Progress 09/01/22 to 08/31/23
Outputs
Target Audience:We continued to work with numerous commercial hemp companies and hemp growers to source germplasm and to discuss the objectives of our project during this reporting period. Through one-on-one contacts and field days at all locations, we reached hemp growers, other farmers interested in possibly growing the crop, extension specialists, seed growers, and others interested in hemp and hemp products. Changes/Problems:The project is proceeding as planned. The only meaningful change is that we have added several evaluation locations with interested scientists who brought their own support to run evaluation trials of the same germplasm, using the same methods, the the PIs are growing. This will add considerable value to the germplasm collection. What opportunities for training and professional development has the project provided?Numerous graduate and undergraduate students participated in data collection, harvest, and sample preparation on these trials across all locations. These students included some working with hemp on other research projects and others whose primary research was in another crop. These students learned about hemp genetic variation, about methods of characterization, and other aspects of hemp biology and agronomy. Dr. Daniel Pap, a postdoctoral scientist in the Brummer lab at UC Davis, has taken the leadership role for this project, coordinating all aspects of the trial with the other participants and working very closely with co-PI Stansell to ensure all the work dovetails with the needs of the NPGS Hemp Germplasm Collection. How have the results been disseminated to communities of interest?The results of this project are disseminated through field days at all locations and also through one-on-one discussions/emails/phone calls with interested hemp growers, processors, and seed companies. We also make our data available to the public through the GRIN-Global database at the USDA National Plant Introduction Station. Any plant breeder with an interest in hemp can access the data and use it to help make decisions about which germplasm to request. What do you plan to do during the next reporting period to accomplish the goals?Plans for the remainder of the project Our future plans remain largely unchanged from the initial proposal. While we provide a brief overview of several key items below, we emphasize that the comprehensive details of each objective can be found in the original proposal. Objective 1. We will maintain an ongoing effort to acquire additional germplasm for our collection, both from local growers and companies as well as from international germplasm collections. We will also keep increasing seed production in our greenhouses located in OR, CA, and NY. Some of these newly acquired accessions will be included in the evaluation trials in the coming years. Objective 2. The initial season provided valuable insights into our data collection capabilities in the field, allowing us to determine the pace at which we can gather data, identify the most relevant traits, and explore the potential for developing improved collection methods to enhance the accuracy and efficiency of characterizing accessions. This season's results will demonstrate the effectiveness of our streamlined system, potentially enabling us to expand our phenotyping efforts across all six sites in subsequent years. All the data obtained from these accessions will be compiled and shared with Co-PI Stansell for integration into the GRIN-Global database. Co-PI Stansell's team at PGRU is pursuing whole-genome sequencing on every hemp accession held within the Hemp Germplasm Repository. These data should become available by the end of 2023 and will be used to run Genome-Wide Association Study (GWAS) analysis against datasets generated during the SAC trials. Objective 3. We are continuing to develop broad-based pools of germplasm for essential oil, seed, and fiber types. Co-PI Baluch at Oregon State University provided bulked seed and novel germplasm to the NPGS Germplasm Collection. Some critical developments in regenerating and distributing these genetic resources have occurred during the year 1 SAC trials, specifically, important improvements in pollen collection, pollen storage, and pollen delivery have been made. In year 2 SAC trials, we will be evaluating field regeneration cages made with 10-micron pore mesh to allow higher throughput seed regeneration and bulking, while retaining the genetic integrity of a given accession.
Impacts
What was accomplished under these goals?
