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 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** 2020 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 major goal of this project is to back-up the National Plant Germplasm System’s (NPGS) plant genetic resource collections in secure facilities using state-of-art preservation techniques to ensure long-term survival on plant and microbial germplasm. This goal is accomplished by curation activities and research to support curation, as well as, collection composition and utility. The fiscal year 2019 consolidation of two plant units brought new perspectives to both curation and research in fiscal year 2020.
ARS participated in a congressionally mandated survey of NPGS sites to reveal the status of plant genetic resources within NPGS. This information will provide the basis for a plan over the next 5 and 10 years. ARS focused its response, in this self-analysis, on the composition and handling of crop genetic resources in long-term storage, as well as the impact of collection growth and increasing demand for skilled human labor on our abilities to effectively meet Best Practices for maintaining scientific collections. The analysis revealed that 436,518 accessions of a total 592,448 NPGS accessions (~74%) are duplicated at Fort Collins, Colorado, but that there is wide disparity among crops in what is preserved. For example, excellent representation of the small grains collections and low representation of many trees and nuts. Moreover, ARS leads the national effort to send samples to the Global Trust seed bank in Svalbard, Norway which now houses 140,124 seed samples from ARS. However, these statistics reflect “duplicated” samples, which do not necessarily meet standards of quality and quantity. An assessment of the number of samples that meet established quality and quantity standards for back-up is substantially lower at 100,265 (17%). This low proportion should prompt a discussion about acceptable risks of losing genetic resources and actions required to maintain an acceptable risk level.
The analysis also showed that between 5000-6000 accessions are received by ARS in Fort Collins, Colorado, each year, about 200 of which are vegetatively propagated germplasm (i.e., clones). Currently, this is the capacity Fort Collins, Colorado, has to process clonal materials, which partially explains the very low representation of some crops (such as trees and nuts) mentioned above. Research efforts are focused on increasing capacity to cryopreserve clonal materials as well as other germplasm forms that do not respond well in conventional freezer (-20C) storage (Objective 2). Progress was made in developing protocols to cryopreserve grape shoot tips, embryos of oak, hazelnut, sassafras, and Torreya. As of April 2020, 50,512 (8.5%) plant samples were stored cryogenically. Many of these samples can be maintained less expensively in conventional storage to make space for a larger group of germplasm (clones, seeds, and pollen) that require cryogenic storage to survive.
Assessing viability is a core activity because germplasm must be alive to fulfill its role as a genetic resource. Currently, ARS technicians in Fort Collins, Colorado, conducts about ~13,000 viability assessments per year (Objective 1). About 7500 of these viability assessments monitor health of seeds in long-term storage. The number is insufficient to maintain a 20-year monitoring interval, which is generally believed optimum without reliable predictors of longevity. Research has provided some new methods to monitor aging using small sample sizes. Moreover, a new viability assay, using intensity of gene expression, appears promising in identifying seeds that are alive but on the brink of death. We are beginning to test implementation of these new methods to see how well they fit into workflows and allow more efficient monitoring. The remainder of viability assessments are conducted on incoming materials: seeds that may or may not have known germination requirements, and clonal materials in which recovery procedures are highly genotype-specific and require a lot of optimization. These are labor intensive procedures that require highly skilled technicians. When a successful viability assessment is available, then a new crop can be tested for survival following cryo-exposure. 
Advances that improve effectiveness of cryogenic storage (in liquid nitrogen) (Objective 2) come in a number of directions that include new crops added into the repertoire of cryopreserve-able materials (e.g., breadfruit, grapes, pineapple and some cherries/apricots) as well as material that is continually processed to increase the number of NPGS accessions that are backed up at Fort Collins, Colorado, (e.g., citrus, mint, banana, strawberry, and apple).
Similarly, the most effective protocols for long-term storage of specific agriculturally important microbial cultures are often unknown. With other USDA-ARS cooperators, the effectiveness and relative costs of long-term storage at -196° C (liquid nitrogen) versus -80° and -20° C refrigerated storage were compared, to ascertain best management practices for long term preservation of those microbes.
