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                    U.S. DEPARTMENT OF AGRICULTURE
 Agricultural Genetic Resources Preservation Research: Fort Collins, CO
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ARS Home Plains Area Fort Collins, Colorado Center for Agricultural Resources Research Agricultural Genetic Resources Preservation Research Research





- Walters, Christina
- Richards, Christopher
- Volk, Gayle
- Jenderek, Maria
- 2023
- 2022
- 2021
- 2020
- 2019
- 2018
- Publications
- Plant Genetic Resources, Genomics and Genetic Improvement (301)

Research Project: Efficient and Effective Preservation and Management of Plant and Microbial Genetic Resource Collections **Location:
                            Agricultural Genetic Resources Preservation Research** 2023 Annual Report **Objectives**
                                

Objective 1:  Efficiently and effectively preserve and back-up plant genetic resource collections under conventional (freezer) conditions.
Objective 2:  Efficiently and effectively cryopreserve and back-up plant and microbial genetic resource collections using liquid nitrogen as the cryogen.
Objective 3:  Design and test methods and strategies for exploiting genomic data to enhance the efficiency and effectiveness of the NPGS’s plant genetic resource management projects.
Objective 4:  Formulate and validate methods and strategies for efficiently and effectively sampling, preserving, and using the genetic diversity of selected crop wild relatives. **Approach**
                                

Genetic resources are the foundation of the United States’ agricultural future; their safety, health, and genetic integrity must be safeguarded. The USDA/ARS National Laboratory for Genetic Resources Preservation (NLGRP) will safeguard the U.S. National Plant Germplasm System (NPGS) base seed collection, designated non-NPGS seed collections, cryopreserved NPGS clonal accessions, microbial collections, and information associated with those genetic resources. With almost a million accessions, it will be responsible for preserving the world’s largest collection of plant and microbial genetic resources stored under one roof. This is particularly challenging for the NLGRP, and all of the NPGS, because the size of the genebank holdings continue to expand. Effective priority-setting can partially address that challenge, but it alone is not a solution. Research that generates effective strategies and methods for progressively improving the efficiency of plant genetic resource management is also critically important for successfully attaining the NPGS’s mission under challenging fiscal conditions. This project will provide long-term plant and microbial genetic resource storage. Genetic resources will be backed-up, monitored, and maintained with up-to-date, documented, best management practices, so that vigorous, pathogen-free seeds, propagules, and microbial cultures can be distributed when needed to other NPGS sites and other active collections. With other NPGS cooperators, this project will capitalize on its substantial capacity for seed testing and genetic resource storage in liquid nitrogen to assist NPGS genebanks which manage “active collections.” **Progress Report**
                                

The National Laboratory for Genetic Resources Preservation (NLGRP) in Fort Collins, Colorado is charged with ‘backing-up’ all accessions within the National Plant Germplasm System (NPGS) using state-of-the-art preservation methods. One facet of this mission is the distinction between duplication and true back-up (meaning the ability to replace a sample should it be lost or destroyed at the primary site), and another facet is the development of tools and concepts to maintain viability of plant germplasm during conventional storage in the freezer (Objective 1) and cryogenic storage at liquid nitrogen temperatures (Objective 2), as well as successful recovery of stored germplasm into healthy plantlets. NLGRP is responsible for diverse germplasm that includes about 20,000 species of domesticated or wild origin and uses both sexual propagules (seeds and pollen) and vegetative (i.e., clonal) propagules (e.g., shoot tips and dormant buds) in preservation work. A cross-cutting issue is maximizing access to diversity of preserved collections by increasing the number of species that can be genebanked or identifying novel or untapped genes. Genebanking efficiencies increase when a uniform preservation treatment delivers high initial survival, as in the case of orthodox seeds that are all dried in a similar manner. In this case, diversity is expressed as variation in how long seeds survive in storage (i.e., longevity). Many of the plant propagules (i.e., germplasm) preserved at NLGRP, especially vegetative tissues (i.e., clones), do not tolerate the drying treatments used in orthodox seed protocols and rather require complex treatments that vary among species, and even individuals, to maximize initial survival. Reaching a balance between uniformity of treatment and high survival requires collaboration between curation and research activities. An administrative reorganization during this Project Plan cycle facilitated the necessary interactions that allow this collaboration within seed and clonal teams, though there may be fewer interactions between teams.
