce Example crosses between Color Class cannabis varieties dure, selected lines can be chosen so as to have a total THC and other CBS (THCV/CBDV, CBC, CBG) parental lines. contents 90.0%, a total CBS content >1.5%, and a desirable THVO1 Crosses CBCO1 Crosses CBGO2 Crosses 30 aroma and flavor profiles. In another embodiment of the AZURE XTHVO1 AZUREx CBCO1 AZURE x CBGO2 present invention, regardless of the exact selfing/selection LACKXTHVO1 LACKX CBCO1 LACKX CBGO2 procedure, the selected lines can be chosen so as to have a LUE XTHVO1 LUEx CBCO1 LUE X CBGO2 total THC:CBs ratio of greater than 9:1.8:1,7:1, 6:1, 5:1, 4:1, RONZE XTHVO1 RONZEx CBCO1 RONZEx CBGO2 ROWNXTHVO1 ROWN X CBCO1 ROWN X CBGO2 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:9, and lower, and a USCIA XTHVO1 USCIA X CBCO1 USCIA X CBGO2 35 desirable aroma and flavor. OLD XTHVO1 OLD X CBCO1 OLD X CBGO2 According to the present invention, the lines can also be REENXTHVO1 REEN X CBCO1 REEN X CBGO2 REY XTHVO1 REY X CBCO1 REY X CBGO2 further selected for a specific content of certain other cannab JADE XTHVO1 JADEx CBCO1 JADEx CBGO2 inoids and/or of certainterpenes/terpenoids, and/or for addi LEMONXTHVO1 LEMON X CBCO1 LEMON X CBGO2 tional phenotypic and genotypic characteristics. Desirable MAGENTAX THVO1 MAGENTAX CBCO1 MAGENTAX CBGO2 40 NAVY XTHVO1 NAVY X CBCO1 NAVY X CBGO2 phenotypic characteristics includebutare not limited to larger OLIVE XTHVO1 OLIVE X CBCO1 OLIVE X CBGO2 plant size (i.e., greater bulk or biomass), higher production of ORANGE XTHVO1 ORANGEx CBCO1 ORANGEx CBGO2 flower buds, larger flowers, more trichomes, shorter plant PINKXTHVO1 PINKX CBCO1 PINKX CBGO2 stature, ability to tolerate lower and/or higher growing tem PURPLE XTHVO1 PURPLE X CBCO1 PURPLE X CBGO2 peratures, greater germination percentage, greater seedling RED XTHVO1 RED X CBCO1 RED X CBGO2 SEA XTHVO1 SEAx CBCO1 SEAx CBGO2 45 vigor, more efficient water usage, disease resistance, pest SILVERXTEHVO1 SILVERX CBCO1 SILVERX CBGO2 resistance, and other desirable agronomic and production TANXTHVO1 TAN X CBCO1 TAN X CBGO2 traits. For an overview of diseases and pests of importance to VIOLETXTHVO1 VIOLET X CBCO1 VIOLET X CBGO2 WHITE XTHVO1 WHITEx CBCO1 WHITEx CBGO2 Cannabis production see Clarke et al. (2000) Hemp Diseases YELLOW XTHVO1 YELLOW X CBCO1 YELLOW X CBGO2 and Pests. Management and Biological Control. An 50 Advanced Treatise (CABI Publishing). In an alternative version of this breeding regime the In one representative version of this breeding regime the selected F2 progeny are backcrossed to the Class variety as resultant F1 progeny can be selfed to produce F2 seed which the recurrent parent. Selection for desirable phenotypes and/ are grown to produce F2 progeny. Selection for desirable or genotypes can be conducted after this initial backcross, phenotypes and/or genotypes can be conducted within the F1, 55 after any Subsequent backcross (e.g., progeny obtained after F2, or Subsequent progeny since the selections can be main 2, 3, 4, 5, 6, 7, 8, 9 or more backcrosses). In some embodi tained (i.e., fixed) via asexual reproduction. Alternatively, the ments, selected lines will have a total THC contents 90.0%, a F2 progeny can be crossed among themselves to produce a total CBs content a 1.5%, and an aroma and flavor profiles bulked F3 population from which desired progeny can be typical of its class. In other embodiments of this breeding selected and/or further generations of crossing can be con 60 scheme selected lines can be chosen to have a total THC:CBs ducted. In another embodiment, the resultant F1 progeny can ratio of greater than 8:1 and approaching 1:1 and lower, and by backcrossed to the THC class or CBs variety to further an aroma and flavor(s) typical of its class. The lines can also reinforce the traits of other parent. In yet another representa be further selected for a specific content of certain other tive version of this breeding scheme F1, F2, or subsequent cannabinoids and/or of certain terpenes/terpenoids, and/or progeny may also be crossed to additional CBS varieties to 65 for additional phenotypic and genotypic characteristics. create even more complex cannabinoid combinations. For The progeny resulting from any selection stage of either the example, Color Class X THVO1 F1's can be subsequently selfing or backcrossing versions of the breeding regimes of US 9,370,164 B2 111 112 the present invention can be asexually reproduced so as to fix These crosses result in the production of two F1 and maintain the desirable THC content, CBS content, the populations=CBD-GOLD Lines 1A, 1B. Individuals from the F1 lines of each F1 population can be analyzed via aroma and flavor(s) typical of the desired class, and the other GC/MS to determine their respective chemotypes. It is desirable phenotypic and/or genotypic characteristics. The expected that the F1 populations will comprise individuals resultant selected lines will be designated as Specialty Can that show a Chemotype II cannabinoid distribution, with nabis Varieties. intermediate levels of both tetrahydrocannabinol (THC) and The progeny resulting from any stage of either the selfing cannabidiol (CBD). or backcrossing versions of this regime can also be crossed to Plants with suitable terpene