HEMP

Cornell University CBD trial conducted in 2018 in Gates West field 018 in Geneva, NY.

Cornell University pollination trial conducted in Geneva, NY in 2018.

Cornell University pollination trial conducted in Geneva, NY in 2018.

Cornell University pollination trial conducted in Geneva, NY in 2018.

Cornell University CBD trial conducted in Geneva, NY.

Cornell University pollination trial conducted in Ithaca, NY, Bluegrass Lane, in 2018

Cannbinoid analysis of feral accessions collected in Illinois in 2019

Hemp trials conducted at Cornell University in 2019 evaulating environmental stress and season-long cannabinoid production at the Wegmans Organic Farm

Hemp trials conducted at Cornell University in 2019 evaulating fungicide resistance.

Hemp trials conducted at Cornell University in 2019 evaulating environmental stress and season-long cannabinoid production under high-tunnel production systems

Hemp trials conducted at Cornell University in 2019 evaulating environmental stress and season-long cannabinoid production

Hemp trials conducted at Cornell University in 2019 evaulating environmental stress and season-long cannabinoid production

Hemp trials conducted at Cornell University in 2019 evaulating environmental stress and season-long cannabinoid production

Hemp trials conducted at Cornell University in 2019 evaulating environmental stress and season-long cannabinoid production

Hemp trials conducted at Cornell University in 2019 evaulating environmental stress and season-long cannabinoid production

Hemp trials conducted at Cornell University in 2019 evaulating environmental stress and season-long cannabinoid production

Hemp trials conducted at Cornell University in 2019 evaulating environmental stress and season-long cannabinoid production

Hemp trials conducted at Cornell University in 2019 evaulating environmental stress and season-long cannabinoid production

Hemp trials conducted by Cornell University in 2020 to evaluate CBD production.

Hemp trials conducted by Cornell University in 2020 to evaluate CBD production.

Hemp trials conducted by Cornell University in 2021 to evaluate CBD production.

Hemp trials conducted by Cornell University in 2021 to develop mapping populations

Hemp trials conducted by Cornell University in 2021 to develop mapping populations

Hemp trials conducted by Cornell University in 2021 to develop mapping populations

Hemp trials conducted by Cornell University in 2021 to develop mapping populations

Hemp trials conducted by Cornell University in 2021 to develop mapping populations

Hemp trials conducted by Cornell University in 2021 to develop mapping populations

Hemp trials conducted by Cornell University in 2021 to develop mapping populations

Hemp trials conducted by Cornell University in 2021 to develop mapping populations

Hemp trials conducted by Cornell University in 2021 to evaluate…

https://www.biorxiv.org/content/biorxiv/early/2025/03/13/2025.03.10.642411/DC1/embed/media-1.docx?download=true

Routine germplasm testing.

Hemp trials conducted by Alabama Agricultural and Mechanical University in 2022 at the Winfred Thomas Agricultural Research Station (WTARS; 372 Walker Ln, Hazel Green, AL 35750; 34.901410, -86.560777; Location: Back of the main building, WTARS, AAMU (Hazel Green, AL)) by Dr. Ernst Cebert, Xianyan Kuang, and Xinhua Xiao. PGRU sent AAMU seed from 9 accessions (NY and Iowa feral germplasm: G 33199 21UO SD, G 33240 21UO SD, G 33316 21UO SD, G 33322 21UO SD, G 33323 21UO SD, G 33329 21UO SD, G 33330 21UO SD, G 33331 21UO SD, G 33332 21UO SD). These trials evaluated stand count, plant height, biomass, plant architecture parameters, and sex ratio. This work is part of Research Project: Strengthening Hemp Research and Educational Capacity at Alabama A&M University by Morphological Evaluation of Diverse Hemp Germplasm. Location: Plant Genetic Resources Unit (PGRU). Project Number: 8060-21000-030-003-S. Project Type: Non-Assistance Cooperative Agreement. Start Date: Sep 1, 2021. End Date: Aug 31, 2023

FLD2022-38819 Alabama Department of Ag & Industries, Food and Drug Laboratory. DEA Reg #PA0132340. Testing conducted to ensure State compliance. Inspector Mandy Wilson. AAMU license 01_22-20006. FSA Farm 8022, FSA tract 4484. Samples run by Joe Basile.

Flavinoid analysis by Dr. Korey Brownstein at USDA-ARS NCAUR.

