Skip to main content
ARS Home » Research » Research Project #434353

Research Project: Innovative Strategies and Methods for Improving the Management, Availability, and Utility of Plant Genetic Resource Collections


2019 Annual Report

Obj. 1: Develop, assess, and validate protocols and strategies for more efficiently and effectively storing plant genetic resources under conventional (i.e., freezer) conditions. 1.A: Revise current seed banking “standards” for improved performance. 1.B: Assess variation of seed longevity and develop methods to measure early aging effects. Obj. 2: Develop and test methods and strategies for more efficiently and effectively cryopreserving plant genetic resources. 2.A: Improve effectiveness or efficiency of cryogenic storage for dry propagules (i.e., no “freezable” water). 2.B: Improve effectiveness of cryoprotection and recovery for hydrated propagules (i.e., containing “freezable” water). 2.C: Develop cryoexposure as a possible tool to identify presence/absence of endophytes. Obj. 3: Design and test methods and strategies for exploiting genomic data to enhance the efficiency and effectiveness of the NPGS’ plant genetic resource management projects. 3.A: Develop strategies to collect and analyze genomic data for improved collection management. 3.B: Demonstrate use of genetic tools for sample identification. 3.C: Characterize the influence of environment on genomic diversity held in collections. Obj. 4: Formulate and validate methods and strategies for efficiently and effectively sampling, preserving, and using the genetic diversity of selected crop wild relatives. 4.A: Strategies to use genetic data to inform collection management about identity, diversity, integrity, and usefulness of CWRs. 4.B: Identify improved conservation methods for securing CWR collections, especially at clonal repositories.

The Plant Germplasm Preservation Research Unit (PGPRU) of the USDA-ARS National Laboratory for Genetic Resources Preservation (NLGRP) embraces its unique mission to develop strategies and technologies for improving the management, availability and utility of plant genetic resource (GR) collections, especially those of the National Plant Germplasm System (NPGS). NPGS collections at active sites are vulnerable to abiotic and biotic threats, making it imperative that our national agricultural genetic resource treasures are safeguarded for future generations. Our challenge is to identify, develop, and document optimal methods for securing more than 15,000 species of cultivated and wild plant germplasm in the most efficient way possible, taking into account the type of propagule (seeds, pollen, dormant buds, shoot tips) as well as the storage platform (usually in a mechanical freezer or in liquid nitrogen). We will also leverage genetic and genomic tools to inform collection management and to improve delivery of associated data to customers and stakeholders. This project will have worldwide impact by delivering new technologies, standardized Best Practices, novel approaches, and new perspectives for improved genebank management. This unit works closely with the co-located project, Plant Genetic Resources Preservation Program (PGRPP), to transfer and implement technologies that increase the capacity to preserve diverse plant genetic resources and to reduce genebanking costs while increasing quality and accessibility of samples and associated information.