contents and profiles can be other Cannabis plants/varieties within, between or among the self-fertilized to create a series F2 segregating populations various classes of Cannabis so as to produce additional plants 10 or families; all non-desirable lines can be rejected from the for selection and maintenance through asexual reproduction. breeding regimen. In this way, a series of F2's can be In this way, specialty Cannabis varieties with various, desired created=1 AF2a, 1 AF2b, 1 AF2c, 1 BF2a, 1 BF2b, 1 BF2c, etc. flavor combinations can be produced and Subsequently main F2 families can be propagated and screened via GC/MS to tained through asexual reproduction. determine individual chemotypes; it is expected that in the F2 The resultant specialty Cannabis plants of the present 15 segregating populations we will see chemotype I, chemotype invention also generally have more terpene essential oil con II, and chemotype III plants. Chemotype I plants can be tent per plant than contemporary marijuana varieties. More discarded and only chemotype II and chemotype III plants essential oil per plant means less plant matter is required per can be retained and again screened by GC/MS to evaluate treatment/administration, thereby also further minimizing their suitability in terms of terpene content and profile. any health risks for medical and recreational Cannabis Smok It may also be desirable to mate selected F1 lines via a ers. This would also further increase production efficiency. backcross Scheme to the P1 GO13 to reinforce the GOLD The inventors of the present invention hypothesized that genetic background, although doing so will re-introduce B(t) breeding plants with increased CBD, or THCV content would alleles (i.e., the alleles that encode for THC production) into alleviate most of the commonly recognized real and per the breeding population, resulting in a population of chemo ceived adverse effects of high THC Cannabis. According to type I and II plants. the present invention, a direct result of increased CBD is 25 lower THC content because THC synthase and CBD synthase Similar breeding schemes may be followed to obtain addi are allelic. Thus, another objective of the present invention tional class-CBD progeny by repeating the steps described was to create specialty Cannabis varieties with an optimal for GO13 with other class varieties and/or CBD parental dose of THC and resulting in the most efficacious ratio of lines. THC:CBD or THCV:THC. 30 Example 6 According to the present invention, it is possible to apply dosage data to creating custom blended granular mixes for Development of THC:CBD Specialty Cannabis rolled delivery, pellets for bowls and house one-hitters, Varieties extracts for dabs, etc. with the flowers of these highly resinous Unique parental THC and CBD lines from Examples 2-4 newly-developed varieties with designed cannabinoid con 35 were selected and one of the parental cultivars was treated tent so as to reduce adverse effects associated with THC. with silver thiosulfate to coax the pistillate plant to produce Gold Class Breeding Regime for THC:CBD Producing staminate, pollen-bearing flowers. The THC and CBD lines Plants. were then crossed, the resulting progeny were screened by Example Basic Breeding Scheme. TLC to identify plants producing both THC and CBD, or The initial cross for the Gold Class CBD Breeding Regime CBD alone. Progeny exhibiting the desired chemotype II and that can be conducted as follows: P1 (GOLD Line (GO13)x 40 III profile were allowed to reach maturity and the flowers P2 (CBD Line (CBD1). The hybrid cross between Parent 1 were harvested and processed. In general, field observations (P1) and Parent 2 (P2) could only beachieved by induction of could detect the crosses with the desired characteristics, how staminate flowers on the pistillate plants by an exogenous ever this was verified by chemotype analysis and the final application of the chemical silver thiosulfate. This process flower was analyzed for cannabinoid and terpene content. allows otherwise pistillate (female) plants to be coaxed to 45 Table 25 outlines the initial crosses performed with THC produce staminate, pollen bearing flowers. During this pro class varieties and CBD parental lines. The crosses produced cess, to investigate and exclude the possibility of maternally progeny approaching ratios Supporting the single locus model inherited genetic factors, reciprocal crosses can be made for THC and CBD synthase. TLC results described in the where both P1 can be induced to produce pollen and fertilize table show the field-determined chemotype of the progeny P2 (Line 1A), and P2 can be induced to produce pollen and (chemotype II THC and CBD producing, and chemotype fertilize P1 (Line 1B). III CBD only). TABLE 25
Crosses performed between class cannabis varieties and other CBD parental lines. TLC result indicates chemotype I, II or III. P Donor CBDOS P Acceptor
CBDOS CBDO3 CBDO2 CBDO2 CBDO4 YELO3
TLC TLC TLC TLC TLC TLC Code Result Code Result Code Result Code Result Code Result Code Result
1 CBDOSXP- III CBDO3XP- III CBDO2xP- - CBDO2xP- II CBDO4xP- - YELO3XP- - O1 O1 O1 31 O1 O1
US 9,370,164 B2 115 116 TABLE 25-continued Crosses performed between class cannabis varieties and o her CBD parental lines. TLC result indicates chemotype I, II or III. P Donor CBDOS