Collections of wild species, particularly those that are relatives to crops (CWR) and native to the United States are supported by ARS. Many of these activities occur in collaboration with land manager agencies and conservation groups such as SOS (Seeds of Success) program led by the Bureau of Land Management (BLM) and Center for Plant Conservation (CPC), the United States’ authority on genebanking rare and endangered plant species (Objective 4). Conservation status and geographic location of species and populations that should be targeted for inclusion in NPGS collections was analyzed this year. Seeds are usually the propagule form to collect and preserve this germplasm. Important strides were made to understand germination requirements of seeds from CWR as well as their storage behavior, to include response to cryopreservation or duration of survival at -20C. Some accomplishments to note are first-time successful germination of endangered species Persea borbonia (CWR to avocado) and Torreya taxifolia as well as advanced studies on sassafras (threatened by Laurel wilt disease), hazelnuts, willow, oaks and citrus CWR.
ARS scientists are using genome sequence data to characterize collections, identify diversity with dense or low representation in NPGS collections and to develop tools to quickly locate diversity that correlated with a trait of interest (Objective 3). The progress in preservation and genomic technologies were applied to benefit and resolve some problems associated with CWR genetic resource collections.
Curriculum and training resources that capture key information about genebanking are being developed to educate the next generation of genebank managers; several learning objects have been created and are available online. **Accomplishments**
                                

1. Crop wild relatives in the United States chronicled to guide conservation action. Industry and university plant breeders rely on plant species that are “close cousins” (called crop wild relatives) that have naturally occurring traits that may hold the key to improving crop yields, drought tolerance, and disease and insect resistance. Many of these crop wild relatives are native to North America and have only recently been targeted in research and collection efforts; therefore, it is important to better understand, collect, and preserve these species and their valuable traits. To meet this critical need, ARS scientists in Fort Collins, Colorado, recently completed a comprehensive study of 600 crop wild relatives. They identified where these native species occur and their conservation status and showed that about 7% are critically endangered, 50% are endangered, and 28% are vulnerable in their natural habitats. Further, 60% are categorized as “high priority” for further action to secure them in seed collections, botanical gardens, and nature. This study provides essential information critical for developing a strategic plan to secure and make available these precious genetic resources. 2. Using a metabolic signal to identify seeds that will soon die of old age. There is no warning when seeds are near death, as they can still germinate normally. However, this period can be very brief resulting in dead seeds at the time of planting, encumbering substantial economic loss to seed companies and genebanks. To identify such seeds, ARS scientists in Fort Collins, Colorado, showed that metabolic slow-down, which is quantified by the rate that genes are expressed when seeds take up water, is an excellent indicator of imminent mortality in soybean. This discovery improves standard viability monitoring tests, which only indicate either dead or alive, and enables genebanks to proactively schedule seed regenerations before seed mortality, saving time, money, and valuable genetic resources. 