FFAR Hemp Consortium: Multistate Screening of Triploid CBD Hemp Agronomic Performance and Pollen Sensitivity

Multistate characterization of agronomic performance of hemp cultivars, including sterility of new triploid cultivars DURATION OF PROJECT Start Date: 05/01/21 End Date: 4/30/22 Duration: 12 months BUDGET TOTALS Total FFAR Ask: $78,576 Total Cash Match: $52,826 Total In-Kind Match: $25,750 (value of seed donations) Total Match: $78,576 Total Project Budget: $157,152 PROJECT COLLABORATORS Check all institutions that will participate in the proposed work ?Cornell ?NC State ?University of Kentucky List all institutional PIs participating in the proposed project. If other consortium members are participating, please list them as well. Dr. David Suchoff – NC State Dr. Hsuan Chen – NC State Dr. Lawrence Smart – Cornell Dr. Jocelyn Rose - Cornell Dr. Bob Pearce – University of Kentucky 2 PROJECT ABSTRACT/SUMMARY Include a project abstract/summary of 200 words or less suitable for dissemination to the public. Increased field production of grain and fiber hemp results in significant amounts of winddispersed pollen. Pollination of floral hemp grown for cannabinoid extraction can result in drastic yield loss and unmarketable quality due to the presence of seeds. Consequently, farmers growing floral hemp require tools to minimize the threat of pollination. The proposed project seeks to characterize newly developed triploid (3n) hemp cultivars for sterility and agronomic traits over two growing seasons. Field trials will be conducted in North Carolina, New York, and Kentucky. Each location will have a pollen-challenged and pollen-free site. Three triploid hemp cultivars and their corresponding diploid (2n) equivalent (n=6 cultivars) from Oregon CBD will be used in these field trials. Cornell will test three additional triploid cultivars that do not have a direct diploid equivalent. Data generated in these trials will include time of flowering and harvest, seed production, floral biomass, and cannabinoid concentrations. The results generated from these trials will allow for a better understanding of the use of triploid cultivars in hemp production and lead to improved production recommendations for farmers across the nation. PROJECT CONTENT PROJECT EXECUTIVE SUMMARY The executive summary should be an overview of the proposed project that is informative to others working in the same or related fields and understandable to a technical reader. It should be a brief, high-level description of the full scope of work, including project objectives, anticipated outcomes, potential impact, budget, collaborators, and timeline. (1 page limit). As new genetics and processing capabilities become available, the agricultural landscape is poised to contain diverse hemp production systems including floral, grain, and fiber. The latter two types of hemp produce significant amounts of wind-dispersed pollen that poses a serious risk to farmers producing floral hemp for cannabinoid extraction. Optimum floral biomass and cannabinoid concentrations are obtained by producing only female plants in the absence of males/pollen. For example, Meir and Mediavilla (1998) demonstrated that unpollinated female plants produced approximately 2.5-fold more extractable oils than pollinated plants. Also, the presence of seeds is unacceptable in smokable flower, so even a small amount of pollination and seed set will cause smokable floral hemp to be unmarketable. Consequently, floral hemp farmers will require tools to avoid inadvertent pollination and help ensure maximum productivity and profitability. Induction of polyploidy (>two sets of chromosomes per cell) is both a naturally occurring phenomenon and a technique often used by plant breeders. Plants containing more than two sets of chromosomes (>2n) often show enhanced traits such as improved yield and/or quality (Sattler et al., 2015). The production of triploid (3n) cultivars by crossing a tetraploid (4n) parent with a 3 diploid (2n) parent is a strategy used to create sterile and seedless crops, some of which are still capable of producing a seedless fruit, such as watermelon (Citrullus lanatus L.) or banana (Musa). Hemp is diploid (2n = 20) and the development of tetraploid (4n) and triploid lines has been documented (Kurtz et al., 2020); however, the performance and sterility of triploid hemp under field conditions with and without pollen pressure has not been investigated. As such, the objectives of the proposed research are to: 1) determine seed production of three triploid cultivars in comparison with their diploid equivalent (n=6 cultivars) grown in environments with significant pollen pressure; 2) quantify the effects of pollination on floral biomass and cannabinoid production and; 3) characterize agronomic performance, floral biomass yield, and cannabinoid content from those