SAC Germplasm evaluation conducted at OSU (KBREC) by Stephen Baluch. Seeding rate: 10 plants per entry planted 3 ft apart with 6ft between each entry. Plants were planted on 6ft centers. Bed size: 3 ft. Plot size (ft2): 90’*500’ . Area: 1 acre. We used a fertilizer blend, See rates in the attachment. Note: Not all 1 acre was used as entries were reduced to 33. Initial fertilization was calculated and applied based on 1 acre.

SAC Germplasm evaluation conducted at UC Davis: Bed centers are 5ft apart, within bed plants are 3 ft distance. Plot size for one accession: 10ft x 15ft (5 + 5 plant across two beds). White plastic mulch on raised beds. Very abundant weed: Bindweed (weeding about 3 times between rows) Fertilization: 100lbs N/acre during the season. Plus they all got one injection of Coragen at 7oz/acre.

Complete screening of hemp germplasm in REP 1-3 of 2022 USDA phenotyping trial in Research North 010. This method includes individual samples from all Reps. Analysis following UPLC protocol in USDA Hemp Descriptor Manual version 2 (e.g., using shorter run time than in version 1). Samples were run for individual plants with pistillate flowers (3-30 individuals). Samples were taken 5 weeks after terminal flowering initiated following as close as possible to AMS sampling guidelines, although the apical meristem was not always samples (e.g., auxiliary inflorescences were taken because we were also measuring plant height). Technical samples were run as triplicate and this data represents the mean of those three technical values.

Analysis by Phil Bates at Washington State University. Total seed lipid content and fatty acid analysis was carried out by GC-FID and results are expressed as percent of seed mass for FA composition.

Screening of hemp germplasm from 2022 USDA-ARS PGRU phenotyping trial in Research North 010. Trial included 100 accessions, three replications, approximately 10 plants per plot. Evaluation occurred on the individual plant level. Data is presented as means across all individuals.

Preliminary screening of hemp germplasm in REP 1 of 2022 USDA phenotyping trial in Research North 010. This method only includes individual samples from Rep 1 and is used to allow/disallow germplasm for distribution. Analysis following UPLC protocol in USDA Hemp Descriptor Manual version 2 (e.g., using shorter run time than in version 1). Samples were run for individual plants with pistillate flowers (3-10 individuals). Samples were taken 5 weeks after terminal flowering initiated following as close as possible to AMS sampling guidelines, although the apical meristem was not always samples (e.g., auxiliary inflorescences were taken because we were also measuring plant height). Technical samples were run as triplicate and this data represents the mean of those three technical values.

2022 SAC Germplasm evaluation conducted at Washington State University (Othello, WA). Data is presented as means across all individuals. Trial was replicated at WSU, OSU, UC-Davis, and PGRU.

SAC Germplasm evaluation conducted at UC Davis: Bed centers are 5ft apart, within bed plants are 3 ft distance. Plot size for one accession: 10ft x 15ft (5 + 5 plant across two beds). White plastic mulch on raised beds. Very abundant weed: Bindweed (weeding about 3 times between rows) Fertilization: 100lbs N/acre during the season. Plus they all got one injection of Coragen at 7oz/acre.

2023 Germplasm Evaluation for septoria and powdery mildew. Trial run by USDA-PGRU and Cornell University in McCarty 06. Data generated by Jocelyn Schwartz, Chris Smart, Dan Meyers, Tyler Gordon, and Zachary Stansell. [JAS verbatium: “Seeds were planted in flats on 11 May, where they were regularly maintained and watered. Prior to transplanting in the field, Big Yield Blended 19:6:19 Fertilizer was broadcast at a rate of 500 lbs/acre. The transplants were set in the field by hand into 4 ft wide raised beds covered with plastic mulch on 31 May. Each plot consisted of 5 plants with 3 ft spacing in between plants, 3 ft spacing in between plots, and 7.5 ft spacing in between rows. The field was established in a randomized complete block design with 3 repetitions of 81 accessions. Plots were irrigated and fertigated as needed via a drip line with 24-8-16 Miracle Gro all-purpose, water-soluble fertilizer. Plastic weed cloth was applied in between rows and was supplemented with hand-weeding as needed. Inoculum was created with a single conidial isolate of Septoria cannabis collected from Geneva, NY. 100 μl/L of Silwet was added to the inoculum and a final concentration of 4.9 *10^4 conidial suspension was applied using a battery-powered backpack sprayer on 22 June. Approximately 30 mL of inoculum was applied to each plant. Symptoms were first observed 7 days post inoculation. Disease severity ratings were visually estimated on a scale of 0 to 100%. The first disease severity rating was on 29 June, and disease severity continued to be visually estimated each week.”]