3. A state-of-the-art bioinformatic tool to sift through plant genetic resource collections. Genetic traits, such as disease or drought resistance, are the building blocks that plant breeders use to improve crops; however, searching for genetic variation in huge collections of plant genetic resources is like trying to find a needle in a haystack. Additionally, the process requires extensive field evaluation of plants and their response, which is time consuming and expensive. To accelerate crop breeding and make it more cost-effective, ARS scientists in Fort Collins, Colorado, developed an artificial intelligence (AI) program that uses full genetic sequences to identify plants with genetic differences that control important agronomic traits. This important advance in “next generation sequencing,” in which the entire plant genetic code is obtained, provides a faster, less-costly, scientific method to identify specific plants for use in breeding. This AI program identifies a few samples, out of thousands or tens of thousands in a genetic resource collection, which will likely vary for the specific trait needed by the breeder. In addition to accelerating breeding programs, this new tool greatly improves the usefulness of plant gene bank collections by increasing the targeted access of the vast genetic diversity in plant germplasm collections world-wide. **Review Publications**
                            *Volk, G.M., Bretting, P.K., Byrne, P.F. 2019. Plant genetic resources training materials deemed essential by international survey. Crop Science. 59:2308-2316. https://doi.org//10.2135/cropsci2019.05.0324.*
                                *Lebeda, A., Kristkova, E., Kitner, M., Khoury, C.K., Carver Jr, D.P., Widrlechner, M.P. 2019. Research gaps and challenges in the conservation and use of North American wild lettuce germplasm. Crop Science. 59:2337-2356. https://doi.org//10.2135/cropsci2019.05.0350.*
                                *Khoury, C.K., Greene, S.L., Krishnan, S., Miller, A., Moreau, T., Williams, K.A., Rodriguez-Bonilla, L., Spurrier, C.S., Zalapa, J.E., Nabhan, G.P. 2020. Toward integrated conservation of North America’s crop wild relatives. Natural Areas Journal. 40(1):96-100. https://doi.org//10.3375/043.040.0111.*
                                *Khoury, C.K., Carver Jr, D.P., Barboza, G., Jarret, R.L., Van Zonneveld, M., et. al. 2019. Modeled distributions and conservation status of the wild relatives of chile peppers (Capsicum L). Diversity and Distributions. 26(2):209-225. https://doi.org/10.1111/ddi.13008.*
                                *Wang, M., Lambardi, M., Engelmann, F., Pathirana, R., Panis, B., Volk, G.M., Wang, Q. 2020. Advances in cryopreservation of in vitro-derived propagules: Technologies and explant sources. Plant Cell Tissue and Organ Culture. https://doi.org/10.1007/s11240-020-01770-0.*
                                *Volk, G.M., Khoury, C., Greene, S., Byrne, P. 2020. Introduction to crop wild relatives. In: Volk, G.M., Byrne, P., editors. Crop Wild Relatives and their Use in Plant Breeding. Fort Collins, Colorado: Colorado State University. Available: https://colostate.pressbooks.pub/cropwildrelatives/chapter/introduction-to-crop-wild-relatives/.*
                                *Volk, G.M., Richards, C., Walters, C., Dempewolf, H., Byrne, P. 2020. Crop wild relatives in genebanks. In: Volk, G.M., Byrne, P., editors. Crop Wild Relatives in Genebanks. Fort Collins, Colorado: Colorado State University. Available: https://colostate.pressbooks.pub/cropwildrelatives/chapter/crop-wild-relatives-in-genebanks/.*
                                *Williams, K., Volk, G.M. 2020. The USDA plant introduction program. In: Volk, G.M., Byrne, P., editors. Crop Wild Relatives in Genebanks. Fort Collins, Colorado: Colorado State University. Available: https://colostate.pressbooks.pub/cropwildrelatives/chapter/usda-plant-introduction-program/.*
                                *Volk, G.M. 2020. Links to some published best practices for genebanking. In: Volk, G.M., Byrne, P., editors. Crop Wild Relatives in Genebanks. Fort Collins, Colorado: Colorado State University. Available: https://colostate.pressbooks.pub/cropwildrelatives/chapter/published-best-practices/.*
                                *Byrne, P., Richards, C., Volk, G.M. 2020. From wild species to landraces and cultivars. In: Volk, G.M., Byrne, P., editors. Crop Wild Relatives and their Use in Plant Breeding. Fort Collins, Colorado: Colorado State University. Available: https://colostate.pressbooks.pub/cropwildrelatives/chapter/from-wild-species-to-landraces-and-cultivars/.*