S PURO1XP- - WHIO7xP- - SILO8XP- - SILO8XP WHIO4xP- - WHIO1XP- II 05 05 05 35 05 14 6 PURO1XP- - WHIO7xP- - SILO8XP- - SILO8XP WHIO4xP- - WHIO1XP- III O6 O6 O6 36 O6 15 7 PURO1XP- - WHIO7xP- II SILO8XP- - SILO8XP II WHIO4xP- - WHIO1XP O7 O7 O7 37 O7 16 8 PURO1XP- - WHIO7xP- II SILO8XP- II SILO8XP II WHIO4xP- - WHIO1XP- II O8 O8 O8 38 O8 12 9 PURO1XP- - WHIO7xP- - SILO8XP- - SILO8XP WHIO4xP- - 09 09 09 39 09 O PURO1XP- II WHIO7xP- - SILO8XP- - SILO8XP 10 O O 40 1. PURO1XP- - WHIO7xP- II SILO8XP- - SILO8XP 11 1 1 41 2 WHIO7xP- - SILO8XP- - SILO8XP 2 2 42 3 SILO8XP- - SILO8XP 3 33 4 SILO8XP- II SILO8XP II 4 44 5 SILO8XP- - 5 6 SILO8XP 6 7 SILO8XP 7 8 SILO8XP- II 8 9 SILO8XP 9 2O SILO8XP 2O 21 SILO8XP 21 22 SILO8XP 22 23 SILO8XP 23 24 SILO8XP 24 25 SILO8XP 25 26 SILO8XP 26 27 SILO8XP- II 27 28 SILO8XP 28 29 SILO8XP 29 30 SILO8XP- II 30
50 Example 7 included in the table. Similarly, the HPLC cannabinoid analy sis of Table 27 included measurements for CBCA. THCVA, Chemical Analysis of Cannabinoids and Terpenes of CBDVA, CBGVA, CBC, THCV, CBDV, CBGV, CBN and THC:CBD Specialty Cannabis Progeny Delta 8 THC all of which were measured to be less than 55 0.08%, and were therefore not included in the table. The new specialty Cannabis varieties created through Unlike previously available chemotype II or chemotype III crosses described in Examples 5 and 6 were subjected to plants, the specialty Cannabis of the present invention exhibit cannabinoid and terpene chemical analysis as described in chemotype II and III genotypes (B/B, producing both THC Example 1. The levels of cannabinoids were measured by and CBD, or B/B, producing CBD but no THC) while both GC-FID (Table 26) and HPLC (Table 27). Terpenes were 60 producing desirable terpene profiles. That is, the breeding measured using GC-FID and are presented as absolute con program of the present invention has produced chemotype II tent measurements based on the percent content by weight of and III specialty Cannabis plants with desirable terpene pro dry inflorescences (Table 28) and relative content as a percent files in which the myrcene terpene is not dominant. For of the total terpene profile (Table 29). The GC-FID cannab example, the PUR01XP04, PUR01XP10 and PUR01XP05 inoid analysis of Table 26 also included measurements for 65 have limonene-dominant terpene profiles. In some embodi THCV, CBDV, CBGV, CBN, and Delta 8 THC, all of which ments, the limonene terpene is expected to impart the spe were measured to be less than 0.3% and were therefore not cialty Cannabis with a citrusy aroma. In other embodiments US 9,370,164 B2 117 118 the limonene terpene is expected to have added anxiolytic CBD05xP-01 (chemotype III), and SIL08xP-34 (chemotype properties to combat the side of effects of THC. In yet another III) have terpene oil contents greater than 2%. In some embodiment, the reduced myrcene content of the specialty embodiments, the higher oil content of the specialty Can Cannabis will reduce the amount of “couch lock' effect pro nabis varieties provide “smoother aromas and flavors and duced by myrcene. In other embodiments, the terpene profiles 5 will raise the total terpene levels so as increase the pharma of the other chemotype II and III progeny provide diverse cological entourage effects of said terpenes. The higher oil terpene profiles designed to produce desirable aroma/flavors content results in myrcene becoming the dominant terpene, and organoleptic appeal. In other embodiments, the terpene but it remains less than 2/3 of the relative terpene content profiles of the chemotype II progeny allow for terpene entou- providing opportunity for the entourage effects of the other rage effects to reduce the side effects of THC. 10 terpenes to emerge. For example despite having a myrcene The breeding scheme described in Example 6 also pro- dominant profile, the SIL08XP-34 specialty Cannabis of the duced specialty Cannabis plants with increased terpene oil present invention is expected to provide a better organoleptic content. For example, progeny CBD02xP-11 (chemotype experience than that of myrcene dominant chemotype II Vari III), and SIL08xP-30 (chemotype II) have terpene oil con- eties currently available which tend to have very low terpene tents greater than 1.5%. Several other progeny Such as oil levels. TABLE 26 Cannabinoid values as measured by GC-FID for THC: CBD and CBD (chemotype II and III) specialty cannabis varieties. Blank values indicate undetectable levels or 0.
Cannabinoids (GC-FID