There has been an increased emphasis on genebanking standards within both seed and clonal programs during this project plan cycle. Standards include quality and quantity criteria, which are needed to ensure meeting the mission of backing-up genetic resource collections. For the first time, NLGRP researchers took a hard look at how preservation practices measure up to international standards as well as how those international standards measure up to accomplishing the back-up mission. International Standards exist for orthodox seeds as a result of consensus within the global seed genebanking community. Standards for clonal propagules also consider viability and number of propagules, but the actual guidelines vary among institutions. A major outcome of this project (Objectives 1, 2, 4) addressed the benefits of standards in accomplishing the mission (i.e., ensure long-term survival and high fidelity of genetic representation of plant germplasm) as well as the cost of standards when they are too stringent and limit the number of accessions that can be processed or unnecessarily consume valuable propagules when testing is too frequent. From a major query of NPGS holdings at the Fort Collins facility, scientists learned that, despite about 80% duplication in seed-based collections, the proportion of accessions meeting standards is much lower at 18% (backed-up). For clonally propagated crops, 12% of the NPGS collection is backed up. Genebank facilities around the world are also reporting difficulties meeting standards.
NLGRP stores over 460,000 unique seed accessions, receiving about 5000 new or regenerated samples per year, which are tested for initial seed quality (taxonomic verification, germination behavior, vigor, seed mass, absence of embryo). In addition to these tests, NLGRP conducts about 8000 viability tests annually of stored seeds and these data contribute to assessments of seed longevity (Objective 1). In this project plan cycle, we focused on optimizing germination conditions to maximize normal development of seedlings, rather than testing viability using vital staining techniques. While treatments often vary and germination tests are slower than when vital staining is used (tests may take months), there is less processing time, and so analysts are able to nearly double their testing capacity (Objective 1). Even still, germination tests only reveal that a sample has not died; they do not reveal when a sample will die. Progress was made in developing biochemical and biophysical assays to measure seed aging rate. The most significant of these new tests measures the fragmentation of RNA. In this fiscal year, the laboratory will complete a collaborative study relating rate of RNA fragmentation with longevity of seeds from diverse species native to the U.S. (Objective 4). The use of cryostorage of seeds was also investigated (Objective 2). A full inventory of 24 cryotanks storing over 50,000 seed accessions (many for over 30 years) was conducted, and new protocols for testing sensitivity to liquid nitrogen temperatures was implemented. Protocols for cryostoring non-orthodox seeds of papaya and coffee were developed, and routine storage of these crops has begun.
Progress towards cryopreserving clonal accessions of apple, plum, and cherry using a method that harvests dormant buds in deep winter was made throughout this project plan cycle (Objective 2). Shoot tips (meristems) excised from explants grown in vitro or from greenhouses from numerous crops, including potato, citrus, grapes, kiwifruit, strawberry, mint, and pear were also cryopreserved using existing or recently established protocols (Objective 2). Cryoprotecting solutes are required for survival of shoot tips exposed to liquid nitrogen, and a collaborative study with colleagues at Colorado State University demonstrated partitioning of various chemicals within different regions of the cytoplasm. Research on cryotherapy continues to demonstrate that pathogens can be eliminated from apple shoot tips; this technology is being transferred to ARS apple rootstock breeding and Animal and Plant Health Inspection Service (APHIS) programs to efficiently sanitize infected materials. FY 2023 saw a major effort to review and augment the database called Genetic Resources Information Network (GRIN-Global) data fields so that critical information documenting clonal collection holdings and recovery methods could be accurately captured.