Standard COA of field evaluated materials. Donor note: Here are some COAs for the Badger genotypes from this year’s field trials. We took a sample from 5 different plants from each Badger variety and did individual COAs for each one.

Prescreening of recently acquired germplasm grown in USDA-PGRU Wellington Farm, Zone 7. \n Non-replicated observation of up to five individuals. Screening for chemotype, cannabinoids, general observations, and some other phenotypic data (TBD). \n Data will be used to inform regeneration priorities. \n Seeds sown 2023-06-04. \n Included on the side of the 2023 SAC Trials run at USDA-ARS PGRU in Geneva, NY, USDA Wellington Farm, Zone 7.

2023 SAC Trials run at Alabama A&M University by Xinhua Xiao and Ernst Cebert. 34 sessions of hemp seeds were planted in jiffy pots in a greenhouse on 4/10/23. 23 sessions were transplanted in the field on 5/4/23 [34°54'2.47N, 86°33'42.77 W], at 372 Walker Lane, Hazel Green, AL, 35750. P fertilization (50lb) was broadcasted in the field before planting on 5/1/23. 3 lb N fertilization (34-0-0) was applied around each plant by hand in the whole field （we have 340 plants in total). Watered the plants with a hose two times during the growing season (5/4/23 planting date; 6/7/23). No other irrigation was applied.. A NO3-N (ppm) test before planting. We collected 2 soil samples for the test in the whole field (110 ft X230 ft =25300 ft^2 =0.58 Acre). Each soil sample was mixed from 5 positions (0-15 cm depth). The NO3-N test result was 2.35 ppm and 4.32 ppm. See https://portal.nifa.usda.gov/web/crisprojectpages/1027227-development-and-characterization-of-diverse-hemp-germplasm-for-submission-to-the-npgs.html

USDA-ARS PGRU 2023 field trials at Wellington Farm in Geneva, NY. Trials are part of the NIFA-SAC grant led by Dr. Charlie Brummer of UC-DAVIS.

Part of SAC HEMP PATHOLOGY, UKY GERMPLASM TRIAL, FUSARIUM HEAD BLIGHT, 2024 USDA-PGRU/UKY CA (https://www.ars.usda.gov/research/project/?accnNo=445654). Trial in Lexington Kentucky. Trial Planting July 11, 2024, with last rating Sept 25, 2024. Hurricane Sept 27, plants fell over and were not ratable. Note, disease severity increased dramatically after the hurricane (6” rain in two days), but plants were broken and fallen into adjacent plots. General weather 2024: excessive heat and drought. July heat index max 104.5, mean 92.2; high temperature max 94.7, mean 87.4; high RH mean 92.8%, Precip 2.5 inch. August heat index max 101.5, mean 89.5; high temperature max 97.5, mean 85.8; high RH mean 94.1%; Precip 3.3 inch. September head index max 95.5, mean 81.0; high temperature max 91.0, mean 80.1; high RH mean 92.5%; Precipitation 6.2 inch. FHB Disease Severity: Plot 144 disease emergence Aug 28, final ratings 20-40% severity Plot 145 disease emergence Aug 28, final ratings 0-30% severity Plot 163 disease emergence Aug 28, final ratings 20% severity Plot 204 disease emergence Aug 7, final ratings 20-30% severity Plot 219 disease emergence Sept 11, final ratings 10-20% severity Plot 254 disease emergence Aug 14, final ratings 0-30% severity Plot 323 disease emergence Aug 28, final ratings 20-30% severity Plot 353 disease emergence Sept 11, final ratings 10-20% severity Plog 385 disease emergence Sept 25, final ratings 0-10% severity *all other plots, disease 0% incidence, 0% severity To assess accessions for resistance to Fusarium head blight, seed of each accession will be sown in 144-cell trays and grown for 3 to 4 weeks before transplanting into the field at the University of Kentucky North Farm Complex near Lexington, KY. Transplants will be planted into conventionally tilled soil using the production model typically practiced for burley tobacco production (no raised beds and no plastic) in rows spaced 1.07 m apart with 0.3 m between plants within the row. Drip irrigation will be provided as needed. Accessions will be planted in the field in a randomized complete block design with 10 plants per plot and 3 replications. Previous research at this location has shown that Fusarium head blight is endemic in this area so we will use natural inoculum to assess resistance. Wheat will be planted in early spring to increase inoculum in the area, and artificial inoculations will supplement natural infections, as needed. Fusarium disease will be assessed for incidence and severity using a quantitative rating scale (Gauthier, unpublished). Because accessions are expected to have varied and inconsistent maturity, ratings will take place every two weeks beginning at flowering. Disease assessments and plant development stage will be recorded. The area under the disease progress curve (AUPDC) will be calculated and means separated using Tukey’s HSD. All statistics will be performed using R statistical packages. Samples of plants with delayed disease development or signs of tolerance to FHB will be sent to the USDA PGRU greenhouses for genotyping and hybridizations.