same diploid-triploid pairs of hemp cultivars in a pollen-free environment. This work has the potential to allow farmers to produce floral hemp without concern for inadvertent pollination and consequent degradation of final product. This issue has already led to numerous lawsuits (“Oregon industrial hemp litigation”, 2018), which could be avoided through the use of triploid hemp cultivars. Collaborative field trials will be initiated May 2021 in North Carolina (Suchoff and Chen) and Kentucky (Pearce) and in June 2021 in New York (Smart). Three floral hemp diploid-triploid pairs will be provided by Oregon CBD for investigation: ‘Lifter’ (2n), ‘Lifter Seedless’ (3n), ‘White CBG’ (2n), ‘White CBG Seedless’ (3n), ‘Suver Haze’ (2n), and ‘Suver Haze Seedless’ (3n). All six cultivars will be planted in a pollen-free and pollen-challenged environment in replicated small plot trials within each state in methods typical of the growers in each region. Pollen challenge will be provided by grain and fiber hemp planted as a border around the trial or in an immediately adjacent trial. Data collected will include periodic height measurements, time of flower initiation, whole plant wet weight, dry floral biomass yield, and cannabinoid content. When present, total seed count and weight per plant will be determined. Floral samples (including seed if present) will be collected at 21 days after initiation of flowering, at the time of harvest (full maturity), and from a subset of whole plant dried, stripped floral biomass. Plant samples will be dried and milled at each site using a common protocol and subsamples will be sent to Cornell for cannabinoid analysis in the lab of Joss Rose. Results will be analyzed within six months after harvest of each trial. Data collected will help determine if triploid cultivars remain sterile in the presence of pollen challenge and are an effective means to mitigate inadvertent pollination of floral hemp. We will also characterize the relative agronomic performance of triploid cultivars compared with their diploid equivalents a pollen-free environment. Outcomes generated from this trial will include farmer recommendations for the use of triploid hemp to be integrated within each state’s respective Extension program as well as refereed scientific journal publication(s). Cornell will also be evaluating performance of autoflower varieties direct seeded in three different spacings and photoperiod sensitive varieties direct seeded in one spacing density. Those small plot trials will be harvested with an 8 foot stripper header. Results will be shared with other participants collaborating with Oregon State University with partial funding from USDA ARS. 4 Funds are requested to cover field labor needs, field trial and sampling supplies, cannabinoid analysis at Cornell, travel costs to field sites and Consortium meetings, and publication fees. References: Kurtz, L. E., M. H. Brand, & J. D. Lubell-Brand. 2020. Production of tetraploid and triploid hemp. Hortscience 55:1703-1707. Meier, C. & V. Mediavilla. 1998. Factors influencing the yield and the quality of hemp (Cannabis sativa L.) essential oil. J. Intl. Hemp Assoc. 5:16-20 Oregon industrial hemp litigation: Won’t you be my neighbor? (2018, September 8). Retrieved from https://harrisbricken.com/cannalawblog/oregon-industrial-hemp-litigation-wont-you-bemy- neighbor/ Sattler, M. C., C. R. Carvalho, & W. R. Clarindo. 2015. The polyploidy and its key role in plant breeding. Planta 243:281-296 PROJECT GOALS AND OBJECTIVES List the objectives by month or year for the duration of the project along with specific measurable actions that will be taken to achieve the corresponding goal. Objective 1: Determine seed production of three triploid cultivars in comparison with their diploid equivalent (n=6 cultivars) grown in environments with significant pollen pressure . Goal 1: Assess triploid cultivars as a means to reduce the risk of inadvertent pollination and seed production. Measurable Actions: Floral samples will be collected at harvest and any seed produced will be counted and weighed. Objective 2: Quantify the effects of pollination on floral biomass and cannabinoid production. Goal 2: Improve our understanding of the effects of seed production on floral hemp yield and quality. Measurable Actions: Floral biomass yields will be determined and cannabinoid content analyzed with or without seeds present. Objective 3: Characterize agronomic performance, floral biomass yield, and cannabinoid content from those same diploid-triploid pairs of hemp cultivars in a pollen-free environment. Goal 3: Determine if triploid cultivars have agronomic benefits over diploid relatives in pollenfree environments. Measurable Actions: Agronomic performance, floral biomass yields and cannabinoid content will be analyzed. APPROACH 5 A detailed account of the procedures/methodology