Fort Collins scientists participated in an international effort to assess vulnerabilities of apple and citrus genetic resources and develop strategies to explore and enhance genetic diversity in genebanks. Our laboratory was involved in collaborative work with more than 40 collections to use a newly developed SNP (single nucleotide polymorphisms) array to characterize apple accessions. Historic apple cultivars within U.S. National Parks and on public lands were identified using a similar genetic marker system and has led to in situ conservation efforts (Objective 3). In addition, the discovery of wild Malus as Malus domestica hybrids is another important outcome of the newly developed molecular marker system in apple (Objective 4).
Accessions representing wild populations are usually comprised of many individuals representing an array of genotypes creating a genetic pool of largely untapped genetic variation for desirable agronomic traits. However, incomplete characterization of phenotypes, accession heterogeneity and presence of undesirable “wild” traits hampers the use of genetic resources from wild populations. Scientists at the Fort Collins laboratory developed foundational genomic resources that will improve access to novel variation from crop wild relatives (Objective 3). Using ‘pangenome’ concepts and methods, individuals from populations were pooled and genotyped to generate a core set of genes representative of the species’ range. Using Beta vulgaris ssp. maritima as a test case, a new reference genome was generated and shown to be comparable to a standard reference for sugar beet EL10 that was published by another group in 2020. The Beta vulgaris ssp. maritima pangenome resource was provided to ARS sugar beet breeders through a local BLAST (Basic Local Alignment Search Tool) server allowing them to probe sequences for variation at any genomic position. The approach also allowed detection of variation among domesticated Beta vulgaris sequence data at any desired locus, and this was demonstrated in an examination of the diversity and distribution of functional alleles of rhizomania resistance (RZ2) and the bolting gene (BvBT).
Recognizing the need for educational resources for training and outreach, ARS and Colorado State University colleagues spearheaded efforts to build a publicly available platform (GRIN-U.org) and develop content relating to plant genetic resources management and use. GRIN-U.org now has nearly 200 items available as e-books, infographics, videos, webinars, articles, and podcasts and 95 subscribers to its quarterly newsletters. An e-book describing the process of clonal cryopreservation had 11,000 visitors between February and July 2023. An online five-week 1-credit plant genetic resources management course was developed and released in 2022 as part of a three-course series and will be offered again in fall 2023. **Accomplishments**
                                

1. Developed a global conservation strategy for citrus. The first “Global Strategy for the Conservation and Use of Citrus Genetic Resources” was developed by ARS scientists in Fort Collins, Colorado, and Riverside, California, and colleagues at the University of Florida (DOI 10.5281/zenodo.7757226). An extensive survey distributed to curators of citrus genetic resource collections from around the world collected data to document the diversity, availability, security, and vulnerability of citrus collections. This information was used to propose priority actions that will unify the citrus genebanking community by having access to shared online resources, training opportunities, increased standardization of data collection processes, and identified opportunities to improve the health and security of plant collections representing citrus genetic diversity. This research demonstrates the key role of the USDA National Plant Germplasm System collection with regard to citrus conservation and distribution on an international scale. 