Classification of Common California Cannabis Cultivars via Secondary

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

Several Years to Achieve Several Goals: 1. 2. 3. 4. 5. 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 Raw Materials Garbage In = Garbage Out Finished Products

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

Stress Free Environment = Good Data Production Facility Example Studio A Studio B Studio C Production/Consumer Interface Meaningful & Useful Information Studio D Studio E Studio F Studio G 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 Δ 0 Genotype (G) + Environment (E) → Phenotype (P)

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

Material and Data Flow Tier II Extract 1 Sample Plant Extract 2 Homogenized Mass Data Moisture Data Extract 3 Terpenes 0. 016 -1. 5% Mass Data Cannabinoids 2. 3 -36% Morphological Data Resource Data Sample Prep Worksheet Metabolites B Raw Sample Data Field Reports Metabolites A Raw Metabolite Data Cannabinoids 0. 14 -2. 5% Raw Terp and Cannab Data 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.

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 Gx. E 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 (R 2 = 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.

56 genotypes: Total Terp & CB Content

Terpene Traits Visualized with COLOR Phyto. Print. TM 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 Phyto. Print. TM • 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 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.

Phyto. Prints. TM of Cannabis Archetypes terpinolene limonene ocimene α-pinene & α-pinene myrcene 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 Super. Classes

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 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. LCM 1 G Chemotype (I, 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 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.

Single Genotype: Classic OG Kush • MCL 1 T class • Myrcene, caryophyllene, limonene • >2% terps, >25% CB’s

Classic OG Kush: Multiple Classes

GOD 8 1311 A 9 W 8 13 K 1 GOD 1 A 9 WK 7 8 13 7 b 1 GOD 1 F 06 SLV 8 D 7 W 9 K 5 SLV 0 O 8 140 4 F 8 SLV 0 1404 A 089 112 8 14 © 05 B SLV 0 1 SLV 0 8 1406 02 © D 09 8 14 7 0 SLV 0 6 D 09 © SLV 0 8 Y 026 7 b © 140 81 SLV 0 406 D 0 703 97 c 8 14 © 07 SLV 0 8 14 A 121 © SLV 0 08 D 11 7 8 © 140 SLV 0 8 A 8 SLV 0 1407 B 133 110 8 14 © COM 09 B 120 PSLV 0 141113 © _0 8_ SLV 0 1411_D 01 8 1 SLV 0 _1412_ 22 F 1 8_ SLV 0 1502_D 12 8_ 13 SLV 0 1502_F 2 1 SLV 0 8 1504 27 A 1 8 SLV 0 _1504_ 47 D 1 8_ SLV 0 1506_ 43 A SLV 0 8_1506 152 _F 8 SLV 0 _1506_ 131 A 8_ 15 LEG 1506_A _3 A_ 15 LEG 1507_C _4 A_ 13 LEG 1507_C _3 A_ 13 LEG 1507_C _4 A_ 13 LEG 1507_C _7 A_ 13 LEG 1508_A _8 A_ 16 LEG 1509_B _0 A_ 14 LEG 1508_D _0 A_1 15 LEG 509_A _7 A_1 51 16_9 LEG A_ 1_A 17 LEG 1511_D _2 A_1 16 LEG 512_B _0 A_ 15 LEG 1512_C _9 A_1 15 LEG 601_D _2 A_1 602 17_0 _B 1 6_2 Weight % 20. 0% 15. 0% 24. 9% 25. 1% 24. 8% 25. 0% 24. 0% 23. 4% 22. 0% 20. 5% 29. 4% 28. 9% 27. 3% 28. 0% 26. 9% 26. 0% 1. 5% 1. 4% 1. 9% 1. 7% 1. 8% 1. 6% 1. 7% 1. 9% 2. 7% 2. 5% 2. 4% 2. 2% 2. 1% 1. 9% 1. 8% 1. 6% 1. 3% 1. 4% 1. 6% 1. 5% 24. 0% 23. 3% 22. 5% 24. 1% 2. 5% 2. 3% 2. 4% 2. 2% 2. 5% 2. 7% 2. 4% 2. 3% 2. 2% 2. 6% 2. 4% 2. 2% 2. 4% 2. 0% 2. 6% 2. 4% Baseline 18. 2% 15. 8% 25. 0% 13. 9% 16. 0% 30. 0% 28. 3% 2. 0% 1. 9% 2. 5% 24. 7% 23. 2% 25. 5% 24. 0% 24. 4% 22. 6% Weight % 3. 0% 24. 6% 21. 4% 22. 4% 20. 1% 24. 0% 23. 9% 25. 0% 22. 4% 24. 1% 23. 4% 25. 1% GOD 8 1311 A 9 W 8 13 K 1 GOD 1 A 9 WK 7 8 13 7 b GOD 11 F 06 SLV 8 D 7 W 9 K 5 SLV 0 O 8 140 4 F 8 SLV 0 1404 A 089 112 8 14 © 05 B SLV 0 1 SLV 0 8 1406 02 © D 09 8 14 7 SLV 0 06 D 09 © SLV 0 8 Y 026 7 b © 140 81 SLV 0 406 D 0 703 97 8 SLV 0 1407 A c © 121 8 14 © 0 8 D SLV 11 SLV 0 08 1408 7 © 8 14 A 13 3 07 B SLV 0 11 8 COM 1409 B 0 © P-14 120 © 111 SLV 0 3_0 8_ SLV 0 1411_D 01 8_1 412 122 SLV 0 _F 1 8_ SLV 0 1502_D 12 8_ 13 SLV 0 1502_F 2 1 SLV 0 8 1504 27 A 1 8 SLV 0 _1504_ 47 D 1 8_ SLV 0 1506_ 43 A SLV 0 8_1506 152 _F 8 SLV 0 _1506_ 131 A 15 8_ LEG 1506_A _3 A_ 15 LEG 1507_C _4 A_ 13 LEG 1507_C _3 A_ 13 LEG 1507_C _4 A_ 13 LEG 1507_C _7 A_ 13 LEG 1508_A _8 A_ 16 LEG 1509_B _0 A_ 14 LEG 1508_D _0 A_ 15 LEG 1509_A _7 A_ 16 LEG 1511_A _9 A_ 17 LEG 1511_D _2 A_ 16 LEG 1512_B _0 A_ 15 LEG 1512_C _9 A_ 15 LEG 1601_D _2 A_1 602 17_0 _B 1 6_2 Classic OG Analysis: Total Oil Content 3. 5% Total Terpenes 1. 0% 0. 5% 35. 0% Cannabinoids (HPLC) 10. 0% • 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 TXX 1 G class • Terpinolene Dominant

TXX 2 T Class Hybrid Cannabinoid Data

TXX 2 T Class Hybrid Terpene Data

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