Project : USDA ARS

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Research Project: Curation and Research to Safeguard and Expand Collections of Plant and Microbial Genetic Resources and Associated Descriptive Information

Location: Agricultural Genetic Resources Preservation Research

2024 Annual Report

Objectives
Objective 1. Apply preservation technologies to back-up NPGS accessions in long-term storage, in alignment with the overall NPGS Plan. Sub-objective 1A: Preserve orthodox seeds Sub-objective 1B: Preserve intermediate seeds Sub-objective 1C: Preserve recalcitrant seeds Sub-objective 1D: Preserve dormant buds & shoot tips of clonal germplasm Sub-objective 1E: Preserve pollen Objective 2. Conduct research to develop and apply sampling procedures and ex situ genetic resource management practices based on information about the genetic resources’ diversity. Sub-objective 2A: Strategically acquire accessions and characterize variation within and among accessions through taxonomic, geo-spatial and phenotypic observations Sub-objective 2B: Characterize and access variation in heterogeneous accessions Sub-objective 2C: Adjust Genebanking Standards for seed accessions backed-up at NLGRP to account for new technologies and within-accession variation Objective 3. Expand and improve information systems to better document plant genetic resources and communicate genetic resource management activities. Sub-objective 3A: Maintain, expand, and improve data storage and access Sub-objective 3B: Expand and improve data processing Sub-objective 3C: Expand and improve customer outreach and experience

Approach
The National Laboratory for Genetic Resources Preservation (NLGRP) provides essential infrastructure for the National Plant Germplasm System (NPGS). Unique in its mission, facilities, and staffing expertise, NLGRP houses the world’s largest collection of plant genetic resources (PGR), which are stored in freezer or cryogenic platforms. Germplasm (or propagules), such as seeds, shoot tips, dormant buds, and pollen, come from NPGS sites to back-up USDA’s PGR collections in a centralized genebank dedicated to keeping germplasm alive for decades, even centuries. NLGRP also provides back-up services for other institutions, who, like USDA, promise to make their PGR available for research. Genebanking, itself, can be viewed as a huge experiment in trying to make time stand still, not changing the viability or genetic identity of samples. This challenge is addressed by research on how to achieve and maintain survival in diverse germplasm that respond differently to preservation. Skilled staff conducts time-consuming work to identify, precisely dissect, cryopreserve, and recover tiny propagules, often without a priori knowledge of germination or growth requirements and often using in vitro techniques. New tools will be developed to find novel diversity and characterize diversity that may be susceptible to genetic erosion in genebanks. This will contribute to revised Best Practices that improve genebank performance. For NPGS’s massive collection, data management is a key feature to ensure information generated by NLGRP is available and linked to the physical sample and to evaluate potential changes to the samples due to preservation. As more genebanks are established globally, NLGRP supports the steady need for scientific support related to new approaches and technologies and provides training through on-site interactions and online materials.