2. Identified climate change impacts for the USDA National Plant Germplasm System. Climate change is affecting plant genetic resource collections throughout the United States. ARS researchers in Fort Collins, Colorado, published the first assessment of the possible and real impacts of climate change on plant genetic resources conservation in the USDA National Plant Germplasm System (NPGS). Specific examples describe how temperature and drought have affected crop collection management. In addition, a web-based application was developed to provide NPGS collection managers with climate predictions for their specific locations. A complementary e-book was released that describes the importance of plant collections as society must adapt to climate change and provides information for climate adaptation planning. The recommendations and tools provided in these documents are a critical first step needed to safeguard NPGS plant collections to ensure long-term access for research and breeding. 3. Cryopreservation of sweet cherry dormant buds. Maintaining the genetic identity of sweet cherry cultivars requires clonal propagation, usually by using buds that can be grafted. Dormant buds acclimate to low temperatures during winter and can be cryopreserved if they become sufficiently cold hardy. Unfortunately, dormant buds of sweet cherry are difficult to preserve because they do not acquire sufficient cold-hardiness during warm winters. ARS researchers in Fort Collins, Colorado, developed a new method to supplement the acquired cold-hardiness of sweet cherry buds by soaking them in antioxidants prior to exposing them to liquid nitrogen. This method enhances bud survival, making it possible to cryopreserve the sweet cherry collection efficiently. This method is being implemented at NLGRP to preserve the sweet cherry collection in Davis, California. 4. Genetic approaches for improving germplasm collections of lost crops. Hordeum pusillum, “little barley”, was domesticated by Native Americans beginning 3800 years ago, but it was lost 500 years ago, most likely supplanted by maize. Wild populations of Hordeum pusillum are common and widespread throughout the United States, but plants exhibiting domestication syndrome traits (e.g., low shattering, larger and lighter seeds, few branching) are no longer found on the landscape. The USDA National Plant Germplasm System (NPGS) has only six accessions of this crop wild relative, which contrasts with 3248 wild accessions of common barley, Hordeum vulgare. To improve the representation of little barley in the NPGS collection, ARS scientists in Fort Collins, Colorado, collected samples from 18 broadly dispersed sites across the U.S. The population structure of this species was ascertained using genomic markers (simple sequence repeats, SSRs), which guided the probable locations of novel diversity, prompting 51 additional collections made in 2023. These efforts led to three new NPGS accessions that showed distinct genetic variation. Moreover, genes conferring domestication traits, known from cultivated barley, were found in little barley populations and raise the possibility of re-domesticating this once important lost crop. Little barley is likely to have genes of agronomic importance such as drought or heat tolerance, which can be used in breeding domesticated barley. **Review Publications**
                            *Araujo De Oliveira, A., Ledo, A., Polek, M., Krueger, R., Shepherd, A.N., Volk, G.M. 2023. Methods for cryopreserving of date palm pollen. In: Rajasekharan, P., Rohini, M., editors. Pollen Cryopreservation Protocols. New York, NY: Humana. p.519-525. https://doi.org/10.1007/978-1-0716-2843-0_49.*
                                *von Zonneveld, M., Volk, G.M., Dulloo, M.E., Kindt, R., Mayes, S., Quintero, M., Choudhury, D., Achigan-Dako, E.G., Guarino, L. 2023. Safeguarding and using fruit and vegetable biodiversity. In: von Braun, J., Afsana, K., Fresco, L.O., Hassan, M., editors. Science and Innovations for Food Systems Transformation. New York, NY: Springer Cham. p. 553-567. https://doi.org/10.1007/978-3-031-15703-5_30.*
                                *Volk, G.M., Peace, C.P., Henk, A.D., Howard, N.P. 2022. DNA profiling with the 20K apple SNP array reveals Malus domestica hybridization and admixture in M. sieversii, M. orientalis, and M. sylvestris genebank accessions. Frontiers in Plant Science. 13. Article e1015658. https://doi.org/10.3389/fpls.2022.1015658.*