2024 Germplasm Evaluation of diverse hemp germplasm in an outdoor field in Geneva NY for Septoria leaf spot and powdery mildew, see USDA-ARS PN 8060-21000-034-031-A [https://www.ars.usda.gov/research/project/?accnNo=445656]. One hundred and five accessions were selected for evaluation during the summer of 2024. Seeds were sown indoors on May 15th, 2024, and seedlings were transplanted to the McCarthy 001 field on May 28th of 2024, using a randomized complete block design with three blocks and a target number of five plants per block. Transplants were spaced three feet apart on raised beds, and landscape fabric was laid between rows to reduce weed pressure. Drip irrigation was used for watering when needed. An initial inoculation with a spore suspension of Golovinomyces ambrosiae spores was conducted on June 27th, 2024. The first powdery mildew colonies were observed on leaves in mid-July, and differential levels of disease were quantifiable by the end of that month. Disease was assessed visually as percent disease on a scale from 0-100% of a whole plant leaf area. Individual plants were rated on August 1st, 7th, 12th, 20th and 28th. Flowering time and sex were evaluated on a weekly basis. For plants that hadn't flowered by October 5th, one or two weeks in the future were estimated based on visual floral development. Although many plants of Chinese origin are among the least susceptible ones as of the last rating, many showed signs of disease as they approached flowering”

2024 Germplasm Evaluation of diverse hemp germplasm in an outdoor field in Geneva NY for Septoria leaf spot and powdery mildew, see USDA-ARS PN 8060-21000-034-031-A [https://www.ars.usda.gov/research/project/?accnNo=445656]. Trial run as a collaboration between Cornell University (Larry and Chris Smart laboratories) and USDA-PGRU Hemp Germplasm Laboratory in McCarthy 01. Pathogen data and associated information generated by Jocelyn Schwartz (lead author), Chris Smart, Larry Smart, Tyler Gordon, and Zachary Stansell and others [Add additional names given by JAS]. JAS verbatim: “Seeds were planted in flats on 15 May, where they were regularly maintained and watered. The transplants were set in the field by hand into 4 ft wide raised beds covered with plastic mulch on 30 May. Each plot consisted of 5 plants with 3 ft spacing in between plants, 3 ft spacing in between plots, and 7.5 ft spacing in between rows. The field was established in a randomized complete block design with 3 repetitions of 105 accessions. Plots were irrigated and fertigated as needed via a drip line with 24-8-16 Miracle Gro all-purpose, water-soluble fertilizer. Plastic weed cloth was applied in between rows and was supplemented with hand-weeding as needed. Inoculum was created with a single conidial isolate of Septoria cannabis collected from Geneva, NY. 100 μl/L of Silwet was added to the inoculum and a final concentration of 6.4 *10^5 conidial suspension was applied using a battery-powered backpack sprayer on 3 July. Approximately 30 mL of inoculum was applied to each plant. Symptoms were first observed 7 days post inoculation. Disease severity ratings were visually estimated on a scale of 0 to 100%. The first disease severity rating was on 10 July, and disease severity continued to be visually estimated each week.