Cannabs THC: CBD THC CBD CBG CBC by GC by GC Sample W9% 95% CI Wt 90 95% CI Wt 90 95% CI Wt 9%. 95% CI Wt 9%. 95% CI Wt 9%. 95% CI Chemotype WHIO1XP-15 O.13% 4.90% O.05% O.38% S.46% O.O3 III CBDO2xP-11 O.25% O.10% 8.88% O.68% O.15% O.05% O.S5% 0.22% 9.83%, 1.04% 0.03 O.O1 III CBDO3XP-O1 O.16% O.04% S.40% 1.81% O.06% O.05% O.38% 0.07% 6.00% 1.97% 0.03 O.OO III CBDO3XP-10 O.26% 8.35% O.23% O.60% 9.43% O.O3 III CBDO3XP-07 O.17% 6.22% 1.03% O.09% O.03% O.47% 0.03% 6.87% 0.87% 0.03 III CBDO4xP-O1 O.21% O.04% 8.34% O.95% O.16% O.00% O.39% 0.11% 9.10% 0.79% 0.03 O.O1 III CBDO4xP-09 O.22% 8.34% O.18% O.42% 9.17% O.O3 III CBD05XP-O1 O.31% O.06%. 11.05% 1.94% 0.27% 0.17% O.56% O.12%, 12.19% 1.59% 0.03 O.O1 III CBDOSXS-13 O.21% O.01% 7.69% 1.92% O.27% O.11% O.36% 0.08% 8.53%. 1.95% O.O3 O.O1 III PURO1XP-06 1.59% 4.20% O.12% O.28% 6.18% O.38 PURO1XP-04 2.20% O.64% 6.00% 1.82% O.21% O.02% 0.42% 0.03% 8.83%, 2.51% 0.37 O.OO PURO1XP-10 1.57% 4.02% O.21% O.32% 6.12% O.39 PURO1XP-OS 1.55% 2.43% O.09% O.32% 4.40% O.64 SILO8XP-O1 1.95% 6.20% O.18% O.40% 8.73% O.31 SILO8XP-08 S.60% O.S3% 5.05% O.71% O.19% O.02% O.33% 0.04%. 11.17%. 1.19%. 1.11 O.OS SILO8XP-30 6.20% 4.71% O.21% O.35% 1.47% 1.32 SILO8xP-14 2.43% O.40% 8.57% 1.31% O.29% O.06% 0.42% 0.14%. 11.71% 1.51% 0.28 O.OO SILO8XP-18 2.33% 7.18% O.35% O.44% O.30% O.32 SILO8XP-34 7.659 6.56% O.27% O.S3% S.O1% 1.17 SILO8XP-03 3.86% 10.75% O.49% 0.65% 5.75% O.36 SILO8XP-37 3.09% 8.34% O.44% O.S1% 2.37% 0.37 SILO8XP-38 4.40% 3.57% O.08% O.31% 8.36% 1.23 WHIO4xP-O2 2.68% 8.10% O.31% O.S1% 1.60% O.33 WHIO7xP-07 4.62% 4.11% O.17% O.33% 9.27% 1.12 WHIO7xP-11 2.20% 4.62% O.15% O.35% 7.32% O.48 WHIO7XP-O1 6.14% S.26% O.25% O.47% 2.13% 1.17 WHIO7XP-08 3.27% 3.05% O.15% O.32% 6.82% 1.07 WHIO7XP-O2 2.12% S.28% O.22% O.42% 8.03% O.40 YELO3XP-23 3.52% O.S1% 6.70% 1.38% O.24% O.05% O.S.0% O.06%. 10.97%. 1.78% 0.53 O.O3 YELO3XP-26 2.99% O.35% 7.23% 1.04% O.25% O.02% O.S9% 0.06%. 11.06%. 1.32% 0.41 O.O1 WHIO1XP-22 2.60% 6.20% O.23% O.S.6% 9.58% O.42 WHIO1XP-12 3.94% 7.67% O.16% O.62% 2.39% O.S1 WHIO1XP-14 3.48% 6.09% O.23% O.S3% O.34% 0.57 WHIO1XP-19 3.89% 6.78% O.18% O.S9% 1.44% 0.57 WHIO1XP-23 48% 4.63% O.09% O.36% 6.56% O.32 CBDO2xP-15 2.29% 5.76% O.25% 0.66% 8.96% O.40 CBDO2xP-16A 2.93% 9.63% O.33% O.84% 3.72% O.30 CBDO2xP-17 41% O.34% 4.89% O.12% O.20% O.05% O.31% 0.11%. 6.81% 0.62% 0.29 O.O6 CBDO2xP-10 2.62% 7.07% O.30% 0.55% O.S4% 0.37 CBDO2xP-12 2.39% 7.11% O.42% O.S.0% O.42% O.34 CBDO2xP-14 .83% 6.40% O.33% O.44% 9.00% O.29 CBDO2xP-18 2.48% 6.42% O.15% 0.55% 9.61% O.39 CBDO2xP-31 .79% 4.69% O.16% O.38% 7.02% O.38 CBDO2xP-OS 56% O.31% 5.80% 1.98% O.28% O.16% O.40% 0.07% 8.04%. 2.38% 0.27 O.04 CBDO2xP-30 S8% S.00% O.25% O.44% 7.26% O.32 CBDO2xP-32 2.14% O.11% 4.67% O.S2% O.15% O.06% O.40% 0.13% 7.35% 0.57% 0.46 O.O3 CBDO2xP-40 .86% 3.85% O.23% O.37% 6.31% O.48 CBDO2xP-53 .64% 3.47% O.16% O.32% 5.59% O.47 CBDO2xP-09 .93% 7.15% O.36% O.46% 9.90% 0.27 CBDO2xP-28 2.06% O.74% 6.50% 1.82% O.19% O.14% O.40% 0.21% 9.15% 2.91% 0.32 O.O3
US 9,370,164 B2 135 136
55TETET US 9,370,164 B2 137 138
55TETET US 9,370,164 B2 139 140 Example 8 Due to the short growth season, relative comparisons were used for assessing progeny for future production and/or Phenotypic Analysis THC:CBD and CBD Specialty breeding schemes. Cannabis Progeny Floral Cluster Density—Floral cluster density is a measure of how tightly packed floral buds are in a plant inflorescence. For this example, floral cluster density was visually estimated The new specialty Cannabis varieties created through and assigned a score of 1-10 with higher numbers corre crosses described in Examples 5 and 6 were subjected to sponding to denser clusters. Due to the short growth season, phenotypic analysis as described in Example 2. Seeds were relative comparisons were used for assessing progeny for allowed to germinate in indoor facilities for 10 days and were future production and/or breeding schemes. then transferred to grow in an outdoor growing facility.- Plants 10 Ripening Mode Ripening Mode was determined by were allowed to grow for 120 days after germination until tracking the ripening of mature inflorescences. All progeny maturity and were analyzed as described in Example 2. Mea- exhibited relatively short and uniform ripening times. The Surements were conducted as described in Example 2 unless r1pen1ng for all progeny was even among all the inflores noted otherwise CCS. Thi1S 1S 1 COaStrast W1with Oother COFFIS bis W1Cwhich Ca The progeny of this example were grown during the 'short& G exhibit staged ripening in which various inflorescences ripen defined as November through February in California at different times. SeaSO O e 9. ry Average Calyx Length—Calyx length was measured in (-36.67° N). The 2013-2014 short was marked by record centimeters from the base of the calyx to the tip of the leafbut lows and a run of cloudy days that dramatically reduced not the pistil. Measurements were taken from mature plants at growth, flower production, trichome formation. These factors 20 120 days post germination. combined with low light angles reduced yields and oil pro- Initial selections were conducted based on measured phe duction significantly. However, the cooler temperatures com- notypes and chemical analysis described in Example 7. Cut bined with higher precipitation weather also provide excel- tings of desirable progeny were preserved for Subsequent lent conditions for fungal pathogens and provide researchers growth trials during a longer warmer season. These cuttings with a great environment to select for resistance to both cold 25 are also being used for Subsequent breeding as described in weather, low light levels and fungal pathogens. Table 30 Example 5, 19, and 20. Phenotypic results for these cuttings outlines the results of the phenotypic analysis. and their F2 and S2 progeny will be grown outdoors during Node Branching Node branching was visually deter- the upcoming season as described in this example or through mined by inspecting nodes and determining the amount of indoor growth as described in Example 2. Year round produc branching at plant maturity at 120 days post transfer. For this 30 tion to maximize natural light production is greatly dependent example branching was notated with a Y to indicate branch- upon short season trials to select progeny that perform well in ing at nodes and N to indicate low or no branching at nodes. the conditions outlined above. Many of the selected progeny Apical Inflorescence Size For this example, inflores- of several lines are being propagated and flowered in con cence size was visually estimated and assigned a score of 1-10 trolled indoor environments to determine more standardized with higher numbers corresponding to larger inflorescences. growth metrics. TABLE 30