                                *Volk, G.M., Gmitter, F., Krueger, R. 2023. Conserving citrus diversity: From Vavilov’s early explorations to genebanks around the world. Plants. 12(4). Article e814. https://doi.org/10.3390/plants12040814.*
                                *Moreau, T., Denning, S., Byrne, P., Volk, G.M. 2023. Climate change impacts agricultural productivity and food security. In: Volk, G.M., Moreau, T.L., Byrne, P.F., editors. Conserving and Using Climate-Ready Plant Collections. Fort Collins, Colorado: Colorado State University. Available: https://colostate.pressbooks.pub/climatereadyplantcollections/chapter/agricultural-productivity-and-food-security/*
                                *Volk, G.M., Byrne, P., Moreau, T. 2023. Importance of plants for mitigating and adapting to the effects of climate change. In: Volk, G.M., Moreau, T.L., Byrne, P.F., editors. Conserving and Using Climate-Ready Plant Collections. Fort Collins, Colorado: Colorado State University. Available: https://colostate.pressbooks.pub/climatereadyplantcollections/chapter/importance-of-plants/*
                                *Volk, G.M., Moreau, T. 2023. The need for plant genetic resource collections in a climate change context. In: Volk, G.M., Moreau, T.L., Byrne, P.F., editors. Conserving and Using Climate-Ready Plant Collections. Fort Collins, Colorado: Colorado State University. Available: https://colostate.pressbooks.pub/climatereadyplantcollections/chapter/the-need-for-pgr-collections/*
                                *Irish, B.M., Volk, G.M. 2023. Climate change affects plant interactions with pollinators, pathogens and pests. In: Volk, G.M., Moreau, T.L., Byrne, P.F., editors. Conserving and Using Climate-Ready Plant Collections. Fort Collins, CO: Colorado State University. Available: https://colostate.pressbooks.pub/climatereadyplantcollections/chapter/pollinators-pathogens-and-pests/.*
                                *Volk, G.M., Moreau, T. 2023. Climate change impacts management of genebanks and botanic gardens. In: Volk, G.M., Moreau, T.L., Byrne, P.F., editors. Conserving and Using Climate-Ready Plant Collections. Fort Collins, Colorado: Colorado State University. Available: https://colostate.pressbooks.pub/climatereadyplantcollections/chapter/genebanks-and-botanic-gardens/*
                                *Volk, G.M., Moreau, T. 2023. Climate adaptation planning for plant collections and conservation. In: Volk, G.M., Moreau, T.L., Byrne, P.F., editors. Conserving and Using Climate-Ready Plant Collections. Fort Collins, Colorado: Colorado State University. Available: https://colostate.pressbooks.pub/climatereadyplantcollections/chapter/climate-adaptation-planning/*
                                *Groeten, D., Farias-Soares, F., Rogge-Renner, G.D., Pereira, M.L., Walters, C.T., Silveira, V., Catarina, C.S., Guerra, M.P., Steiner, N. 2023. Carbohydrate and dehydrin-like protein profiles during Araucaria angustifolia seed development provides insights toward ex situ conservation. Trees. https://doi.org/10.1007/s00468-023-02419-z.*
                                *Goeten, D., Elias, R.A., Giacomolli Polesi, L., Walters, C.T., Guerra, M.P., Steiner, N. 2022. Effect of water content and biochemical cell state on the germination rate of cryopreserved Butia eriospatha embryos (Arecaceae). Plant Cell Tissue and Organ Culture. 152:339–356. https://doi.org/10.1007/s11240-022-02411-4.*
                                *Nadarajan, J., Walters, C.T., Pritchard, H., Ballesteros, D., Colville, L.P. 2023. Seed longevity-the evolution of knowledge and a conceptual framework. Plants. 12(3). Article e471. https://doi.org/10.3390/plants12030471.*
                                *Broccanello, C., Bolton, M.D., Secor, G., Richards, C.M., Ravi, S., Concheri, G., Campagna, G., Squartini, A., Stevanato, P. 2022. Bacterial endophytes as indicators of susceptibility to Cercospora Leaf Spot (CLS) disease in Beta vulgaris L. Scientific Reports. 12. Article e10719. https://doi.org/10.1038/s41598-022-14769-8.*
                                *Jenderek, M.M., Ambruzs, B.D., Tanner, J.D., Bamberg, J.B. 2023. High regrowth of potato crop wild relative genotypes after cryogenic storage. Cryobiology. 111:84-88. https://doi.org/10.1016/j.cryobiol.2023.03.006.*


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