Objective 1: Germplasm Acquisition The team has acquired 415 accessions from various donors and international sources. These accessions encompass feral germplasm and internationally recognized cultivars, as well as breeding stocks and crosses generously provided by various public and private breeding programs. All of these accessions have been added to the NPGS hemp collection at the Plant Genetic Resources Unit in Geneva, NY, curated by co-PI Stansell. From this extensive collection, we carefully selected 29 new accessions for which an ample quantity of seeds was available for evaluation in the field in 2023. These accessions will not only be used for our multilocation screening but will also be made immediately accessible to the public through PGRU. Our selection criteria focused on ensuring a diverse representation of major hemp product types (fiber, grain, and flower) while minimizing redundancy. We excluded synonyms originating from different sources. In cases where multiple feral accessions were available from a single location, we retained only one for our field trials. This approach allows us to conduct a comprehensive evaluation of a wide range of germplasm and establish a core initial database for PGRU. Objective 2: Germplasm Evaluation In 2022, we conducted a comprehensive evaluation of 26 accessions and ten checks across four locations: Prosser, WA; Davis, CA; Geneva, NY; and Klamath Falls, OR. Each trial included 10 plants per accession in each of four replications. Our primary objective in the inaugural trial year was to establish a robust evaluation system and refine the process of characterizing and comparing phenotypic accessions of this versatile and multifaceted crop. We successfully implemented cultivation methods that were harmonized across all locations, ensuring consistency in crucial practices such as seeding, transplanting, field spacing, and fertigation. We recorded experimental practices, conducted detailed soil analyses, recorded daylength and temperature, and made the collected data accessible through the GRIN-Global database (Figure 1). Throughout the growing season, we monitored the flowering stages and sex of each individual on a weekly basis. At individual flower maturity, we measured the height, maximum canopy diameter, height of the maximum diameter, trunk length, trunk diameter, opposite and alternate branching, and internode length (Figure 2). We also made notes of stem color, leaf variegation, pest or pathogen pressure, and took high-quality photographs of the whole plant. Additionally, at flower maturity, we placed a mesh bag over the main cola to determine the amount of seed shattering. At seed maturity, we measured the wet biomass, harvested a seed sample, and sampled a one meter section of the main branch for subsequent analysis. We also harvested the mesh bag with the flower inside it. The weight percentage of seeds collected from the mesh bag, along with the seeds threshed from inside the mesh bag's cola, provided us with an estimate of seed shattering. The several hundred flower samples were dried and ground, seed samples were dried and threshed, and stem samples were pruned and dried before all material was shipped to their respective analytical labs. High-Pressure Liquid Chromatography (HPLC) was used for cannabinoid profiling. The cannabidiol production of the accessions evaluated in the NY and CA sites showed a good correlation. Seed analyses were conducted to generate data on the nutritional aspects of the hemp, including the seed oil, protein, and fatty acid content of the mature seeds. Stem analysis was performed after decortication to estimate the bast and hurd fiber quality, fiber length and strength, and other pertinent characteristics. For the 2023 season, we established collaborations with Dr. Ernst Cebert from Alabama A&M University (Huntsville, AL) and Dr. Babitha Jampala from Louisiana State University (Baton Rouge, LA). They have joined our team (using their own funding) and will participate in evaluating the same germplasm as our existing sites, allowing us to gain a better understanding of the performance of this germplasm in Southern states. In preparation for the 2023 season, we have standardized and simplified our data collection methods, focusing more on key agronomic traits. As a result of the good similarity between our field trials, we have adjusted our experimental design. We have reduced the number of checks from ten to six, and we have also decreased the number of plants in each field replication. Previously, we had 10 plants of each accession in four replications and in four different states. Now, we will have 5 plants of each accession in four replications per location, with testing locations in six states. These modifications will enable us to collect data in a more reliable and efficient manner. To ensure consistency in plant characterization, we have developed a practical manual for the selected traits based on the Hemp Descriptor and Phenotyping Handbook created by Co-PI Stansell. These changes and improvements for the 2023 season aim to enhance our understanding of hemp performance, facilitate efficient data collection, and promote consistency in plant characterization across different locations. All six locations in six states successfully established the experimental fields in May-June of 2023 and began collecting flowering data to determine the ideal harvest dates. The first harvest and phenotyping of day length neutral plants are conducted this month, in mid to late July (Figure 3). Currently, this project is supporting generating whole-genome sequencing for the entire UDSA-ARS Hemp Germplasm collection. This will be accomplished in two phases. Phase 1 is underway and is a comparison between short-read whole genome sequencing (Illumina) and long-read approaches (Pac-Bio/Oxford Nanopore) to determine optimal parameters before sequencing the whole collection. In phase two, two individuals (an XX and XY) from each accession will be sampled for low-coverage whole-genome sequencing. All data will be publicly available on NCBI and linked with the appropriate accession on GRIN-Global. This work should be complete by the end of 2023. Objective 3: Sustain and manage newly obtained germplasm and pre-breeding Co-PI Stansell and Tyler Gordon at the USDA-ARS Plant Genetic Resources Unit in Geneva, NY have developed a high-throughput selfing system for hemp and are generating S1, S2, and S3 lines based on phenotypic data collected by the year one SAC trials. These lines will be evaluated in subsequent SAC trial years and environments for agronomic performance and will be publicly shared with breeders as inputs to develop locally adapted cultivars.