Phenotype table of THC: CBD and CBD (chemotype II and III) progeny. Plant Apical Plant Diameter Aveti Number Inflor- Flower Height at at Number of Node of escence Cluster Tri maturity maturity of Leaf Inter- Branch- Inflor- Size Density chome Ripening Flower Color & Cultivar ID (cm) (cm) Leaflets Type nodes ing escence (1-10) (1-10) Density Mode Notes SILO8XP-O1 65 45 7 B 7 N L 2 9 8 Short, Even Normal color SILO8XP-03 78 72 7 B 9 Y M 2 9 7 Short, Even Normal color SILO8XP-08 96 62 7 B 8 Y M 3 8 7 Short, Even Normal color SILO8xP-14 74 39 5 B 7 N M 3 9 8 Short, Even Normal color SILO8XP-27 8O S4 5 B 8 Y H 3 10 9 Short, Even Normal color SILO8XP-30 72 40 5 B 8 N M 4 9 9 Short, Even Normal color SILO8XP-34 120 49 7 B 8 Y M 2 8 8 Short, Even Normal color, Vigor-- SILO8XP-37 97 48 5 B 9 N M 3 9 8 Short, Even Normal color SILO8XP-38 97 47 5 B 11 Y M 3 8 9 Short, Even Normal color YELO3XP-16 99 48 9 B 10 Y M 6 7 8 Short, Even Normal color YELO3XP-23 110 8O 11 B 9 Y M 5 6 8 Short, Even Normal color YELO3XP-26 92 39 9 B 9 N 6 6 7 Short, Even Normal color YELO3xP-27 95 60 9 B 9 N M 4 7 7 Short, Even Normal color PURO1XP-04 65 48 7 B 7 N H 1 5 5 Short, Even Purple Flowers PURO1XP-06 50 30 5 B 8 Y M 2 4 6 Short, Even Normal color PURO1XP-10 62 51 9 B 8 N M 3 6 7 Short, Even Black Leaves, Vigorous growth KRYA-1 82 42 6 B 8 Y M 5 7 Short, Even Leaf Serrations WHIO7-02 90 39 7 B Y H 5 5 8 Short, Even Normal color, Vigorous growth WHIO7-03 93 50 7 B 7 Y M 3 5 6 Short, Even Normal color WHIO7-07 8O 60 7 B 9 Y H 5 6 6 Short, Even Normal color WHIO7xP-11 45 28 5 B 6 N M 5 6 6 Short, Even Normal color SILO4xP-O1 47 40 7 B 7 Y M 7 8 7 Short, Even Normal color, Sweetterpene Smell SILO4xP-O2 50 31 5 B 6 N L 6 8 8 Short, Even Normal color US 9,370,164 B2 141 142 TABLE 30-continued
Phenotype table of THC: CBD and CBD (chemotype II and III) progeny. Plant Apical Plant Diameter Ave # Number Inflor- Flower Height at at Number of Node of escence Cluster Tri maturity maturity of Leaf Inter- Branch- Inflor- Size Density chome Ripening Flower Color & Cultivar ID (cm) (cm) Leaflets Type nodes ing escence (1-10) (1-10) Density Mode Notes CBDO4xP-1 70 64 5 B 9 Y H 4 5 8 Short, Even Normal color, Sweet and mint terpene Smells CBDO4xP- 74 40 5 B 6 Y M 4 8 8 Short, Even Normal color CBDO4xP- 77 40 7 B 7 Y M 6 6 7 Short, Even Normal color CBDO4xP- 83 67 7 B 8 Y H 4 5 7 Short, Even Normal color, Production, High Yield CBD04XP-6 96 46 7 B 6 Y H 3 8 6 Short, Even Normal color, Vigorous growth CBDO3XP-O1 55 27 5 B 6 N M 6 4 5 Short, Even Normal color CBDO3XP-O3 1OO 100 7 B 9 Y M 4 8 7 Short, Even Normal color, Vigorous growth CBDO3XP-OS 82 76 5 B 10 Y H 6 7 8 Short, Even Normal color, Vigorous growth CBDO3XP-07 73 56 7 B 8 Y H 7 6 7 Short, Even Normal color CBDO3XP-09 96 70 5 B 8 Y M 6 7 8 Short, Even Normal color, Vigorous growth, Sweet smell CBDO3XP-10 93 42 5 B 6 Y M 7 6 8 Short, Even Normal color CBDO3XP-11 84 42 7 B 7 Y M 5 5 5 Short, Even Normal color CBDO2xP-OS 1OO 74 7 B 8 Y 7 5 7 Short, Even Normal color, Vigorous growth CBDO2xP-10 8O 61 7 9 Y 5 7 6 Short, Even Normal color CBDO2xP-11 78 62 7 10 N M 5 5 7 Short, Even Blue flower color CBDO2XP-12 8O 69 7 9 Y 5 6 6 Short, Even Blue flower color CBDO2xP-15 87 85 11 11 Y 7 6 6 Short, Even Normal color, Production, High yield CBDO2xP-16a 78 60 7 10 Y 7 6 6 Short, Even Normal color CBDO2xP-16 84 56 5 8 N M 7 6 6 Short, Even Normal color CBDO2xP-17 81 40 5 6 Y M 4 5 6 Short, Even Normal color CBDO2xP-18 92 64 5 11 Y 4 4 6 Short, Even Normal color, Production, High yield CBDO2xP-28 89 59 9 8 Y M 6 7 6 Short, Even Normal color CBDO2XP-30 76 86 5 8 Y 4 7 6 Short, Even Normal color CBDO2XP-31 81 96 5 9 Y 6 7 TBD Short, Even Blue flower color CBDOSXP-O1 92 81 5 6 Y M 5 9 8 Short, Even Normal color CBDOSXP-O2 120 105 7 9 Y M 7 5 7 Short, Even Normal color CBDOSXP-OS 150 126 7 6 Y M 7 5 8 Short, Even Normal color, Vigor-- CBDOSXS-OS 71 S4 7 B 7 Y M 7 7 7 Short, Even Normal color CBDOSXS-11 86 39 7 B 9 N M 7 8 7 Short, Even Normal color, Cherry Pie CBDOSXS-13 59 33 7 B 7 Y M 7 8 7 Short, Even Normal color CBDO2XP-32 8O 53 7 H 5 N L 6 6 6 Short, Even Normal color, Astringent Cherry CBDO2xP-40 49 38 5 B 6 N M 6 6 6 Short, Even Normal color CBDO2xP-47 72 55 5 B 6 Y M 4 6 6 Short, Even Normal color CBDO2XP-55 73 48 5 B 8 Y H 4 6 6 Short, Even Blue flower color WHIO1XP18 8O 64 5 B 9 Y H 4 5 6 Short, Even Normal color WHIO1XP19 79 59 7 B 10 Y H 3 3 4 Short, Even Normal color, Bubblegum flavor WHIO1XP-22 81 61 7 B 9 Y H 4 3 6 Short, Even Normal color WHIO1XP-23 65 50 5 B 10 N L 32 6 4 Short, Even Normal color CBD24 59 47 5 B 10 Y H 2 3 3 Short, Even Normal color CBD11 61 45 7 H 7 N M 2 4 6 Short, Even Normal color CBD13 60 31 7 H 8 Y M 3 6 4 Short, Even Normal color WHIO1XP-15 1OO 57 5 H 10 Y H 3 7 4 Short, Even Normal color, Production, High yield