Progress Report
The National Laboratory for Genetic Resources Preservation (NLGRP) in Fort Collins, Colorado duplicates collections of plant genetic resources for the National Plant Germplasm System (NPGS) to ensure safety and utility. NLGRP has unique storage facilities (i.e., walk-in freezers at -18C/0F or liquid nitrogen cryotanks held at -180/-292F or -196C/-321F) which are used to preserve germplasm (i.e., plant parts that can be grown out and used). Preserved germplasm requires exacting controls prior, during and following storage, including cell protections to avoid lethal desiccation or freezing stress, insurances that moisture and temperature fall within prescribed limits, and recovery procedures that ensure normal growth upon thawing. NLGRP houses nearly 1.2 million accessions of germplasm from over 18,000 species in diverse forms including seeds, excised embryos, pollen, and explants in the form of dormant buds harvested from over-wintering trees or shoot tips cut from plants grown in vitro or in protected greenhouses. Scientific activity at NLGRP seeks to mitigate the challenges accompanying such a large, diverse biological collection in terms of keeping it alive (Objective 1), ensuring genetic identity (Objective 2) and maintaining accurate and accessible information about NPGS collections (Objective 3). NLGRP’s activities require continuous assessment of the standards used to ensure quality of the physical sample (germplasm) and of the associated data. Objective 1 - In 2024, most of the work involving seed and pollen preservation examined materials that survive drying and either maintain viability during freezer storage (i.e., orthodox seeds) or do not survive in the freezer for extended times (i.e., intermediate seeds and pollens) and must be cryopreserved. NLGRP’s collection is mostly comprised of orthodox seeds (546,659 accessions in the freezer and 50,252 accessions in liquid nitrogen). NLGRP received 11,988 seed accessions in the past year, 453 of which have intellectual property protections. In 2024, progress managing orthodox seed collections was made on numerous fronts: 1) the time taken to process incoming samples decreased from about 2 years to about 3 months; 2) relative humidity (RH) control in seed drying rooms was restored and samples are now moisture-monitored non-invasively using devices that transmit data via Wi-Fi; 3) the number of monitor tests increased from a yearly average of about 3000 to 5000, with a greater emphasis on testing seeds that have been stored cryogenically since the 1990s; 4) germination assays of seeds from wild species, involving dormancy breaking treatments, are now conducted routinely (rather than relying on vital staining) and over 100 new germination protocols have been developed since 2021; 5) the feasibility of extracting RNA from diverse wild species was demonstrated in over 100 species and the potential of using RNA integrity to measure seed aging before mortality remains promising; and 6) data on other seed traits are being collected and incorporated into data sets, including variation of seed mass within an accession, reproductive failure (i.e., absence of embryo), and lipid content. ‘Intermediate’ seeds and pollens do not store well in the freezer, despite initial survival. Methods have been developed to store these materials cryogenically using moisture adjustment to higher relative humidity levels, careful exposure to liquid nitrogen at optimized cooling rates and packaging that allows repeated access to samples without warming them each time. Work with intermediate germplasm is focused on avoiding over-drying samples and monitoring crystallization and melting behavior of lipids, two aspects that provide greater understanding of the mechanisms of damage during freezer storage. Pollen from hazelnut, walnut, pistachio, and pecan was cryopreserved in 2024. Activities involving clonal germplasm (i.e., vegetative cuttings) focused on winter dormant buds or in vitro-grown shoot tips. Over 223 clonal accessions were received in 2024. In winter of 2023-2024, about 80 of these accessions were dormant buds of pear, apple, sour cherry, hazelnut, elderberry, quince, willow and butternut and all were successfully cryopreserved. Other accessions represent numerous crops (pineapple, bamboo, banana, potato, grape and sweet potato) that will be processed for cryopreservation. The effort involves removing endophytes that interfere with survival during cryoexposure, improving the consistency of post-thaw recovery to eliminate callus formation, and developing cryoprotection protocols. Then, cultures need to be multiplied to produce the approximately 150 shoot tips needed to implement a cryopreserved accession. In 2024, about 80 in vitro accessions were placed in cryo-storage, after successfully completing all the necessary steps. Collaborative research with Colorado State University on cryoprotectant permeation into cells provides important insights about the chemicals toxicity and modes of action in preventing lethal freezing transitions. Research on cryotherapy continues to demonstrate that pathogens can be eliminated from apple shoot tips. Objective 2 - Research in Fort Collins continues to focus on how diversity affects genebanking efficiency as well as how genebanking methods impact diversity. Scientists are developing genome sequencing techniques to characterize wild populations using a composite ‘fingerprint’ of individuals. This effort involved collecting seeds and tissues from 110 wild populations of ‘little barley,’ a wild species of cultivated barley that is native to North America from 16 states ranging from the Great Basin to the eastern seaboard. High quality genomic libraries were obtained from both germinated seedlings and field collected, dried leaf tissue. Leaf tissue was the preferred source material because it circumvented the time-intensive procedure of breaking dormancy and growing out seeds (more than 6 months). Whole-genome, pooled sequences were acquired for 61 populations, which allowed us to infer genetic variation of the species across its wide distribution in the United States. One population appeared to have novel diversity which was not already represented in NPGS, and it was added to the NPGS collection. Objective 3 - In 2024, much effort was devoted to ‘cleaning up’ database applications, including user interfaces with the Germplasm Resources Information Network (GRIN) database and information that is generated or heavily used by Fort Collins genebanking staff. The web-based user interface, named ‘smile’ continues to be developed to facilitate check-in, label-making and search functions. Data standards and a more meaningful lexicon have been implemented to reduce undefined jargon and use of numerous names for a single attribute. The changes, as well as increased programming capacity, have led to unprecedented access to accession and inventory information that aid data entry and retrieval as well as troubleshooting for erroneous or missing entries. One outcome of the extensive overhaul in data management is greater clarity in the services provided to genebanking partners that use the storage facilities in Fort Collins to duplicate their collections. Material Transfer Agreements (MTAs) describe this relationship, also known as ‘black box’ storage and these agreements were updated over the last 2 years to provide all partners the same level of service, as well as communicate expectations more explicitly that these services are contingent upon meeting ARS policy: partner institutions must be the primary site for maintenance and curation of their collections and partner institutions must freely distribute (upon research requests) germplasm that they duplicate at NLGRP. Recently modified MTAs have clarified that ‘black box’ is truly black box, and shall not involve Fort Collins genebanking staff in inventory confirmation, moisture-control and repackaging, cleaning, quality testing or data transfer with stakeholders about the status of their accessions. While the duration of MTAs is currently 10 years, an added condition that partners confirm the sustained viability of black boxed samples is being considered; this will ensure that NLGRP’s valuable storage space is not consumed with materials that have been forgotten about and have lost utility. A collaborative project among ARS, Colorado State University, and Iowa State University to release educational resources for training and outreach was completed in May 2024. This project developed the GRIN-U.org website that makes information about plant genetic resources conservation and use publicly available. To date, GRIN-U.org now has 288 items available as eBooks, infographics, videos, webinars, articles, and podcasts and nearly 10,000 unique visitors since its July 2021 launch. An eBook describing the process of clonal cryopreservation had over 37,000 visitors between February and July 2024. A three-course series on plant genetic resources management and use was offered through Colorado State University Online in 2022, 2023, and will be offered again in fall 2024. The project’s YouTube channel has had 362,800 views and 3905 subscribers. Plans are underway to submit a new proposal in 2025 that will continue this outreach effort.

