Project : USDA ARS

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Research Project: Efficient and Effective Preservation and Management of Plant and Microbial Genetic Resource Collections

Location: Agricultural Genetic Resources Preservation Research

2021 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 a secure facility using state-of-the-art preservation techniques to ensure long-term survival of plant and microbial germplasm. This goal is accomplished by integrating research and curation activities that ensure germplasm viability stays high (Objectives 1,2) and that collection diversity and utility increases (Objectives 3,4). A major consolidation of curation and research groups occurred in fiscal year 2019, and activities in fiscal year 2021 continued to implement the unification of plant programs. The number of incoming seed samples from other NPGS sites was a historical high this past year, while amounts of clonal materials available for processing was at an all-time low. This difference probably reflects the seasonality of the seed planting and harvest at sites in 2020 and the slowed activity at sites for obtaining plant cuttings, which require laboratory and greenhouse activities. A congressionally-mandated survey to reveal the status of plant genetic resources within the NPGS is nearing completion. Data from Fort Collins, Colorado, which serves as the duplication site for germplasm in the NPGS, was analyzed and consolidated with other NPGS sites in winter and spring of fiscal year 2021. According to the survey, about 88% and 18% of accessions from seed-based and clonal-based repositories, respectively, are duplicated in secured freezer (Objectives 1 and 4) or cryogenic storage in Fort Collins (Objectives 2 and 4). The analysis queried whether duplicated accessions also met established criteria for sample size and viability that are used globally for plant genebanks. The results revealed that about 20% (ca. 94,000 of 467,000 accessions) and 8% (ca. 4000 of 47,000 accessions) of accessions from NPGS seed-based and clonal-based sites meet the sample size and viability criteria at Fort Collins, Colorado, respectively. Enhanced efforts to monitor viability of stored seeds and calculate seed numbers will boost the proportion of seed accessions meeting international standards (Objectives 1 and 4). The high labor demands for processing germplasm for cryopreservation, influence of endophytes on survival of propagules, and protocol development for small collections, especially of tropical origin, continue to be bottlenecks for rapid progress in backing-up clonally-propagated germplasm (Objective 2). Assessing the viability of stored germplasm continues to be a core activity of the Unit (Objectives 1,2 and 4). A comparison of seed viability assessments across NPGS sites (as part of the survey described previously) showed low redundancy in that effort, despite earlier assessments. The survey did show several areas of possible collaboration that could lead to more reliable predictions of when seeds might lose viability during storage, and this will ultimately lead to more efficient monitoring protocols (Objective 1). In 2021, chemical tests of seed aging during initial storage (before seeds die) focused on changes in RNA both in dry storage and during the early stages of germination (Objective 1). These tests show remarkable promise in identifying aging rate before mortality is detected as well as observing the pathology of mortality during recovery from storage as seed metabolism is jump-started. This work is the foundation for an automated, sequence-based protocol to aid with viability monitoring of stored seeds as well as new insights into embryo rescue of severely deteriorated seeds. The research is part of an effort to evaluate seed longevity of species native to the United States in collaboration with the Bureau of Land Management and Center for Plant Conservation (Objective 4). Cryopreservation of clonally propagated germplasm continued in 2021, with major strides in evaluating robustness of protocol descriptions – that is, the learning success of a skilled technician unfamiliar with a particular protocol when given detailed instructions and videos (Objective 2). As part of a large outreach and instruction initiative, numerous videos of cryopreserving clonal propagules from different species have been produced and are available online. Unlike many seeds that are processed dry, clonal propagules are usually hydrated and require cryoprotection using solutions that osmotically remove water from cells. A recent study demonstrated that a powerful cryoprotectant, dimethyl sulfoxide, sequesters in organelles upon entering cells, which may explain aspects of its mode of action or its toxicity (Objective 2). Additional sucrose applications and air desiccation are also shown to give cryoprotective benefit to fruit tree buds harvested in midwinter (i.e., dormant buds) (Objective 2). Efforts to preserve coffee germplasm, both as seeds and clonal propagules, was stimulated by recent Congressional appropriations. The group in Fort Collins, Colorado, specializes in cryopreservation (Objective 2), and is teaming with several partners including the coffee industry as well as ARS researchers in Hilo, Hawaii, who will develop coffee genetic resource collections, and in Beltsville, Maryland, who will assess the genetic diversity among coffee cultivars using sequenced-based analyses. The genetic diversity of wild plant species provides a wealth of untapped resources for crop improvement. Studies conducted in Fort Collins, Colorado, have shown that flora of the United States includes species that are related to major crops, and are of conservation concern, requiring concerted efforts of habitat conservation and offsite genebanking (Objective 4). Additional studies were conducted to improve management of germplasm from wild populations in genebanks to ensure their viability (Objective 4), to quantify genetic diversity within and among accessions using genomic tools (Objective 3), and to concentrate diversity into core subsets for a particular trait through heuristic algorithms that locate variation within key genes (Objective 3). In collaboration with National Park Service, genetic fingerprinting was used to identify cultivars in one hundred-year-old orchards to assist with site management plans to preserve historical value (Objective 3).

Accomplishments
1. Online tools to train the next generation of genebankers. Both ARS and international genebanks are experiencing an unprecedented wave of retirements among their professionals, which threatens the effective and efficient transfer of institutional knowledge specific to plant genebanking. To stave off the loss of this institutional knowledge and equip plant genebanks for the future, ARS scientists in Fort Collins, Colorado, and Beltsville, Maryland, teamed up to develop and release educational content for plant genetic resources conservation and use. The public website (https://grin-u.org/) developed in this effort provides links for courses, eBooks, videos, and other educational resources for United States and international genebank personnel, universities, industry, and other organizations. These much-needed training resources will accelerate the training of the next generation of plant genebankers, preparing them to address future challenges.

2. Enhanced germination protocols to evaluate seed quality from wild plants. Wild species tend to produce seeds that germinate slowly or at differing times; however, there are few protocols available to guide methods to speed up or synchronize germination for determination of seed viability. This necessitates the use of vital staining methods as a proxy for estimating seed quality. ARS scientists in Fort Collins, Colorado, developed and adapted protocols that can stimulate germination of wild plant species that have been problematic in the past. Treatments combine the use of temperature, hormonal applications, and clay-based media or embryo extraction and growth in culture. This advance increases the accuracy and efficiency of seed quality assessments, especially of stored seeds, and makes it possible to grow out wild-collected seeds for regeneration and restoration programs.

3. Improvements in dormant bud cryopreservation techniques accelerates back-up of sweet cherry and butternut collections. Field collections of sweet cherry (Prunus avium L.) and butternut (Juglans cinerea L.) within the USDA National Plant Germplasm System (NPGS) are at risk of loss because they are not duplicated at a secure secondary site. However, previous efforts to back-up these genetic resources failed because sweet cherry and butternut germplasm did not survive cryoexposure. ARS scientists from Fort Collins, Colorado, developed robust cryopreservation methods for these two species using winter-harvested dormant buds. This new capacity will provide reliable back-up for these NPGS collections, ensuring that these valuable genetic resources are available in perpetuity.

U.S. DEPARTMENT OF AGRICULTURE

Agricultural Genetic Resources Preservation Research: Fort Collins, CO