used to achieve the goals and supporting objectives. Six Oregon CBD cultivars will be started from seed at each institution in early or mid-May for an early or mid-June transplant date. The pollen-challenged site will be located adjacent to each institution’s grain and/or fiber variety trials. The pollen-free site will be incorporated within each institution’s floral hemp variety trials. Both the pollen-free and pollen-challenged sites will be arranged in a randomized complete block with four blocks in a design typical of regional production practices with five- or 10-plant plots of each cultivar randomly assigned within each block. In-field data collection will include periodic height measurements and disease surveys, time of flower initiation, time of harvest, and final plant dimensions. At 21 days after initiation of flowering and again on the day of harvest, the top 20 cm of the main floral stem from three plants per plot will be collected and pooled, dried in the dark at temperatures below 38°C), and milled to an appropriate size for cannabinoid analysis via HPLC or GC-MS and shipped in scintillation vials. The wet weight of five whole plants will be determined for each plot. One plant per plot typical of the performance in that plot will be dried, floral and leaf material stripped to determine total biomass production on an individual plant basis, and if present, any seed cleaned from that biomass with a Clipper seed cleaner or equivalent. That seed will be counted and weighed. Results will be presented at the FFAR Hemp Consortium annual meeting, then at annual national scientific meetings (such as ASA-CSSA-SSSA and/or ASHS), as well as state-wide or county extension meetings and field days. Extension materials will be created for each state to guide farmers on the use of triploid hemp cultivars. Finally, at least one manuscript will be written and submitted to an appropriate refereed journal. ANTICIPATED OUTCOMES OR OUTPUTS Describe the potential impacts of this project on the hemp industry, including any potential commercialization of results. Results from this project will help elucidate the efficacy of triploid cultivars as means to maintain seedless production even in the presence of hemp pollen. If effective, this will allow floral hemp farmers to produce a crop without concern of inadvertent pollination from neighboring grain or fiber hemp. Furthermore, the relative performance of triploid cultivars compared with their diploid equivalents will be determined by this project may help floral hemp producers maximize profitability. BUDGET JUSTIFICATION The amounts requested for each budget line item should be documented and justified NC State: 6 Salaries and Wages $13,602 Summer hourly - 35% FTE ($10,000/year) + 9.05% Fringe x 2 year Technician – 5% FTE + 30.73 Fringe + 3% annual raise x 2 years Supplies: $2,000 Field supplies including, but not limited to, fertilizer, pesticides, stakes, bags. Shipping: $200 Shipping of samples to Cornell for analysis. Travel: $1,000 Gas required for fleet vehicles for travel to field sites. Publications: $600 F&A $1,934 FFAR limited rate of 10% TOTAL $19,336 Oregon CBD in kind match: Seeds $4,500 Cornell University: Salaries and wages: $56,973 Smart – Salary recovery + Fringe (64%) x 2 years Smart - Technician – 25% FTE + Fringe (64%) x 2 years Rose – Technician – 10% FTE + Fringe (64%) x 2 year Supplies: $9,000 Field trial and sample collection supplies, cannabinoid standards, filters, vials sufficient for sample analysis from 6 varieties x 8 plots x 3 sampling times x 3 sites = 432 samples x $8 per sample = $3,456 for sample analysis x 2 years = $7,000 Travel: $4,000 Travel to Raleigh for FFAR Annual Meeting Indirect costs: $7,774 TOTAL $77,747 7 Oregon CBD in kind match: Seeds $18,250 University of Kentucky: Salary & Wages: $14,355 Pearce - 0.50% effort salary + fringe x 2 years Pearce Technician – 2.08% effort salary + fringe x 2years Student Hourly Labor – 20.83% effort salary + fringe x 2 years Supplies (plus 0.9% mandatory recycling fee): $2,825 Field supplies including, but not limited to, fertilizer, pesticides, stakes, bags. Travel: $400 Local travel to plot locations: F&A $1,756 FFAR limited rate of 10% TOTAL $19,316 Oregon CBD in kind match: Seeds $3,000 TIMELINE Provide a brief timeline for project activities including anticipated outcomes or outputs. May 2021: Seeds started in greenhouse June 2021: Hardened transplants planted in field locations. July-August 2021: Height measurements and disease surveys September – December 2021: Harvest data collected, samples prepped and shipped for cannabinoid analysis. January - March 2022: Cannabinoid analysis conducted and data analyzed. April 2022: Results will be shared with Oregon CBD and FFAR consortium and synthesized into scientific publication(s) and appropriate Extension materials.