Brief consolidation of 2024 HudsonAlpha USDA-ARS PGRU Hemp Germplasm Collection Genotyping/Sex-determination/Phenotyping Project. Evaluate and genotype two XX and two XY individual per accession from most of the USDA-ARS Hemp Germplasm Collection, N ~ 2000 genomes. Phenotypic efforts follow established protocols in the USDA Hemp Handbook [https://www.ars.usda.gov/northeast-area/geneva-ny/plant-genetic-resources-unit-pgru/docs/hemp-descriptors/] and Brummer SAC projects. Conducted in Zone 1, in USDA-ARS Wellington Farm (1.13 ha ~ 2.8 ac), 42.535048'N 77.05422'W. Nov 2023 soil testing was most recent test. My understanding is that ENR = 94 lbs./ac. and add 100 lbs. N/Acre to the field. PGRU will share all data and workflow information using Cornell Box with all collaborators invited as editors. In early 2024, the PGRU team will generate a list of accessions for evaluation, pulled germplasm, designed field randomization, established persistent unique identifiers (PUIDs) for every potential individual, procured greenhouse and field supplies, generated plot labels, generate 50 mL tissue sampling tubes with PUID QR codes. Given germination data and seed quantity, we pulled enough seeds to generate 20 individual live plants. Some accessions could not generate all nine live plants from, but they will still be included in the trial. In April 2024, the PGRU team prepared approximately 2-acre field at the USDA-PGRU Wellington Farm in Zone 1 and applied 100 lbs./acre nitrogen before bedding. Field design will be compostable mulch with between row spacing of 7.5’. Between rows w/ landscape fabric for weed suppression. Late April, the PGRU team will fill 161 2x2x3” peat pots with ProMix BT. May 7 sowed seeds from each accession into 2x2x3” peat pots in a greenhouse using ProMix growth media. These seeds were sown in the exact order that they will be transplanted into the field. In rep one, placed a unique pot label in each peat pot. Thin and balance the seedlings in the peat pots such that all three reps have close to the same number of individuals, although the first rep will be prioritized to contain three individuals, as those accessions will be evaluated for sex first. All all flats were thinned by ZJS, TB, DJM 5/15-5/16. ZJS pulled individuals from reps 2 or 3 into rep 1 to bring the number of individuals as close to 3/rep as possible.. Any pot with multiple individuals will be randomly thinned to retain only one individual per pot. Zach conducted a seven day stand count on 5/14/24 and approximately 92% of the accessions had enough plants to proceed with transplanting and genotyping. 88% of the accessions contained at least two live plants, so there is a possibility of recovering an XX/XY pair. Plants were watered as needed in the GH, cold frames, shade house. The field design will be a complete randomized block design of 536 accessions in three replicates, with three individual plants per plot, (i.e., 536 accessions x 3 reps x 3 individuals per plot = 4,824). The true number of plants was lower due to seed quantity and viability. All germination data was recorded. A count of every plant will be made using FieldBook >10 days post sowing (5/17). Several days before transplanting (e.g., ~15-20 days post sowing), these accessions will be moved to a cold frame to harden off (Kevin). Transplanting will occur using a waterwheel at (1.5’ or 2’) spacing, 50 ppm N in tank. Last three weeks of May, DJM ran as many PACE reactions as possible to identify target plants. PGRU collected leaf tissue for DNA extraction for PACE analysis using the sex marker CSP2 in Toth dissertation. Identify two XX and two XY individual per accession (N < 1100). This number will be lower due to multiple reasons (not enough live individuals generated, “feminized”, randomly does not have either an XX or XY [ p = 1 – (2n-2)/ 2n; where n = number of individuals tested]). Data will be processed via SexDescisionTool.Rmd. Drip irrigation will be applied prior to and immediately after to reduce transplant shock. Transplanting occurred on May 23 (P1001-P3194) and May 24 (P3196-3564). The within row spacing is 3’. Fieldbook phenotyping: stand counts taken seven days after transplanting. Stand Vigor counts were taken (StandVigor_20240528, missing = no plant was transplanted, dead = plant died, 1= cotyledon, 2=small, 3 = average, 4=large, 5=very large). The results of stand count generate two slip tags for every plant in the field. Slip tags will be placed on destructive plant every plant with PUID, Accession number, Inventory Number, Inventory ID, Top Name, Plot number, Plant number, and a QR code that captures the PUID. The PUID is concat of Inventory ID, Rep Number, and Plant number. E.g. (24HA_G_33199_21UO_SD_S1004_p2 = “the 2024 Hudson Alpha trial, inventory G 33199,Plot 1004, plant 2”). Prepared 50 mL tubes of 10 g fresh, young leaf tissue, and ship overnight on dry ice. Target 10g fresh tissue. PGRU ran field walkthroughs and phenotyping at least once per week. Phenotyping efforts will mirror trials conducted in the USDA-UC-Davis SAC Trials (publication pending) and the USDA-ARS Hemp Descriptors and Phenotyping Handbook. WEEKLY: phenotypic sex, flowering date, and senescence phenotyping will occured on every individual in the trial (536x3x3). In mid Aug (~100 days after sowing) plant architecture traits were collected. Sequenced plus other key individuals were be selected for additional phenotyping. Phenotype Sex, F/M flowering date, leaf morphometrics. In mid-June, images of every plant will be taken using Field Book. CHEMICAL HARVEST taken ~35 days after terminal flowering via script every Friday to generate the chemical samples as F_FLOW_DATE + 35 days. Take three ~12 cm female inflorescence 35 days after terminal flowering, record date. Seed collection and shattering potential. Affix a seed shattering bag (Midco Global-PQ218, with a 336-um pore diameter, size 10” x 12”) to every sequenced female inflorescence with a printed label so the shattering bags can be tracked after harvest. ONE FEMALE (or Monoecious) PER Sequenced individual. DIA: stem diameter at the ground (mm). HT: plant height (cm). MCD: maximum canopy diameter (mcd) as width of plant at widest set of branches. Measure once between flowering and senescence (cm) MCDH: height evaluated at maximum canopy diameter from ground to max canopy diameter. Measure once between flowering and senescence (cm). Senescence date. Record senescence date weekly during flowering and sex phenotyping. Definition: stem starts to brown + all the fan leaves dry down. Harvest Measurements – at “Date_Seed” DATE_SEED: Take seed sample post-senescence, record date. Only sequenced plants with pistilate flowers. Try to obtain more than 5 grams of seed per plant. Chop sequenced stems at senescence. Cut at ground level with pruning shears. Trim off bottom 12 cm, save 1 m of stem, remove top. Remove branches. Make sure slip tag is on plant. Move stems and seed bags to a warm, dry place with plenty of ventilation—This will likely be the new high tunnels at Wellington. Print labels for each sample that include the "UNIQ-IND" as a QR code.