Accomplishments
1. A user-friendly, modern, web-based user interface for the international GRIN-Global (Germplasm Resources Information Network) database. The Genetic Resources Information Network (GRIN or frequently called GRIN-Global to represent a global stake in sharing genetic resource information) connects the physical sample of plant germplasm with its associated data and makes this data available globally. Over the years, the ARS National Laboratory for Genetics Resources Preservation has relied upon numerous in-house software to liaison with GRIN to enter data and conduct data searches, but these “as needed” programs are troublesome to maintain. ARS researchers and IT specialists in Fort Collins, Colorado, have instituted a configurable web-based user interface for GRIN/GRIN-Global, called 'smile'. Among other advances, 'smile' enables basic genebanking functions like checking-in new inventories, printing labels, assigning storage locations, and making GRIN data queries. This has allowed obsolete software and hardware to be decommissioned and replaced with modern reliable and easily upgradable technology. This accomplishment facilitates more efficient management of the world’s most valuable plant germplasm collection, as genebank collection value is inextricably linked to the data associated with each sample.

2. Locating novel genetic variation in crop wild relatives. Research with a wild relative of barley called ‘little barley’ (Hordeum pusillum) shows the power of a pooled sequencing approach (combining many individuals into one combined sequencing run) developed by ARS researchers in Fort Collins, Colorado. This species is native to the United States, but it was poorly represented in the ARS National Plant Germplasm System collection and had limited descriptive information and no genomic information associated with it. Researchers have now developed a comprehensive genomic dataset for this species using inexpensive whole genome pooled sequencing of dried leaf tissue collected in the field. Over 50 million DNA markers of genetic variation were identified and mapped from 42 wild populations. This promises to be an efficient way for curators to catalog novel genetic variation in wild populations and allows users to identify plant accessions with genetic variation beneficial for crop production.