60 Example 9 terpene and cannabinoid profiles to determine the effect of Volunteer Trials. Using THC:CBD Specialty Cannabis with CBD, effect of higher terpene oil content, and Cannabis. Effect of Added CBD the effect of diverse terpene profiles with reduced myrcene In order to demonstrate the added utility of the specialty contents. The trials were split into two parts. The first part Cannabis varieties of the present invention, volunteer com- 65 (Weeks 1-2) compared volunteer responses to THC-only cul parison trials were conducted. During these trials, Volunteers tivars and cultivars that contained THC plus a small amount of were provided with Cannabis flower blends with varying US 9,370,164 B2 143 144 The volunteer trial for CBD was conducted over 2 weeks. attain the desired analyte levels could be predicted. The Volunteers were split into six groups (1-6). Each volunteer in the group was given two samples (a control and a comparator appropriate amounts of materials were combined in a coffee blend). For instance, they were given a1 and a2, or b1 and b2. grinder. A finer grind was needed during the first four-week or c1 and c2, or d1 and d2, or e1 and e2, or f1 and f2 (see Table section to mask the addition of the kief. The material was split 31 for trial design). In this trial, the control comparator blends into 1.0-1.5 g samples and stored at -20 until distribution were prepared to contain nearly identical levels of THC and terpenes, but each week the comparator had either 1.5% (typically within 24 hours). Enough of each blend was made CBD, or 2.5% CBD added in. For the higher percentages of to analyze the samples in triplicate to Verify the cannabinoid CBD, a cannabinoid rich form of hash known askief was used 10 and terpene levels (See Table 32 and 33 for terpene and rather than flower so a higher concentration could be added without affecting the terpene profile as significantly as adding cannabinoid analysis of blends given to patients). The con whole Cannabis flowers. trols (THC only) are in boldface and it can be seen the levels TABLE 31
CBD Effect Trial Overview for Weeks 1 and 2.
Week
1 2 THC or THC or THC + 1.5% THC + 2.5% Terpene Base Control Comp CBD CBD Class Control and Comparator Terpenes Cultivar ID ID Group 1 Group 6 8. myrcene, pinene GRA8 a1 a2 Group 2 Group 1 b limonene, linalool, caryophyllene, humulene WHI2 b2 b1 Group 3 Group 2 C ocimene, myrcene GRE1 c1 c2 Group 4 Group 3 d terpinolene, Ocimene PUR2 d2 d1 Group 5 Group 4 e myrcene, pinene, ocimene, linalool, caryophyllene PUR5 e1 e2 Group 6 Group 5 f limonene, caryophyllene, myrcene, linalool RED1 f2 f1
The samples were prepared by first assaying the individual 30 of THC are roughly similar within a group. It also can be seen cultivars for their cannabinoid and terpene levels. Once levels that the blending process produced consistent levels of can were determined the mass ratios of the cultivars needed to nabinoids and terpenes that were close to predicted values.
TABLE 32 Cannabinoid levels of cannabis blends for Week 1 and Week 2 trials as measured by GC-FID and HPLC. Blank values indicate undetectable levels or 0.
Cannabinoids (GC-FID Cannabinoids (UHPLC
THC:CBD THCA:CBDA THC CBD by GC THCA CBDA by HPLC
Sample Wto 95% CI Wt 9/o 95% CI Wto 95% CI Wt 96 95% CI Wt 90 95% CI Wt 9/o 95% CI
Week 1 MPCM-13A-002-a1 20.90% 0.91% 24.04% O.44% MPCM-13A-002-a2 18.14% (0.50% 1.82% 0.04% 9.98 O.24 21.32% 0.49% 2.14% O.10% 9.96 O.34 MPCM-13A-002-b S.82%. 1.11%, 1.60% 0.06% 9.90 O.98 19.17% 0.71% 1.90% O.14%. 10.14 1.01 MPCM-13A-002-b2 17.51% 0.58% 20.80% O.65% MPCM-13A-002-c. 9.10% 0.19% 22.91% 0.27% MPCM-13A-002-C2 17.24% 0.66%, 1.65% 0.07%. 10.50 O.86 20.67%. 1.04% 2.02% O.02%. 10.22 O.S8 MPCM-13A-002-d 0.67% 0.31% 1.75% 0.04% 6.11 O16 13.24% 0.23% 2.17% O.17% 6.11 0.44 MPCM-13A-002-d2 12.22% 0.96% 15.27%. 1.15% MPCM-13A-002-e1 20.90% 0.56% 24.69% 0.47% MPCM-13A-002-e 18.41% 0.99%. 1.80% 0.08%. 10.23 O.74 21.86% 0.85% 2.23% O.16% 9.84 O.67 MPCM-13A-002- 5.83% 0.58%. 1.83% 0.07% 8.68 O.45 19.30% O.87% 2.28% O.11% 8.46 0.37 MPCM-13A-002-f2 17.92% 0.55% 21.21% 0.62% Week 2 MPCM-13A-003-a 9.17% 0.84% 25.48%. 1.14% MPCM-13A-003-a2 20.09% 0.68% 2.76% 0.18% 7.28 O.26 25.45%. 1.00% 3.31% O.33% 7.72 O.67 MPCM-13A-003-b 6.81% 0.19% 2.69% 0.04% 6.26 O.OS 21.46% O.76% 3.18% O.14% 6.75 O.S3 MPCM-13A-003-b2 16.60% 0.69% 22.28% 0.98% MPCM-13A-003-c. 8.92% 0.55% 24.57% 0.35% MPCM-13A-003-c2 18.59% 0.34% 2.93% 0.30% 6.37 O.63 23.56% O.11% 3.53% O.43% 6.72 O.85 MPCM-13A-003-d 4.50% 0.67% 2.93% 0.27% 4.97 O.48 18.51% 0.71% 3.47% O.40% 5.37 O.62 MPCM-13A-003-2 11.45% 0.63% 16.09% 0.60% MPCM-13A-003-e1 20.97%. 1.09% 27.28% 0.60% MPCM-13A-003-e2 20.18% 0.53%. 2.80% 0.15% 7.21 O.21 25.58% 0.78% 3.24% O.22% 7.90 O.30 MPCM-13A-003- 7.72% 0.24% 3.05% 0.10% S.82 O.13 22.96% 0.24% 3.60% O.20% 6.39 O4O MPCM-13A-003-f 17.07% 0.16% 22.80% O.26%
US 9,370,164 B2 147 148