Progress Report
This is the first and last report for this new project, which began in October 2018, continuing research from the previous project. In May 2019, the two plant programs at the USDA National Laboratory for Genetic Resources Preservation (NLGRP) were consolidated in order to strengthen complementary research and curatorial efforts for plant and microbial genetic resources, and so this project has been terminated. The major goals of this project are to improve the management and availability of National Plant Germplasm System’s (NPGS) plant genetic resource collections by developing innovative strategies for preserving germplasm, locating and capturing novel diversity, and using genomic tools to identify potentially useful traits. Preserving genetic resources from natural populations that are threatened, including many of North America’s most iconic species, such as oaks and ash trees and species that are congeneric with crops (i.e., crop wild relatives) is a critical task of genebanks such as NLGRP. The promise of these genebanks is predicated on their ability to maintain germplasm (also called propagules) viability, which involves ‘deep-freeze’ storage in either single (-18C, 0F) or dual-stage (-80C, -112F) mechanical freezers or in liquid nitrogen (-170 to -196C, -274 to -321F); these low temperatures are lethal without plant cell adaptations and exacting specialized treatments. The scientists utilize natural tolerances of the plant or develop protective treatments for propagules (e.g., seeds, pollen, winter-acclimated dormant buds, or shoot tips, often grown in micro-culture) to preserve genetic resources from about 16,000 species of cultivated and wild plants. The choice of propagule largely depends on reproductive biology of the plant as well as the desired ‘conservation target,’ which may be preserving populations, individuals, genes, or traits. Cultivars of many of our fruit crops and forage grasses represent a specific genotype (i.e., individual) and preserving that individual (i.e., a clone) is exceptionally labor-intensive. On the other hand, preserving germplasm (usually seeds) from non-domesticated plants carries challenges including unknown reproductive biology and growth requirements, poor health, high heterogeneity, and sample sizes too small for current curation methods. NPGS collections continue to grow at about 2% per year. This growth should occur to increase available genetic diversity, and that new diversity needs to be explored for potential usefulness that can be incorporated into modern crops. Significant progress was made towards Objective 1 (Improving efficiency of conventional seed storage practices at -18C). An extensive review of standard seed banking collection, processing, and storage protocols for wild-collected samples was developed in collaboration with the Center for Plant Conservation and has culminated in a manual and several learning videos soon to be online. These will serve as background and talking points as NLGRP reviews its Standard Operating Procedures (SOPs). Critical aspects of temperature maintenance have been reviewed, and a revised SOP was drafted. In preparation for more SOP development at NLGRP, staff have been trained using Six Sigma approaches to identify and solve bottlenecks in accomplishing core mission-critical tasks. A critical feature of NLGRP operations is measuring deterioration of samples before evidence of lost viability; the unit has successfully developed methods that correlate aging rate and chemical changes using very small sample sizes. Some of these methods are being tested in wild collected samples to determine if they provide more reliable longevity predictions while reducing number of seeds used to monitor viability. Advances that improve effectiveness of cryogenic storage (in liquid nitrogen) (Objective 2) come in a number of directions that include first-ever reports of high survival in cryo-exposed shoot tips (clonal germplasm) of Prunus (peach, plum, apricot, sweet cherry, sour cherry, and almond) and Vitis (grape) and continued success cryopreserving Citrus, Solanum, and Mentha (citrus, potato and mint). Though counter-intuitive, desiccation-tolerant seeds, pollen, and fern spores that exhibit low survival during conventional freezer storage (-18C) were shown to have excellent survival in liquid nitrogen if drying is less stringent than SOPs for conventional storage. Freezing properties of water in cryoprotected embryonic axes of oak and maple were explored using differential scanning calorimetry and suggest tissue-specific sensitivity to desiccation and low temperature in recalcitrant embryonic axes. Collaborative work continues to demonstrate that selective killing of differentiated cells in meristematic tissues by cryo-exposure (i.e., cryotherapy) is effective in eradicating obligate plant pathogens from infected shoot tips. More accurate genome sequencing and annotation contributes to progress in Objective 3 (Using genomic data to support genebank operations). Chloroplast sequence data displayed genetic relationships within Maleae taxa as reticulate, rather than traditional branching patterns used in Systematics, and demonstrated a complex evolutionary pattern in Pyrus (pear) speciation. Collaborative work using new PacBio© single-molecule real-time sequencing provided high quality genome assembly for apple and revealed a transcriptional activator for anthocyanin biosynthesis associated with the important agronomic trait of red-skinned fruit. Genomic markers in apple are also being used to identify historic homestead and orchard cultivars that may represent novel diversity not currently represented in the NPGS collection. Genomic analyses showed that common pea was domesticated in two separate events, and that an important seed trait for cultivation, non-dormancy, became fixed. The progress in preservation and genomic technologies described above were applied to benefit and resolve some problems associated with crop wild relative (CWR) genetic resource collections (Objective 4).