                                *Volk, G.M., Krishnan, S. 2020. Case study: Coffee wild species and cultivars. In: Volk, G.M., Byrne, P., editors. Crop Wild Relatives in Genebanks. Fort Collins, Colorado: Colorado State University. Available: https://colostate.pressbooks.pub/cropwildrelatives/chapter/case-study-coffee-wild-species-and-cultivars/.*
                                *Volk, G.M., Krueger, R., Moreland, B., Bonnart, R., Shepherd, A. 2020. Citrus shoot tip cryopreservation. In: Volk, G.M., editors. Training in Plant Genetic Resources: Cryopreservation of Clonal Propagules. Fort Collins, Colorado: Colorado State University. Available: https://colostate.pressbooks.pub/clonalcryopreservation/chapter/citrus-cryopreservation/.*
                                *Volk, G.M., Bonnart, R., Shepherd, A. 2020. Citrus micrografting for regrowth after shoot tip cryopreservation. In: Volk, G.M., editors. Training in Plant Genetic Resources: Cryopreservation of Clonal Propagules. Fort Collins, Colorado: Colorado State University. Available: https://colostate.pressbooks.pub/clonalcryopreservation/chapter/citrus-micrografting/.*
                                *Volk, G.M., Bonnart, R. 2020. Prunus shoot tip cryopreservation. In: Volk, G.M., editors. Training in Plant Genetic Resources: Cryopreservation of Clonal Propagules. Fort Collins, Colorado: Colorado State University. Available: https://colostate.pressbooks.pub/clonalcryopreservation/chapter/prunus-cryopreservation/.*
                                *Volk, G.M., Jenderek, M., Chen, K. 2020. Cryopreservation of dormant apple buds. In: Volk, G.M., editors. Training in Plant Genetic Resources: Cryopreservation of Clonal Propagules. Fort Collins, Colorado: Colorado State University. Available: https://colostate.pressbooks.pub/clonalcryopreservation/chapter/apple-dormant-bud-cryopreservation/.*
                                *Bauchet, G., Bett, K.E., Cameron, C.T., Campbell, J.D., Cannon, E., Cannon, S.B., Carlson, J., Chan, A., Cleary, A., Close, T., Cook, D., Cooksey, A., Coyne, C.J., Dash, S., Dickstein, R., Farmer, A., Fernandez-Baca, D., Hokin, S., Jones, E., Kang, Y., Monteros, M., Munoz-Amatriain, M., Mysore, K., Pislariu, C., Richards, C.M., Shi, A., Town, C., Udvardi, M., Wettberg, E., Young, N., Zhao, P. 2019. The future of legume genetic data resources: Challenges, opportunities, and priorities. Legume Science. 1(1):e16. https://doi.org/10.1002/leg3.16.*
                                *Zhang, L., Hu, J., Han, X., Li, J., Gao, Y., Richards, C.M., Zhang, C., Tian, Y., Liu, G., Gul, H., Yang, C., Meng, M., Yuan, G., Kang, G., Wu, Y., Wang, K., Zhang, H., Wang, D., Cong, P. 2019. A high-quality apple genome assembly reveals the association of a retrotransposon and red fruit colour. Nature Communications. https://doi.org/10.1038/s41467-019-09518-x.*
                                *Khoury, C.K., Greene, S.L., Moreau, T., Krishnan, S., Miller, A. 2019. A road map for conservation, use, and public engagement around North America’s crop wild relatives and wild utilized plants. Crop Science. 59:1–6. https://doi.org/10.2135/cropsci2019.05.0309.*
                                *Khoury, C.K., Kates, H.R., Carver Jr, D.P., Achicanoy, H.A., van Zonneweld, M., Thomas, E., Heinitz, C.C., Jarret, R.L., Labate, J.A., Reitsma, K., Nabhan, G.P., Greene, S.L. 2019. Distributions, conservation status, and abiotic stress tolerance potential of wild cucurbits (Cucurbita L.). Plants, People, Planet. 2(3):269-283. https://doi.org//10.1002/ppp3.10085.*
                                *Lima, L.W., Stonehouse, G.C., Walters, C.T., El Mehdawi, A.F., Fakra, S.C., Pilon-Smits, E.A. 2019. Selenium accumulation, speciation and localization in Brazil nuts (Bertholletia excelsa H.B.K.). Plants. 8(8):289. https://doi.org/10.3390/plants8080289.*
                                *Walters, C., Maschinski, J. 2019. Conventional seed banking to support species survival in the wild: Introduction. In: Falk, D., Holsinger, K., Wieland, G., Olwell, P., Millar, C., Guerrant, E.O., Havens, J.K., Maunder, M., Haskins, K., editors. CPC Best Plant Conservation Practices to Support Species Survival in the Wild. Escondido, CA: Center for Plant Conservation. p. 1-9.*
