Source: UNIVERSITY OF CALIFORNIA, DAVIS submitted to
DEVELOPMENT AND CHARACTERIZATION OF DIVERSE HEMP GERMPLASM FOR SUBMISSION TO THE NPGS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
EXTENDED
Funding Source
Reporting Frequency
Annual
Accession No.
1027227
Grant No.
2021-38624-35739
Project No.
CA-D-PLS-2688-CG
Proposal No.
2021-05523
Multistate No.
(N/A)
Program Code
HW
Project Start Date
Sep 1, 2021
Project End Date
Aug 31, 2023
Grant Year
2022
Project Director
Brummer, E. C.
Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
Plant Sciences
Non Technical Summary
Hemp (Cannabis sativa L.) has been grown as a crop for thousands of years for its essential oil, seed, and fiber. The renaissance in domestic and global hemp cultivation requires intensification of crop improvement for various characteristics, including end-product traits, agronomic characteristics, and broad stress tolerances. Currently, no public hemp germplasm collection exists in the US. We propose to acquire, characterize, and evaluate relevant germplasm across four diverse environments in WA, OR, and CA and to provide germplasm and associated data to the National Plant Germplasm System. Co-PD Stansell is the curator of the hemp collection and this collaboration will ensure our data conforms to the GRIN-Global hemp data standards under development. Hemp refers to any Cannabis germplasm with a concentration of D9-tetrahydrocannabinol (THC) < 0.3% dry weight and all evaluated materials will adhere to that standard. In addition to evaluation of germplasm itself, we will develop pre-breeding germplasm pools that represent broad-based populations facilitating recombination among the individual accessions, rapidly introducing wide phenotypic diversity for downstream breeder selection. These pools, or synthetic populations, one for each of the three main market classes, will be deposited in the NPGS system and made freely available for distribution. Additionally, we will also assess various methods to store pollen to facilitate hybridization and seed production of many hemp accessions and populations at once. This project addresses priority area B. Industrial Hemp:1. Germplasm collection for inclusion in the USDA Hemp Germplasm Repository.
Animal Health Component
0%
Research Effort Categories
Basic
(N/A)
Applied
50%
Developmental
50%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20217301081100%
Knowledge Area
202 - Plant Genetic Resources;

Subject Of Investigation
1730 - Hemp;

Field Of Science
1081 - Breeding;
Goals / Objectives
Our immediate objectives in this proposal are as follows:Obj. 1. Acquire hemp germplasm for deposit into the National Plant Germplasm System.Obj. 2. Evaluate hemp germplasm in multilocation trials on the west coast of the US.Obj. 3. Conduct pre-breeding to develop broad-based hemp germplasm populations for selection.These objectives fit within our larger collaborative project of improved hemp production and cultivar development. The trials proposed here will dovetail with our ongoing tri-state cooperative breeding program, adding value to that project without duplicating effort.
Project Methods
Objective 1: Acquire germplasm for deposit into NPGS (CA, OR, WA, ARS)Obtaining germplasm. We have sourced a large number of germplasms from private companies, interested individuals, and our own breeding programs. In addition to genetic resources identified by co-PD Stansell, the Global Hemp Innovation Center at Oregon State University currently has a collection of 90 hemp accessions that are targeted for preliminary evaluation prior to deposition into the NPGS hemp collection, with more accessions expected to be added to the GHIC collection in coming years.All PDs will continue to search for germplasm useful for the NPGS as a part of our ongoing breeding programs.Obj. 2. Evaluate germplasm in multilocation trials on the west coast of the US (CA, OR, WA)Genetic marker evaluation. (ARS, CA, OR) All germplasm will be characterized using genome-wide SNP markers to assess genetic relatedness with other germplasm in the NPGS system.Greenhouse evaluation. (OR, CA, WA) Germplasm will be evaluated for 25 priority traits including day-length sensitivity, plant architecture, and leaf morphology, among others. Traits amenable to characterization on seedlings will be a particular focus of greenhouse data. Data on traits also measured in the field, such as flowering time, will be compared between the two environments to assess genotype × environment interactions.Field evaluation. (CA, OR, WA) We will conduct annual field evaluations at four locations (Fig. 3). Due to anticipated seed limitations, we will evaluate transplanted seedlings rather than direct seeded plots. Seedlings will be germinated in the glasshouse approximately two weeks prior to planting in the field, and hardened in a lath house for several day before transplanting. This project will evaluate the genotype × environment interaction for various compositional traits as well as agronomic, morphological, and phenological traits. Germplasm trials will contain 20-30 accessions each year, including a standard check control cultivar for each market class - essential oil, seed, and fiber. The checks will be determined based on our past experiences and in consultation with industry partners. The same checks will be used every year at all locations, to facilitate comparisons among germplasm accessions. Each year, we will evaluate 15-25 new