US 9,370,164 B2 151 152 The controls (a1, b2, c1, d2, e1, and f2) had only THC to comparator samples with added CBD cannabinoid (see while the comparators (a2,b1, c2, d1, e2, and fl) had approxi Table 34 and FIG. 3). Several feedback trends can be seen in mately the same amount of THC plus a small percentage of the comparison of the two samples. Most notably, there CBD. In Week One approximately 1.5% of CBD was added, appeared to be an obvious decrease in the level of “mind in Week Two 3% of CBD was added. These non-THC can- 5 high”, “body high”, “intoxication”, “sedation', and “dura nabinoids have demonstrated pharmacology (such as CBR tion' for Cannabis blends containing CBD. There also antagonist and 5HT-1a agonist) that were hypothesized to appeared to be an increase in the ability to “function nor attenuate some of the negative side effects of THC by block- mally” for Cannabis blends containing CBD. There was also ing the action of THC itself or by activating alternative path- a decrease in “anxiety” and an increase in “energy’ level for ways. 10 these blends. Each comparison control and comparator The controls and comparators in Weeks 1-2 were also sample contained equal amounts of THC and nearly identical blended to have very similar terpene profiles in order to terpene profiles. Thus the differences outlined in Table 34 and ensure both samples had similar aroma, flavor, and putative FIG. 3 are attributed to the relatively small amount of CBD entourage effects, so as not to predispose the Volunteer to added to comparator samples. thinking one or the other would be different based on orga- 15 The observed trends suggest that the addition of a non noleptic properties. Both Table 33, and FIG. 1 (with compari- THC cannabinoid, such as found in chemotype II cultivars, son pairs indicated with brackets) of relative terpene content can help reduce the feelings associated with being “high”. show the precision with which the blends were engineered to reduce intoxication, reduce the duration, reduce sedation, and have comparable terpene profiles. The blends were always improve the ability to function normally while under the prepared so the myrcene content was below 60% and the total 20 influence of THC. Thus in some embodiments, the specialty identified essential oil content was about 1.5%. Cannabis of the present invention with CBD has the potential The amount of added CBD was kept below 3% since add- to reduce adverse effects and provide a larger margin of safety ing more of the myrcene-rich CBD cultivar would have sig- for a number of applications wherein the specialty Cannabis nificantly altered the terpene profile and all the groups would is provided as a blendoras flower material from an individual have become myrcene dominant. Adding more of the 25 variety. In some embodiments, the CBD containing specialty myrcene-rich CBD cultivar would have also diluted the rela- Cannabis can be used at times when users wish to still be able tive amount of THC, and at this stage it was desired to ensure to function after Smoking. In other embodiments the specialty any changes in effect were due to the addition of CBD rather Cannabis of the present invention can be used for medicinal than a significant reduction in THC content. applications. Many times patients attempting to use Cannabis Thirty volunteers were recruited and asked to fill out demo- 30 for medical treatment discontinue use due to the aforemen graphic Surveys. Each week the Volunteers were given a con- tioned “negative' side effects, such as being “high” or intoxi trol and a comparator, two corresponding surveys (FIG. 2), cated, and these ratios have demonstrated a clear potential to and asked to fill out the survey forms as they administered the mitigate these effects. samples over the following week. This was designed to be a The decrease in flavor feedback for the CBD blends was head-to-head comparison and the results were then tabulated 35 likely due to the addition of unpalatable CBD-rich plant mate in Excel and analyzed both as absolute ratings and as differ- rial and kief to reach the desired cannabinoid levels. This ences between the control and comparator. The results of result further reinforces our original hypothesis of the need Weeks 1 and 2 (control vs. comparator) are summarized in for specialty Cannabis varieties which contain CBD with Table 34 as averages followed by 95% confidence intervals. desirable terpene profiles to create pleasing aromas/flavors TABLE 34
Combined feedback results for Week 1-2 trials. Average Differences for Weeks 1-2 Question A. 95% CI B 95% CI C 95% CI D 95% CI E 95% CI F 95% CI TOTAL 95% CI Aroma -1 42 O O.98 -0.9 59 -O.75 27 2 76 -0.5 2.07 -0.23 O.65 Flavor -1.86 98 -0.13 .14 -1.6 55 -1 .74 0.857 45 -O.83 .71 -0.8 0.67 Mind -1.57 .12 -0.11 .40 -1.6 61 -1.63 O.74 -0.43 27 O.429 70 -O.85 O.S8 Body -0.43 0.72 O.222 52 -1.2 62 -1.38 .33 1143 .93 -1.14 .79 -0.5 O.65 intoxication -1.14 OO -0.56 27 -1 13 -1.5 O.74 O.286 O2 -0.57 41 -0.77 O46 Calmness -O.71 2.13 0.444 O9 -0.6 O6 2.25 73 1143 0.79 O.286 O.70 O438 O.S9 Alertness O 42 -0.33 27 1 87 -0.38 52 1143 OO 0.571 53 O.333 O.62 Anxiety -0.29 O.93 O.333 O3 O6 .14 -3 74 -0.86 O.79 -0.71 95 -0.58 O.61 Focus O O.96 O.222 O.91 -O.7 40 0.75 .65 0.857 O.S1 0.857 45 O.271 O.S1 Mood -2.43 70 O.222 O.79 -1.4 61 -0.13 .14 O.714 O.S6 1 2.01 -0.38 O.63 Energy O.143 31 1.111 O.89 -0.3 34 O2S O3 O.286 O2 1143 84 O417 O.S1 Hunger -O.71 2.17 1.222 30 0.4 81 -0.38 23 O .54 -0.43 2.45 O.083 O.71 Thirst -0.14 2.16 -0.56 .14 -O.7 .94 -1.5 23 -0.57 34 -0.29 90 -0.65 O.66 Physical -0.57 53 O.667 22 -0.1 O.85 O.S 48 1143 OO -0.29 2.21 O.229 O.S6 Emotional -1.43 34 O O.86 -0.3 .24 O.625 28 1.429 .04 1143 2.03 O.208 0.57 Function O.714 .33 -0.33 22 O.8 O.96 -0.38 .11 1.429 .04 1.286 .85 O.S.42 O.S2 Sedation -0.57 70 -0.22 O.63 -1.7 73 -1.13 59 -0.57 53 -0.43 81 -0.81 O.61 Duration -1 54 O 70 -0.44 O.93 -0.29 .85 -0.29 46 -0.67 31 -0.43 0.57 Positive -O.71 2.08 0.778 S6 -0.6 68 0.75 47 1 O.74 1143 2.20 O3S4 O.69 Negative O.286 2.83 -0.67 O.92 1.6 92 -0.5 .85 O.143 S1 -0.43 2.93 O.12S O.82