Publications
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Progress 09/01/21 to 08/31/22
Outputs
Target Audience:We have worked with numerous commercial hemp companies and hemp growers to source germplasm and to discuss the objectives of our project during this reporting period. We have not had any field days or other extension activities to discuss in this reporting period, although we have plans to hold outreach events at each location before the end of the growing season. These will be reported in next year's progress update. Changes/Problems:At this point, the project is proceeding as expected. We have expanded the number of accessions we are able to evaluate and characterize. What opportunities for training and professional development has the project provided?One postdoctoral scientist, Daniel Pap, at UC Davis has taken on overarching coordination of research for this project. He has been intimately involved in all aspects including desiging field trials, collecting data, andn demonstrating leadership of the group. In addition, numerous graduate and undergraduate students have worked on the project collecting data at the various locations. Staff at all locations have learned about cultural practices growing hemp and in conducting trials on diverse hemp germplasm. How have the results been disseminated to communities of interest?Not yet, although field days are planned for later in 2022 (and subsequent years) at all locations. Scientific publications will follow after data are collected over 2+ years. Information from this trial is being imported into the GRIN-Global germplasm information system and will be accessible by GRIN users. What do you plan to do during the next reporting period to accomplish the goals?Our future plans are essentially unchanged from the initial proposal. Below, we highlight several items, but we intend the full details of each objective to follow that in the initial proposal. Objective 1. As mentioned earlier, seed quantities of many of the accessions we currently have in had are very limited. Consequently, beginning later in 2022, we will increase seed in the greenhouses in OR, CA, and NY. Some of these accessions will be added to the evaluation trials in subsequent years. We intend to continue to identify new germplasm accessions to add to the project through our contacts with various hemp growers, companies, and interested individuals. As we obtain germplasm, we will work with Co-PI Stansell to move this into the collection. We have discussed sharing germplasm with Dr. Shelby Ellison and her feral hemp project; this will occur for the 2023 field season. Objective 2. In the near term, we will collect data from our first-year field trials as well as companion trials in the greenhouse. As the field season ends later this summer/early fall, we will send samples as specified in the proposal for fiber, oil, cannabinoid, and other end product analyses. This first season will also help us better gauge how rapidly we can collect data in the field, which traits are most relevant, and if better collection methods can be developed to more accurately or efficiently characterize accessions. All the data on these accessions will be compiled and provided to Co-PI Stansell for inclusion in the GRIN-Global database. In future years, we intend to run field trials much as we are doing this year. We will assess the germplasm we have available through our project and consider any new germplasm that Co-PI Stansell has obtained, decide on a short-list of germplasm to be included in our trial, and determine which check cultivars should be continued and which, if any, deleted or added. Data collection will be streamlined based on our experiences in 2022. Some accessions will be retested to get a better understanding of year × location × genotype interactions for various traits. Genetic marker analysis will be continuing throughout the project. Using a standardized hemp marker array would be an ideal way to develop a common basis for evaluating accessions. We are aware of a couple efforts to create a community array, but at this point, haven't identified one to focus our genotyping on. This is something we hope to resolve by the end of 2022. Objective 3. Later in 2022, and continuing through the rest of the project, we will begin to develop broad-based pools of germplasm for essential oil, seed, and fiber types. The pools will be kept separate by market class, at least initially. As we generate data on the accessions we are evaluating, we can more effectively allocate them to pools. The pools will be completely IP free, and will be provided to the NPGS for curation and distribution as they are developed. Additional germplasm will be added to the pools each year as we identify additional accessions of a given market class. One of the challenges of germplasm regeneration, as well as of developing unique germplasm pools, is keeping accessions and populations isolated from one another. One avenue toward streamlining the crossing and development of germplasm pools is to prescreen seedings for sex. Males could be kept in one greenhouse, where they are individually covered to exclude pollen and harvested for pollen as they mature; and females kept in a separate house and hand pollinated as desired. Stored pollen from existing populations could be used to hybridize with female plants of new germplasm sources, one way that the germplasm pools could be easily expanded with limited need for full isolation of the entire population.