accessions.Trials will be planted between February (El Centro) and June, with planting dates chosen to reflect the local environment at each location. We will evaluate numerous traits in field trials. The traits and methodology to measure them will be based on the standards being developed by co-PD Stansell so that our data fits smoothly into the GRIN-Global database for hemp that he is developing. Traits we will measure during the growing season include: plant height pre-flowering, at first flower, and at maturity; plant architectural traits including number of branches, branch angle, node number on main stem, and others; primary stem diameter at ground level; primary and secondary cola length and diameter; stigma color; plant color; trichome density; and others. We will identify the date of first flower and the length of the flowering period. At harvest, we will assess plant dry weight, primary and secondary cola weight, seed yield, and others. We will finalize our trait list and the standardized methodology to assess them prior to planting our first trials in 2022. We will be evaluating all accessions for a full suite of end product traits.Cannabinoid analysis. (WA) Cannabinoid (and seed protein and oil) analyses will be performed by Co-PD Gang at the Tissue Imaging, Metabolomics and Proteomics Laboratory (TIMPL) at WSU, of which he is Director. Cannabinoid profiles will be determined using our standard LC-PDA-MS/MS-based methodsProtein and amino acid analysis (WA) Total protein levels in the seed will be measured using standard protein tests (e.g., Bradford reagent) to obtain total protein yield values. For particularly interesting accessions (those that appear to be novel accessions) detailed analysis of the protein composition of the seed will also include established proteomics approaches.Hemp seed lipid compositional analysis (WA) Seed lipid content analysis will involve whole seed quantification of total fatty acid amount and composition, and subsequent analysis of seed TAG and membrane lipid amounts and fatty acid compositions. 1000 hemp seed samples (from each variety and location) will be screened for seed oil content by weight, per seed, and for fatty acid composition. 10 seeds from each biological replicate (~100-200 mg dry weight) will be weighed, and then total lipids extracted and simultaneously derivatized to Fatty Acid Methyl Esters (FAME), which will be analyzed by gas chromatographyFiber analysis. (Dr. Chris Delhom, ARS-New Orleans) Yield and qualities of bast fiber and hurd materials will be measured by a gravimetric method and the Hurd:Bast ratio calculated.Samples of bast fiber length, stiffness, and strength will be tested. Hurd will becharacterized for particle size distribution, % fiber, strength and stiffness, hurd density, bulk density, and water sorption.Statistical analyses. Data will be recorded on either individual plants (morphology, phenology) within plots or on a plot basis (yield, composition). Data on individual plants will be averaged to produce a plot value, which will be used in subsequent analyses. We will assess effects due to locations, accessions, and location×accession interactions for the various traits using linear mixed models. Means and/or best linear unbiased predictors (BLUPs) will be computed. Based on common check cultivars used in all trials across years and on individual accessions repeated in multiple years, we will also conduct analyses across years in order to assess year×accession and year×location×accession interactions. Traits for which interactions are statistically significant will be analyzed by location, by year, or by location-year combination as appropriate. Relationships among accessions for all traits will be conducted using Pearson correlation coefficients computed from mean values of each trait. Using individual plant data, we will evaluate differences among accessions based on their coefficients of variation.Obj. 3. Conduct pre-breeding to develop broad-based germplasm populations for selection.Developing pools. Crosses will be made between individual genotypes of two or more germplasm sources for a given market type. We will include as many different accessions as possible for a given market class in each pool. Populations will be developed by crossing female plants of each accessions with pollen from males different accessions. Thus, we will ensure recombination among accessions, generating as broad based populations as possible, segregating for many traits and importantly, uncovering useful genetic variation that is hidden by virtue of linkage to adaptation related traits. We anticipate making three broad-based pools for essential oil, seed, and fiber germplasm. Pools will be expanded each year with additional germplasm.Pollen longevity experiments. To facilitate development of pre-breeding synthetic populations of hemp for each market class (essential oils, fiber, and grain), it would be beneficial to be able to harvest pollen from each parent selected for the population and be able to store it until pollen has been collected from all parents to allow for mixing of pollen prior to pollination. For initial pollen storage studies, pollen will be harvested by hand over a five-day period. Pollen storage studies will test pollen viability, pollen germination, and pollen cryopreservation. Pollen cryopreservation will be evaluated over a six month storage period with testing of pollen germination monthly.