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

2022 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. Currently about 88% and 18% of accessions from seed-based and clonal-based repositories, respectively, are duplicated in Fort Collins. However, only about 20% (ca. 94,000 of 467,000) accessions from seed-based sites and 8% (ca. 4,000 of 47,000) accessions from clonal-based sites meet the criteria for sample size and viability needed for true back-up according to international standards for crop genebanks. Improving back-up status of NPGS collections requires enhanced research and curation activities to ensure that germplasm viability stays high (Objectives 1, 2) and that collection diversity and utility increases (Objectives 3, 4). Activities in fiscal year 2022 continued to implement the unification of plant research and curation programs that were consolidated in fiscal year 2019. Outreach activities continue to play a significant role in our programs. In addition, a one credit course on plant genebanking was developed and will be offered through Colorado State University (CSU) Online. Objective 1. Approximately 4,500 seed accessions from wild and domesticated populations were processed and stored at -18C (0F). Seed processing is hampered by frequent malfunction of infrastructure, particularly relative humidity (RH) and freezer controls, the latter being repaired in 2022. Assessing the viability of incoming and stored germplasm is a core activity and about 11,000 NPGS accessions received germination tests in 2022 (4,000 initial tests and 7,000 monitor tests). De-emphasis of fast, but laborious, techniques such as vital staining, in favor of longer tests that break seed dormancy, has nearly doubled seed analyst productivity since 2019. Even still, international standards of monitoring ARS’s ~500,000 seed accessions every 15 years requires about 3 times current capacity. Research efforts to optimize monitoring frequencies based on predicted longevity are expected to increase efficiency and reduce risks of germplasm dying during storage. One promising test measures fragmentation of RNA, which can be detected before viability declines. Databasing tools represent another serious bottleneck. Development of a new web-based User Interface (‘smile’) to input and extract data generated by this project is underway. Components will support the daily routine tasks conducted at the facility as well as interface with the Genetic Resources Information Network (GRIN). Objective 2. Cryogenic storage (in liquid nitrogen (LN)) of orthodox seeds was curtailed in 2019 to review the relevant policies and procedures as well as reduce the backlog of processing seeds in Objective 1. Revised processing methods are being vetted and standard operating procedures (SOPs) will be prepared. The policy of what orthodox seed species are stored cryogenically has been completely revised, and research needed to support new policy has been proposed in the next five year project plan. Methods to cryopreserve seeds such as papaya, coffee, hazelnuts and walnuts, which exhibit so-called intermediate storage behavior, are recently available and will be implemented for routine curation. This new capacity is expected to increase the proportion of accessions at clonal-based sites that can be backed up. Approximately 250 clonal accessions were cryopreserved in 2022 using either dormant buds (apple, pear, sweet cherry, and plum) or shoot tips (grape, potato, banana, citrus, and kiwi fruit). Three virtual tour videos and multiple ebook chapters and videos about clonal cryopreservation methods were released on the NPGS training website GRIN-U.org. Shoot tip cryopreservation methods were modified and combined with thermotherapy techniques to eradicate two viruses and one viroid from four USDA apple rootstocks, which can now be released to industry. The presence of endophytes in plant cultures prevented successful shoot tip cryopreservation in some crops (date palm, walnut) and strategies to identify Citrus endophytes were published. High labor demands for processing clonal 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. There is renewed interest in preserving genetic resources as pollen, and ongoing work seeks to optimize moisture conditions and viability testing for diverse collections including Prunus (cherries, plums, and peaches), walnut, date palm, pecan, cotton, oak, hazelnut, citrus, avocado and hemp. Objective 3. The research project uses genomic tools to quantify genetic diversity within and among accessions, to predict sources of novel diversity that should be included in NPGS collections and to identify unused accessions with genes of agronomic importance in allele mining projects. In 2022, a new approach that genotypes a pool of individuals within a heterogeneous sample was introduced to provide a more accurate snapshot of the genetic identity and utility of accessions, especially crop wild relatives. Objective 4. The international collection survey for the Global Conservation Strategy for Citrus was conducted and analyzed, with plans to release the final Strategy document in Fall 2022. Accessions of Hordeum pusillum, a broadly distributed North American relative of cultivated barley, have been collected from the Desoto and Holly Springs National Forest area in Mississippi, Comanche and Pawnee National Grasslands of Colorado, and Ft. Pierre National Grasslands of South Dakota. These and existing accessions within the NPGS will be used to characterize the genetic structure among populations and search for signatures of past domestication. Seeds are the preferred germplasm form for accessions from wild populations, but they are more difficult to genebank than seeds from domesticated plants. Much progress was made in 2022 towards characterizing seed traits from wild populations including seed mass, germination requirements, storage behavior and longevity in storage. Much of this work was conducted in collaboration with conservation groups and land managers, particularly the Bureau of Land Management and Center for Plant Conservation.