65 The results are presented as the difference in feedback and reduced side effects. Patients may discontinue use of scores between control samples with just THC cannabinoid, previously available medicinal CBD marijuana due to US 9,370,164 B2 153 154 unpleasant aromas and poor organoleptic feel. Currently ratio of the samples being tested was ~1:2 (Harlequin). On existing THC:CBD cultivars have terpene profiles and total Week six, the THC:CBD ratio of the samples was ~2:1 oil content that result in organoleptic properties and entou (BLU6 and CBD1 blend). The terpene profile of the control rage effects that are less appealing than the THC-only culti was the typical low oil myrcene-dominated profile of the vars. In one embodiment, patients wishing to use the specialty 5 mixed cannabinoid cultivars, while the comparators had Cannabis of the present invention for medicinal purposes will higher oil content representative of the specialty Cannabis prefer the improved aroma and flavor. plants of the present invention. While it has been known that CBD is an antagonist to the Samples for the trials were generated as described in CB1 and CB2 receptor (Mechoulam et al., 2007 “Canna Example 9. However, for this example, samples were ground bidiol-recenties between CBD advances producing Chem varieties biodivers have 4(8) often 1678-92), compared stud. 10 by hand. As before, each sample was analyzed via GC-FID varieties with- high- - CBD contents and varying THC contents. and HPLC before being provided to volunteers in order to Thus it has been difficult to distinguish the effects of the ensure consistency (Tables 35 and 36, and FIG. 4). The- - same- - addition of CBD, to that of the reduction of THC. In this questionnaire that was used in Example 9 (i.e., as provided in study, we have shown that beneficial trends can be seen with 15 FIG. 2) was used to assess the volunteer feedback on the the addition of small amount of CBD, and that, unexpectedly, tested blends. ------these effects do not require a substantially diminished THC The sample ID of the control sample is highlighted (Tables COntent. 35 and 36) and the relative terpene profile is labeled in FIG. 4. Week 5 of this study compared a typical low oil 1:2 THC: Example 10 20 CBD variety (Harlequin in this case) to higher oil blends prepared from a parental CBD line (CBD01) and various Volunteer Trials Using Specialty Cannabis. Effect of parental THC lines. Because the mass ratios required to create Added High Terpene Oil the 1:2 THC:CBD ratio were approximately 1:4, the terpene profiles were all dominated by myrcene from CBD01, and The fifth and sixth week of the trials were designed to test 25 this is observed in the analytical results. While all the relative the effect of higher terpene oil content on Cannabis plants. terpene profiles were similar and dominated by myrcene, the For this trial, the same groups (1-6) used in Example 9 were absolute content was significantly different, with the control asked to compare the more oil rich profiles of (a-f) to the (Harlequin) having less than 1% and all of the comparators “typical terpene profile of (g) found in currently existing having greater than 1.5%. THC:CBD or CBD varieties (Tables 35 and 36, and FIG. 4). 30 Week six compared a typical low oil 2:1 THC:CBD variety This “typical profile was represented by a known chemotype (mimicked by blending BLU06:CBD01) to higher oil blends II variety “Harlequin” (Week 5), or by mimicking the terpene prepared from a parental CBD line (CBD01) and various profile with a blend of CBD01 and BLU06 varieties (Week 6), parental THC lines. More diversity can now be seen in the which allowed the THC:CBD ratio to be adjusted. For each terpene profiles of the comparators and the control. The con week, the control and the comparator samples had nearly trol samples had lower terpene oil contents of ~1%, while the identical ratios of THC:CBD. On Week five, the THC:CBD comparators were generally between 1.5-2%. TABLE 35 Cannabinoid levels of cannabis blends for Week 5 and 6 trials as measured by GC FID and HPLC. Blank values indicate undetectable levels or 0.
Week 5
MPCM-13A-OO6-b 3.49% 0.53% 8.69% MPCM-13A-06-c 3.65% 0.09% 8.87% MPCM-13A-006- 3.42% 1.38% 10.41% O.33 MPCM-13A-006-e 4.04% 0.48% 9.26% 0.44
4.57% 0.12% 9.97% Week 6 MPCM-13A-007-a 9.79% 0.35% 4.58% 0.15 14.66% 2.10 0.14
MPCM-13A-007-c 9.63% 0.41% 5.90% MPCM-13A-007- 7.39% 0.31% 5.14% 1.44 MPCM-13A-007-e 10.83% 0.71% 7.14% 0.18 14.61% 1.52 0.18 MPCM-13A-007-f 8.06% 0.57% 5.27% 8.13% 0.37% 4.10%