2024 SAC Trials run at Alabama A&M University by Xinhua Xiao [lead], Xianyan (Justin) Kuang [collaborator PI], Abiodun Adeniyi [graduate student], and additional students and technicians at AAMU. Experiment followed standard SAC single-plant protocol. See https://portal.nifa.usda.gov/web/crisprojectpages/1027227-development-and-characterization-of-diverse-hemp-germplasm-for-submission-to-the-npgs.html. Donor notes for single plant hemp data collected in 2024: "Seeds received 3/11, sown 4/10 in peatpots, 4/26 transplanted. 100 g N ferilizer (46-0-0) was applied in the water and sprayed to the jiffy pots before planting. Collected samples (Collected flower samples, take architecture data, flower rate evaluation) on 8/19. Single plant images taken 8/20.

2024 SAC Germplasm evaluation conducted at Louisiana State University. Data is presented as means across all individuals of accession_inventory. Trial was replicated at WSU, OSU, UCD, AAMU, and partially at ARS-PGRU Geneva, NY. Seedout date: March 27th, 2024, Transplanting date: April 18, 2024, Location Zip Code 70820. Soil analysis (for N content) : 2690 pp. Fertigation info (if applicable): no fertigation. See https://portal.nifa.usda.gov/web/crisprojectpages/1027227-development-and-characterization-of-diverse-hemp-germplasm-for-submission-to-the-npgs.html

2024 SAC Germplasm evaluation conducted at Oregon State University by Dr. Everald McLennon. Data is presented as means across all individuals of accession_inventory. Trial was replicated at LSU, WSU, OSU, UCD, AAMU, and partially at ARS-PGRU Geneva, NY. Seedout date: May 16, 2024, Transplanting date: June 10-11, 2024, Location Klamath Falls at Klamath Basin Research & Extension Center. Additional trial notes from Dr. McLennon: "Trial conditions were generally good and no major production issues were encountered. A few varieties were late maturing as up to 130 days after transplanting they had not yet flowered. Some varieties produced seeds before the chemical harvest (35 days after terminal flowering) period, so flower samples were not collected for these varieties. All varieties were susceptible to freezing conditions while some varieties were damaged (broken branches etc.,) by strong breeze. Beet curly top virus affected a few varieties. Bird predation was a challenge during the trial. Pictures at vegetative stage (8 weeks after transplanting) showed good phenotypic differences. Taking pictures of whole plant flowering at chemical harvest (35 days after terminal flowering) was difficult because plants grew so well, and plot area was very dense." See https://portal.nifa.usda.gov/web/crisprojectpages/1027227-development-and-characterization-of-diverse-hemp-germplasm-for-submission-to-the-npgs.html