Accomplishments
1. Cryotherapy eradicates pathogens from apple plant genetic resources. Apple stem grooving virus and apple chlorotic leaf spot virus are serious and prevalent apple viruses that are difficult to eradicate. In a collaboration with USDA-ARS Geneva, New York, Animal Plant Health Inspection Service (APHIS), and University of California-Davis, ARS scientists in Fort Collins, Colorado, used a combination of thermotherapy and cryotherapy to eliminate these viruses and apple hammerhead viroid from shoot tips of four apple cultivars from the Cornell-Geneva apple rootstock breeding program. The methods are now used to eliminate pathogens from commercial apple rootstocks as well as apple genetic resources introduced into the U.S. The research allows expansion of the apple genetic resource collection, evaluation of traits in disease-free germplasm, and most importantly, large-scale nursery propagation of apple trees in areas where these viruses are found. This technique has produced virus-cleaned apple rootstocks that have been transferred to micropropagation nurseries to support the U.S. apple industry.

2. Tapping the genetic diversity of wild plants for crop improvement. The samples of wild relatives of important crops in plant genebanks often contain heterogeneous plant populations rather than single genotypes (individuals). This is because samples of crop wild relative species are typically taken from diverse populations in the wild and are mixtures of genotypes. Analysis of a single seed, therefore, cannot represent the genetic variation and may miss agronomically important traits. Alternatively analyzing many individuals separately can be cost prohibitive. As a result, important genetic variation carried by crop wild relatives is often undetected and unused. ARS scientists in Fort Collins, Colorado, developed a ‘pooled’ sequencing technique to ‘fingerprint’ the population in a single genetic analysis, which greatly improves genetic characterization. This technique provides users with information on the full range of genetic variation, and the resulting composite genotype or ‘pan genome’ can be searched using standard tools to identify accessions with agronomically important genes. This research greatly increases the value of genebanked plant samples by accurately characterizing the population. Ultimately it delivers on the hidden genetic treasures in crop wild relatives highlighting novel traits with agronomic importance to plant breeders.

3. Genebanking seeds that do not survive in genebanks. Many of the USDA National Plant Germplasm System (NPGS) collections are not backed up in long-term, preserved collections because the seeds do not survive conventional, freezer storage. Examples are papaya, willow, and many tree nuts, especially those containing high levels of nutritious monounsaturated oils. ARS scientists in Fort Collins, Colorado, developed robust methods to cryopreserve these types of seeds in liquid nitrogen at much lower temperatures for survival for 15 years and likely much longer. To store these seeds, they are processed quickly to avoid rapid aging; water content is carefully adjusted; and cooling and warming rates are optimized to prevent crystallization of the oils. The research provides a generalized method applicable to several thousand NPGS accessions, potentially doubling the proportion of backed-up accessions in some of NPGS’s most vulnerable collections over the next few years, especially at tropical and subtropical locations. This will provide assurance to breeders, researchers, and industry that these previously difficult to preserve seeds are backed up and available for use for many decades.

U.S. DEPARTMENT OF AGRICULTURE

Agricultural Genetic Resources Preservation Research: Fort Collins, CO