Classification of Common California Cannabis Cultivars via Secondary Metabolite Characterization Mark A. Lewis President, Napro Research California, USA

Several Years to Achieve Several Goals: Validated assays & analysis to communicate data Collect data & valid survey of ‘chemoscape’ Analysis 1: Class cultivars based on chemotype Analysis 2: Observe intra-genotypic variability Analysis 3: Asexual vs. hybrid seed comparison Garbage In = Out Raw Materials Finished Products

CEA & Unique Opportunity 56 known cultivars Identified by common name Sourced/grown by experts Stress Free Indoor Environments Organic practices Soil 1000W HPS Many cultivars >40 batches tested

Stress Free Environment = Good Data Production Facility Example Production/Consumer Interface Studio A Studio B Studio C Studio G Studio F Studio E Studio D Meaningful & Useful Information Monitor Grow Conditions and Product Development in Real Time

Data Collection – Climate Metrics Genotype (G) + Environment (E) → Phenotype (P) & (C) Real Time Alarms

Data Collection – Nutrient Metrics Genotype (G) + Environment (E) → Phenotype (P) & (C) Precision Feeding

Data Collection – Phenotypic Metrics Δ0 Genotype (G) + Environment (E) → Phenotype (P)

Data Collection: Chemotype Metrics Lack of guidance period Questions that desperately needed answered: error in sample prep extract stability matrix effects assay performance RSD’s, RSD’s, RSD’s

Raw Terp and Cannab Data Material and Data Flow Tier I Tier II Extract 1 Extract 2 Sample Homogenized Extract 3 Terpenes 0.016-1.5% Plant Mass Data Moisture Data Mass Data Cannabinoids 2.3-36% Morphological Data Resource Data Sample Prep Worksheet Cannabinoids 0.14-2.5% Metabolites B Metabolites A Raw Sample Data Raw Metabolite Data Raw Metabolite Data Raw Terp and Cannab Data Field Reports Sample Data Archive Data Analysis QC Control Charts MCF Sheets

To Name a Few of the 56 Cultivars Examined Sour Diesel OG Kush Purple Erkle Ken’s GDP Trainwreck Snowcap Dutch Crunch OG Legend Hawaiian Skywalker OG Jack Herrer Old Amsterdam Pincher’s Creek Green Candy Purple Kush Bubba Kush Girl Scout Cookies Big Sky Blue Dream Berry White Blueberry SFV OG Congolese Pineapples Express Golden Pineapple Heroijuana Etc., Etc., Etc.

Total Cannabinoid Content Distribution Max 36.5%, Min 10.2%, AVG 19.9% The lot-to-lot RSD’s from 1% to 29% with a 12% AVG Highly sampled cultivars have lot-to-lot variation from 10% to 20%. Does variability reflect GxE interactions?

Total Terpene Content Distribution Max 2.47%, Min 0.67%, Avg 1.57% lot-to-lot RSD from 3% to 41%, average of 21%. Highly sampled cultivars have lot-to-lot variation from 20% to 30%. Cannabinoid variability < terpenoid variability

Cannabinoid-Terpenoid Relationships Higher EO = higher cannabinoid content (R2 = 0.156) Only 15% of variability in terpene content explained by cannabinoid content. 85% of variability due to other explanatory variables not included in this analysis. Pr>F value of <0.0001, thus, correlation real, but very small. Analysis of the Variance Pr>F

56 genotypes: Total Terp & CB Content

Terpene Traits Visualized with COLOR PhytoPrintTM displays active compounds with a list of their different medicinal uses © 2015 BHC Group, LLC. All rights reserved. Any unauthorized use of this document or the images or marks above may violate copyright, trademark, and other applicable laws.

41 Terpenoids Analyzed Only presented 17 on PhytoPrintTM Most ‘relevant’ Intuitively colored, spaced and arranged Result: Bar color and length allow rapid ID of similar fingerprints as well as minor differences that make each cultivar unique Bubba Kush East Coast Sour Diesel Classic Krypto The classification system ignores the “minor” terpenes, which can be defining traits of specific genetic lines. Classic Purps Blue Dream 2 Old Amsterdam Confidential © 2015 BHC Group, LLC. All rights reserved. Any unauthorized use of this document or the images or marks above may violate copyright, trademark, and other applicable laws.

PhytoPrintsTM of Cannabis Archetypes ocimene terpinolene limonene α-pinene & myrcene α-pinene Hybrid with two dominant terpenes myrcene β-caryophyllene © 2015 BHC Group, LLC. All rights reserved. Any unauthorized use of this document or the images or marks above may violate copyright, trademark, and other applicable laws.

Terpene SuperClasses

Indica, Sativa and the Classification System Indica & sativa designations are confusing for ethnobotanical purposes Originally species designations Now there is “mostly sativa”, “mostly indica”, “sativa-like, indica-blah”, etc. Says little to the consumer about the product they are purchasing Class designation conveys most relevant information chemotype LCM1G Most relevant terpenes (up to three) in a fixed (not ranked) order. Rationale is that organoleptic properties and entourage effects are largely dictated by the major analytes, and small changes in ranking will be difficult to discern. Chemotype (I, II, III, etc) implies dominant cannabinoid. This is the major driver of the pharmacology. Final letter represents the second most dominant cannabinoid and indicates “unusual” cannabinoids than can modulate the properties of the first.

PCA & Classification Employed relative terpene profiles for clustering TW OG BD BW Employed relative terpene profiles for clustering Different colors = different classes Lot-to-lot variability does cause some problems. Some genotypes don’t always land in same class. Principal component analysis (PCA) and the resulting biplots provide a manner to determine the defining characteristics of a cultivar, without oversimplifying the analysis by ignoring seemingly insignificant traits… Class system ignores minor terpenes, which can be defining traits of specific genetic lines.

Single Genotype: Classic OG Kush MCL1T class Myrcene, caryophyllene, limonene >2% terps, >25% CB’s

Classic OG Kush: Multiple Classes

Classic OG Analysis: Total Oil Content Baseline Organic practices + biostimulant 2.3% AVG Terps 24.3% AVG CB’s Organic practices – biostimulant 1.8% AVG Terps ↓ 18.9% AVG CB’s ↓ Conventional + biostimulant 1.5% AVG Terps ↓ 28.1 AVG CB’s ↑

Variability & Cannabis Quality Cannabis industry lacks QA/QC Most producers have yet to establish quality metrics Hemp only ‘true’ breeding cannabis seed in market place Unlike corn, you cannot by disease resistant, or bi-color, etc.

Genotype Creation: Type II Trainwreck Trainwreck is TXX1G class Terpinolene Dominant

TXX2T Class Hybrid Cannabinoid Data

TXX2T Class Hybrid Terpene Data

Terpene SuperClasses Indica and sativa may be better suited as an ‘effects gauge’ instead of genealogical nomenclature

Sample Reports © 2015 BHC Group, LLC. All rights reserved. Any unauthorized use of this document or the images or marks above may violate copyright, trademark, and other applicable laws.