2024 SAC Germplasm Trials run at University of California Davis by Dániel Pap and Charlie Brummer according to 2024 SAC protocol for evaluation of USDA-ARS NPGS hemp germplasm. Data is presented as means across all individuals of accession_inventory. Trial was replicated at LSU, WSU, OSU, UCD, AAMU, and partially at ARS-PGRU Geneva, NY. Seeds started: 2024-06-04, Acclimatization: 2024-06-24, Transplanted: 2024-06-26. All rest cultural practice information is same as previous years. See https://portal.nifa.usda.gov/web/crisprojectpages/1027227-development-and-characterization-of-diverse-hemp-germplasm-for-submission-to-the-npgs.html

2024 SAC Germplasm Trials run at Washington State University (Prossor) according to 2024 SAC protocol for evaluation of USDA-ARS NPGS hemp germplasm. Data is presented as means across all individuals of accession_inventory. Trial was replicated at LSU, WSU, OSU, UCD, AAMU, and partially at ARS-PGRU Geneva, NY. All rest cultural practice information is same as previous years.

See https://portal.nifa.usda.gov/web/crisprojectpages/1027227-development-and-characterization-of-diverse-hemp-germplasm-for-submission-to-the-npgs.html

Generic method for donor generated phenotypic data; no protocol specified.

HPLC protocol modified from Brownstein et al for in-house cannabinoid analysis. Note that this assay only includes CBD, CBDA, THCd9, THCA, CBG, CBGA. Developed by Kevin Hernandez, Zachary Stansell, Tyler Gordon, Sadie Kraft, and Korey Brownstein. Will be implemented for routine compliance testing of regenerations and selections. 🧪HPLC cannabinoid evaluation🧪 Sample run time: 20 min. Materials: • Ascentis® Express C18, 2.7 μm HPLC Column • Ascentis® Express Guard Cartridge Holder • Ascentis® Express C18, 2.7 Micron Guard Cartridge • No. 35 sieve • No. 10 sieve • Homogenizer o Mortar and pestle o Geno grinder o Rolling pin on dry ice o Hammer mill o Ninja Blender • 20 mL glass scintillation vial • Analytical balance • 200 proof Ethanol • Sonicator • 0.45 µm membrane filter • 1ml plunger syringe • 1000 ul pipette • 100 ul pipette • 10 ul pipette • 1 ml Autosampler vials • 1000ml Beaker • Computer with Control Panel software • HPLC instrument (The Robin) • 4.0 L amber glass bottle or 1L bottle • 18 M HPLC grade water • 0.1% formic acid • 95% formic acid • 5mM Ammonium formate • Acetonitrile • Syringes (L-1000, green-taped 5000 mL, red-taped 5000 mL) • Pipette (blue box tips) • Labels or Marker Standards: • Cannabidiolic Acid (CBDA; CAS #: 1244-58-2) • Cannabigerolic Acid (CBGA; CAS #: 25555-57-1) • Cannabigerol (CBG; CAS #: 25654-31-3) • Cannabidiol (CBD; CAS #: 13956-29-1) • Δ9-Tetrahydrocannabinol (THCd9; CAS #: 1972-08-3) • Δ9-Tetrahydrocannabinolic acid A (THCA or THCAA; CAS #: 23978-85-0) Standard preparation • All standard solutions are to be prepared at a concentration of 400 µg/mL. • All standards are prepared in methanol. Reagent preparation • 1 L reagent for Mobile Phase A: combine 999 ml of HPLC grade water with 210 ul of formic acid and then add 0.3153g of ammonium formate. Lastly add 1000 ul of formic acid to solution. • 1 L reagent for Mobile Phase B: add 999 ml of acetonitrile to 1 L bottle then add 1 ml of formic acid to solution. Protocol 1. Dry inflorescences at room temperature (20 °C) to < 10% moisture. 2. Grind samples. 3. Sieve through a number 35 sieve (500 μm). 4. Sieve through a number 10 sieve (2000 μm) 5. Place 0.1000-0.1050 grams of ground hemp material in a 20 mL glass scintillation vial. 6. Prepare each sample in triplicate. 7. Add 20 mL of HPLC grade 200-proof ethanol to the sample. 8. Sonicate for 30 min at room temperature. 9. Remove from sonication bath. 10. Allow the sample to sit overnight (18 h) in the dark at room temperature. 11. The next day filter the supernatant through a 0.45 μm Nylon membrane filter into a sample vial for analysis. 12. Inject 1 µL sample volume. 13. Set flow rate to 0.350 mL/min. 14. HPLC gradient program: 0-2 min, 75% B (concave curve 9); 2-5 min, 90% B (concave curve 9); 5-5.1 min, 90% B (linear curve 5); 5.1-11.1 min, 100% B; 11.1-11.2 min, 100% B (linear curve 5); and 11.2-20, 75% B. 15. The column temperature is set to 10°C 16. Ten-point standard curves are developed for the cannabinoids using a setting of 228 nm from the diode array detector. Methods provided by Berhow and Gude (2021) and modified by Dr. Korey Brownstein in 2023. Edited 11/18/2024 by Kevin Hernandez.