                                *Maschinski, J., Walters, C.T., Guerrant, E., Murray, S., Kunz, M., Schneider, H., Affolter, J., Gurnoe, T., Fraga, N., Havens, K., Vitt, P., Heineman, K.D., Horn, K. 2019. Collecting seeds from wild rare plant populations. In: Falk, D., Holsinger, K., Wieland, G., Olwell, P., Millar, C., Guerrant, E.O., Havens, J.K., Maunder, M., Haskins, K., editors. CPC Best Plant Conservation Practices to Support Species Survival in the Wild. Escondido, CA: Center for Plant Conservation. p. 31-42.*
                                *Walters, C.T., Maschinski, J., Havens, K., Vitt, P., Heineman, K., Horn, C. 2019. Cleaning, processing, drying and storing orthodox seeds. In: Falk, D., Holsinger, K., Wieland, G., Olwell, P., Millar, C., Guerrant, E.O., Havens, J.K., Maunder, M., Haskins, K., editors. CPC Best Plant Conservation Practices to Support Species Survival in the Wild. Escondido, CA: Center for Plant Conservation. p. 10-23.*
                                *Maschinski, J., Walters, C.T., Meyer, E., Fitch, R., Havens, K., et al. 2019. Splitting samples for safety duplication storage and testing. In: Falk, D., Holsinger, K., Wieland, G., Olwell, P., Millar, C., Guerrant, E.O., Havens, J.K., Maunder, M., Haskins, K., editors. CPC Best Plant Conservation Practices to Support Species Survival in the Wild. Escondido, CA: Center for Plant Conservation. p. 15-30.*
                                *Pence, V., Westwood, M., Maschinski, J., Powell, C., Sugii, N., Walters, C.T., et al. 2019. Collecting and maintaining exceptional species in tissue culture and cryopreservation. In: Falk, D., Holsinger, K., Wieland, G., Olwell, P., et al., editors. CPC Best Plant Conservation Practices to Support Species Survival in the Wild. Washington, D.C.: Island Press. p. 4-21.*
                                *Maschinski, J., Walters, C.T., Haskins, K.E., Birker, C., Randall, J., Randall, L., Watkins, K., Clarke, M., Davitt, J., Havens, K., Vitt, P., Horn, C. 2019. Curating small samples: Increasing the number of seeds for storage and restoration. In: Falk, D., Holsinger, K., Wieland, G., Olwell, P., et al., editors. CPC Best Plant Conservation Practices to Support Species Survival in the Wild. Washington, D.C.: Island Press. p. 43-47.*
                                *Bettoni, J.C., Bonnart, R.M., Volk, G.M. 2021. Challenges in implementing plant shoot tip cryopreservation technologies. Plant Cell Tissue and Organ Culture. 144:21-34. https://doi.org/10.1007/s11240-020-01846-x.*
                                *Greene, S.L., Carver Jr, D.P., Khoury, C.K., Irish, B.M., Olwell, P., Prescott, L. 2019. Collecting native seed for restoration: Collateral benefits to agricultural crop improvement, research and education. Crop Science. 59(6):2429-2442. https://doi.org/10.2135/cropsci2019.06.0372.*
                                *Jenderek, M.M., Ambruzs, B.D., Holman, G.E., Carstens, J.D., Ellis, D., Widrlechner, M. 2020. Salix dormant buds cryotolerance varies by taxon, harvest year and stem-segment length. Crop Science. https://doi.org/10.1002/csc2.20135.*
                                *Tanner, J.D., Minas, I.S., Chen, K.Y., Jenderek, M.M., Wallner, S.J. 2020. Antimicrobial forcing solution improves recovery of cryopreserved temperate fruit tree dormant buds. Journal of Cryobiology. 92:241-247. https://doi.org/10.1016/j.cryobiol.2020.01.019.*
                                *Aparecida De Sousa, V., Reeves, P.A., Reilley, A.A., Virginia De Aguiar, A., Marcos Stephenon, V., Richards, C.M. 2020. Genetic diversity and biogeographic determinants of population structure in Araucaria angustifolia (Bert.) O. Ktze. Conservation Genetics. 21:217-229. https://doi.org/10.1007/s10592-019-01242-9.*
                                *Hardegree, S.P., Sheley, R.L., James, J., Reeves, P.A., Richards, C.M., Walters, C.T., Boyd, C.S., Moffet, C., Flerchinger, G.N. 2020. Germination syndromes and their relevance to rangeland seeding strategies in the intermountain western United States. Rangeland Ecology and Management. 73(2):334-341. https://doi.org/10.1016/j.rama.2019.11.004.*


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