1. Early detection of seed aging in genebanks through reduced RNA integrity. Plant germplasm stored in genebanks must be monitored for viability to ensure that samples are regenerated before they die. The current ‘gold-standard’ test is a germination assay, which is labor-intensive, consumes a large portion of valuable seeds, and is insensitive during the early, asymptomatic stage of aging. ARS scientists in Fort Collins, Colorado, developed a simple assay, based on degradation of RNA, that detects the rate of aging in seeds during the asymptomatic period. The assay can be developed as a kit to facilitate automated monitoring, and it uses a small number of seeds. This new monitoring tool can accurately predict when seeds should be regenerated while preventing unnecessary consumption of seeds for testing. The fate of RNA in aging dry cells promises to increase the efficiency and automation of plant genebanks as well as reveal key information about the nature of seed aging.

2. Discovered critical sources of genetic diversity in pea by tracing domestication history. When wild plants were domesticated more than 10,000 years ago, some agronomically important traits were captured, but a great deal of useful diversity was also filtered out. That diversity can be recaptured if the origins of domestication are known. ARS scientists in Fort Collins, Colorado, collaborated with numerous scientists around the world to conduct a genomic analysis on Pisum species (pea). Together, they discovered that common pea (Pisum sativum) was domesticated from two wild species native to the eastern Mediterranean region. This finding contrasts with previous-held ideas that common pea was derived from another cultivated pea (P. abyssinicum) that is restricted to Ethiopia. The work highlights the critical need for genomic information to guide collection and management of genetic resources. In particular, the relationship between wild and cultivated pea species now guides the development of a richer collection of pea’s progenitor, P. elatius, where new and useful diversity can be found.

3. Developed a framework for an NPGS genebank training program. About one third of National Plant Germplasm System’s genebank scientists will retire within the next five years, and training programs for the next generation of genebank scientists do not meet contemporary needs. Thus ARS scientists in Fort Collins, Colorado, in collaboration with Colorado State University, developed a training framework for plant genetic resource management based on results of a broad-ranging survey. Advanced training materials that cover a broad range of topics critical to plant genetic resource collections will soon be released to aid the next generation of plant genebankers.