Tested to be compliant with 2021 Department of Agriculture Agricultural Marketing Service Hemp Final Rule (7 CFR Part 990 [Doc. No. AMS-SC-19-0042; SC19-990-2 FR])

MARViTECH Seed Analyzer Protocol By: Daniel Meyers & Anthony Barraco (last updated: June 7, 2023) Part 1: Loading the software. 1. Put on the MARViTech hat for maximum workflow efficiency. 2. Open Marvin software (Marvin 7.0.2). 3. Select the profile you want. Hemp_Test is recommended for general usage. Hemp_Test provides the following data: • Main Seeds: number of seeds • Weight (g) • TGW (g): thousand seed weight in grams • B-Stocking(%): percentage of things recognized as non-seed (including damaged/broken seed) • øArea: mean area of seeds • øWidth: mean width of seeds • øLength: mean length of seeds • Date • Time • Remark: an editable field for text comments • øL/W Ratio: mean length/width ratio of seeds • Formula 1: standard deviation of seed area • Formula 2: standard deviation of seed length • Formula 3: standard deviation of seed width • Formula 5: average seed brightness (“average grey value”) • Formula 6: standard deviation of seed brightness (“standard deviation of grey value”) Note on “grey value” (from the manual): During the measuring process, each pixel from each seed will be categorized in up to 8 customer configured ranges. We get up to 8 results, how many pixels with a brightness level of xx, yy, zz etc. The brightness level, (or grey value) goes from 0 (black) to 255 (bright white). 4. Select File > New to start collecting data for a new dataset. This should open a blank window with “Main Protocol: untitled” at the top. 5. Turn on the attached scale and tare the weigh dish. Part 2: Data collection. In the “Main Protocol: untitled” window, for each sample: 1. Name the sample in the ID column. 2. Place seeds in analyzer tray, spread seeds out so that there is no significant clustering of seeds and close the drawer. 3. Click “Check” button to begin analysis. You should see a white light illuminate the inside of the machine if everything is working correctly. Many of the fields should auto-populate with data. After the white light, the machine will illuminate with colored lights in red, green, and blue sequence. When this is complete, a pop-up window “Save image as” will appear, prompting you to name the generated image & save it in the folder Documents > HempSeeds. As a best practice, create a new folder within this file for each sample set to save your images to for exporting later & so that your pictures all go to the same place. Close this window if you don’t want an image of the seeds or talk to Tony or Dan to help you create a new protocol without this step. 4. To get weight data: 1. Dump seeds from the tray into the weigh dish. The weigh dish should be positioned beneath the funnel that is attached to the machine. There is a catch on the drawer where the tray is lined up with the mouth of the funnel. 2. Click “Weigh” on the software. A new window with “manual input” should pop-up. 3. Backspace out values so the “new value:” field is blank. 4. Click “OK” on the “manual input” window. 5. Click the measure button on the scale. For the Sartorius scale this is the button that looks like a dogeared page in the lower right corner. This should close the pop-up window and auto-populate the remaining fields. 5. Click the “Next” button to proceed to the next sample and generate another row for data collection. Part 3: Exporting data. 1. When you are finished collecting data, you can either save as an .mpr file (basically unopenable without the Marvin software) or export the data in the table by selecting File > Export. Name your file and select the file type as appropriate. Other “exports” (such as “Export all seed data” or “Export all source images”) will be very large files depending on the number of samples you have analyzed. Also, many of these files would be redundant as you already have an image saved for each sample & the raw seed data has already been analyzed within the table. 2. The computer that the seed analyzer is hooked up to WILL NOT be connected to the internet. Save files to a flash drive to get them off the computer and to not take up storage space on the computer. Remember to retrieve your image files if you did save them within the HempSeeds folder on the computer. Please delete any saved files from the computer after you have successfully gotten them off the computer and do not delete the file “.mpr” located in the HempSeeds folder, it is needed to run the Hemp_Test profile.

Data collected during routine regenerations at USDA-ARS PGRU of hemp germplasm. Note sample size is generally between 25-150, and observed under controlled environment conditions.

Test weight analysis of PGRU HEMP seed lots