Impacts
What was accomplished under these goals?
We initially proposed three objectives, and they remain the same: Obj. 1. Acquire germplasm for deposit into the National Plant Germplasm System. Obj. 2. Evaluate germplasm in multilocation trials on the west coast of the US. Obj. 3. Conduct pre-breeding to develop broad-based germplasm populations for selection. During this reporting period, we have made substantial progress on Objective 1 and have planted multi-location field trials for Objective 2. The bulk of Objective 3 will begin later in 2022. Objective 1: Germplasm Acquisition The team has acquired several hundred accessions of hemp from diverse sources, including commercial companies, all of which is free of intellectual property protection and can therefore be donated to the NPGS hemp germplasm collection at the Plant Genetic Resources Unit in Geneva NY curated by co-PI Stansell. Among these germplasms are numerous feminized accessions, which the collection is currently not curating due to the complexities of regeneration, and consequently, we are not including these in our initial trials. Many of the accessions we have acquired have limited seed, so we will begin regenerations in the coming year in the greenhouse or controlled environment growth chambers using isolation cages. Including germplasm from this project, the Plant Genetic Resources Unit (PGRU) in Geneva NY now has over 180 unique accessions, which is likely the largest public repository of hemp genetic resources, globally. Objective 2: Germplasm Evaluation We had proposed to evaluate 20-30 accessions each year in the field and in this first year, we have included 26 accessions for which we had sufficient seed, together with 10 check cultivars comprising medicinal, fiber, and seed end-use traits. Initially we had planned to evaluate these only on the West Coast in WA, OR, and CA, but after the awarding of this project, the PGRU has hired a full-time Cat 1 Hemp Breeder, Dr. Tyler Gordon, who has agreed to evaluate the same set of germplasm along with other accessions he's evaluating and breeding. Thus, we will generate data from NY as well, increasing the number of locations that we proposed to test in and expanding inferences we can draw about our germplasm. We planted trials in Prosser, WA; Davis, CA; Geneva, NY; and Klamath Falls, OR. Our initially planned fourth location in the Imperial Valley in southern California, will not be planted until this coming winter (Nov - Feb). Each trial includes 10 plants per accession in each of four replications. These trials will be planted on white plastic to help with weed control when plants are small; and drip irrigation will be used in the trials in the west. Standard fertilization and other management activities will be applied. The data we will collect will follow that outlined in our initial proposal, based on the Hemp Descriptor and Phenotyping Handbook developed by Co-PI Stansell. The key traits we will assess on all germplasm includes morphological, secondary metabolic, fiber, oil seed, and horticultural quality characteristics. Several critical data points we will include are flowering date, male/female/monoecious ratios, chemotype marker information, plant architecture, morphological variation within plots, high quality images of plants, and analyses of stem fiber, flower cannabinoid and terpene concentrations, and seed oil, protein, and fatty acids. The data from these evaluations will be made available via GRIN-Global and will be analyzed for stability across locations and local adaptation. In addition to field evaluations, we are evaluating these and other accessions in the greenhouse to assess characteristics, such as day neutrality/photoperiod sensitivity, and to collect tissue for DNA extraction. We have extracted DNA from many of the accessions and have started DNA marker analyses for THC/CBC concentration and genetic diversity/relatedness estimation. Objective 3. Pre-breeding, including pollen experiments Co-PI Stansell has been screening materials provided by the SAC for pollen productivity using a novel pollen collection apparatus developed in-house at PGRU. The objective of this work will be to provide pollen alongside seeds as a breeding resource. Regeneration could also be facilitated by collecting pollen, which could then be used to pollinate female plants grown in isolation at a later time. Co-PI Stansell has developed a vacuum system for pollen collection that shows promise. Co-PI Zemetra is beginning experiments to look at various long term storage options.
Publications
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