Review Publications
Bi, W., Hao, X., Cui, Z., Pathirana, R., Volk, G.M., Wang, Q. 2018. Shoot tip cryotherapy for efficient eradication of grapevine leafroll-associated virus-3 from diseased grapevine in vitro plants. Annals of Applied Biology. 173(3):261-270.
Magby, J., Volk, G.M., Henk, A.D., Miller, S. 2019. Identification of historic homestead and orchard apple cultivars in Wyoming. HortScience. 54(1):8-16.
Zhao, L., Wang, M., Li, J., Cui, Z., Volk, G.M., Wang, Q. 2019. Cryobiotechnology: a double-edged sword for obligate plant pathogens. Plant Disease. 103:1058-1067.
Volk, G.M., Jenderek, M.M., Walters, C.T., Bonnart, R.M., Shepherd, A.N., Skogerboe, D.M., Hall, B.D., Moreland, B.L., Krueger, R., Polek, M. 2019. Implementation of citrus shoot tip cryopreservation in the USDA-ARS national plant germplasm system. Acta Horticulturae. 1234:329-334.
Fleming, M.B., Hill, L.M., Walters, C.T. 2018. The kinetics of aging in dry-stored seeds: a comparison of viability loss and RNA degradation in unique ‘legacy’ seed collections. Annals Of Botany. 123:1133-1146.
Ballesteros, D., Hill, L.M., Lynch, R.T., Pence, V., Pritchard, H., Walters, C.T. 2018. Longevity of preserved germplasm: The temperature dependency of aging reactions in glassy matrices of dried fern spores. Plant and Cell Physiology. 60(2):376-392.
Fleming, M.B., Patterson, E., Reeves, P.A., Richards, C.M., Gaines, T., Walters, C.T. 2018. Exploring the fate of mRNA in aging seeds: protection, destruction, or slow decay? Journal of Experimental Botany. 69(18):4309-4321.
Hardegree, S.P., Roundy, B., Walters, C.T., Reeves, P.A., Richards, C.M., Moffet, C., Sheley, R.L., Flerchinger, G.N. 2018. Hydrothermal germination models: assessment of the wet-thermal approximation of potential field response. Crop Science. 58(5):2042-2049.
Volk, G.M., Shepherd, A.N., Bonnart, R.M. 2018. Successful cryopreservation of Vitis shoot tips: Novel pre-treatment combinations applied to nine species. CryoLetters. 39(5):322-330.
Volk, G.M., Jenderek, M.M., Staats, E.R., Shepherd, A.N., Bonnart, R.M., Leado, A., Ayala Silva, T. 2019. Challenges in the development of a widely applicable method for sugarcane (Saccharum spp.) shoot tip cryopreservation. Acta Horticulturae. 1234:335-342.
Volk, G.M., Henk, A.D., Richards, C.M., Bassil, N.V., Postman, J.D. 2018. Chloroplast sequence data differentiate Maleae, and specifically Pyrus, species in the USDA-ARS National Plant Germplasm System. Genetic Resources and Crop Evolution. 66(1):5-15.
Volk, G.M., Namuth-Covert, D., Bryne, P.F. 2019. Training in plant genetic resource management: A way forward. Crop Science. 59(3):853-857.
Magby, J., Volk, G.M., Miller, S. 2019. Heritage apple cultivars grown in homesteads, nurseries and orchards in Wyoming. Journal of American Pomological Society. 73(2):95-101.
Bettoni, J., Souza, J.A., Volk, G.M., Costa, M., Nascimento Da Silva, F., Kretzschmar, A. 2019. Eradication of latent viruses from apple cultivar ‘Monalisa’ shoot tips using droplet-vitrification cryotherapy. Scientia Horticulturae. 250:12-18.
Bettoni, J.C., Bonnart, R.M., Shepherd, A.N., Kretzschmar, A., Volk, G.M. 2019. Successful cryopreservation of Vitis vinifera cv. ‘Chardonnay’ from both in vitro and growth chamber source plants. Acta Horticulturae. 1234:211-218.
Bettoni, J.C., Kretzschmar, A.A., Bonnart, R.M., Shepherd, A.N., Volk, G.M. 2019. Cryopreservation of 12 Vitis species using apical shoot tips derived from plants grown in vitro. HortScience. 54(6):976-981.
Wang, M., Hao, X., Zhao, L., Cui, Z., Volk, G.M., Wang, Q. 2018. Virus infection reduces shoot proliferation of in vitro stock cultures and ability of cryopreserved shoot tips to regenerate into normal shoots in 'gala' apple (malus × domestica). Cryobiology. 84:52-58.
Bettoni, J.C., Bonnart, R.M., Shepherd, A.N., Kretzschmar, A.A., Volk, G.M. 2019. Successful cryopreservation and histological observations of Vitis shoot tips isolated from growth chamber source plants. Vitis. 58(2):71-78.
Bettoni, J.C., Bonnart, R.M., Shepherd, A.N., Kretzschmar, A.A., Volk, G.M. 2019. Modifications to a Vitis shoot tip cryopreservation procedure: Effect of shoot tip size and use of cryoplates. CryoLetters. 40(2):103-112.
Trneny, O., Brus, J., Hradilova, I., Rathore, A., Das, R., Kopecky, P., Coyne, C.J., Reeves, P.A., Richards, C.M., Smykal, P. 2018. Molecular evidence for two domestication events in the pea crop. Genes. 9(11).
Wang, M., Chen, L., Liu, J., Teixeira Da Silva, J.A., Volk, G.M., Wang, Q. 2018. Cryopreservation of apple (Malus spp.): development, progress and future prospects. Plant Cell Reports. 37(5):689-709.
Bi, W., Hao, X., Cui, Z., Volk, G.M., Wang, Q. 2018. Droplet-vitrification cryopreservation of in vitro-grown shoot tips of grapevine (Vitis spp.). In Vitro Cellular and Developmental Biology - Plants. 54:590-599.
Reeves, P.A., Richards, C.M. 2018. Biases induced by using geography and environment to guide ex situ conservation. Conservation Genetics. 19:1281-1293.
Ballesteros, D., Walters, C.T. 2019. Solid-State biology and seed longevity: A mechanical analysis of glasses in pea and soybean embryonic axes. Frontiers in Plant Science. 10:920.