3. Novel method to preserve genetic resources of sweet potato. Sweet potato (Ipomoea sp.) is a valuable crop in the southern United States with increasing use in the baby food and fast-food industries. Yet genetic resources of sweet potato plants are extremely difficult to maintain, due to largely asexual reproduction, and require substantial greenhouse resources as well as vigilance to prevent infection from pathogens. ARS researchers in Fort Collins, Colorado, developed a novel protocol that decreased damage by pathogens and increased recovery of sweet potato shoot tips after liquid nitrogen exposure. This development benefits sweet potato growers, breeders, and related industries who rely on genetic resources for crop improvement, by reducing the needs for maintenance of living sweet potato collections in greenhouses.

4. Assessing health and aging in seeds harvested from wild populations. Seeds from wild species are more difficult to genebank than counterparts from domesticated species. A major problem is low germination of seeds in a sample, which introduces errors in viability assessments, uncertainty about the sustained viability during storage, and failure to meet genebanking standards for viability and seed numbers. ARS researchers in Fort Collins, Colorado, developed the concept of D.E.A.D. seeds to explain why seeds do not germinate – are seeds Dormant, Empty, Aged or Damaged? This construct separates time-dependent physiological features (dormancy and aging) from initial quality traits which may reveal conditions of the natural population that impact its health (empty or damaged). The research represents a major advancement in assessing seed dormancy and aging that has: 1) produced new germination protocols for many wild species, and 2) demonstrated the feasibility of quantifying RNA integrity to accurately track aging rates of seeds before they die. This provides a valuable scientific breakthrough to the global seed bank community, who seek to genebank genetic resources of crop wild relatives, and to plant conservation groups because of needed information to improve vegetation restoration projects.

5. Developed agreement framework to genebank seeds from Federally Recognized Tribes. Seeds are key to Tribal legacies as they are passed between generations. ARS researchers in Fort Collins, Colorado, partnered with the USDA Office of Tribal Relations to develop an agreement framework whereby seeds from Federally Recognized Tribes can be safeguarded at the ARS National Laboratory for Genetic Resources Preservation (NLGRP), which is the back-up storage facility for the ARS National Plant Germplasm System, offering optimal seed storage conditions for long-term preservation. This new agreement framework considers the sensitive nature of tribal seeds and related information and ensures that Tribal Nations maintain sovereign control of their cultural resources. This effort is recognized as important to the “USDA Food Sovereignty Initiative” and will be offered as a model to further sovereignty within Department of Interior’s “Enhance Native Seed Supply” Initiative.

6. Developed and implemented novel methods to cryopreserve pollen. Some of the clonally propagated fruit and nut collections in the ARS National Plant Germplasm System (NPGS) are difficult to back-up as seeds, dormant buds, or shoot tips at the National Laboratory for Genetic Resources Preservation (NLGRP). ARS researchers in Fort Collins, Colorado, developed and implemented novel methods to cryopreserve pollen for NPGS collections of date palm, Prunus, walnut, pecan, pistachio, and hazelnut. Since 2018, NPGS curators have collaborated with ARS to cryopreserve a total of 192 accessions of these crops, including all 37 male date palm trees in the NPGS collection. In 2024, the inventory and in vitro viability data for cryopreserved pollen inventories at NLGRP were uploaded to the ARS plant germplasm database (Germplasm Resources Information Network or GRIN-Global), which represents a significant advancement in meta data availability. This effort increases the security of vulnerable field plant collections in the NPGS and makes cryopreserved pollen available for breeding programs; both of which advance food security and agricultural sustainability in the United States and world-wide.

U.S. DEPARTMENT OF AGRICULTURE

Agricultural Genetic Resources Preservation Research: Fort Collins, CO