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Research Project: Plant and Microbial Genetic Resource Preservation and Quality Assessment


2018 Annual Report

Over the next five years, the Plant Genetic Resources Preservation Program (PGRPP) at the National Center for Genetic Resources Preservation (NCGRP) will focus on the following four objectives that are both hypothesis and non-hypothesis driven. Objective 1: Ensure secure, long-term preservation of the NPGS base collections and associated information and of safety back-up storage for designated non-NPGS plant genetic resources. Objective 2: Facilitate and promote the secure, long-term preservation of plant-associated and other key collections of microbial genetic resources by backing-up collections from ARS and other public-sector institutions. Sub-objective 2a: Provide secure back-up storage of public microbial collections and aid in the development of a U.S. Culture Collections Network. Sub-objective 2b: Develop improved long-term storage systems for selected microbes. Objective 3: Devise, adapt, and/or apply optimal methods for secure long-term preservation of plant genetic resources, and promote research, training, and domestic and international technology transfer of the preceding approaches. Sub-objective 3a: Set priorities to monitor viability of seed collections. Sub-objective 3b: Evaluate effects of LN2 storage on plant germplasm. Sub-objective 3c: Establish optimal harvest time for dormant winter buds used for cryopreservation of selected tree species. Sub-objective 3d.1: Develop protocols for cryopreservation of selected crops. Sub-objective 3d.2: Determine genebanking protocols for crop wild relatives, medicinal plants, and alternative crops. Objective 4: Devise and apply new methods for high throughput phenotyping and genetic analyses of root system architectural diversity in selected crops and their wild relatives.

The changing needs in U.S. agriculture place new demands on farmers and plant breeders for new improved varieties which require access to a wide range of well characterized plant diversity. An increasing global population will require more efficient food production, and a changing climate requires crop varieties adapted to stresses. Limited, and sometimes compromised, water resources are having greater impacts on crop yields. The National Center for Genetic Resources Preservation is one of the largest and most diverse genebanks in the world and the flagship of the U.S. National Plant Germplasm System. Our project’s overarching mission is two-fold: to provide secure long-term preservation, and documentation of diverse genetic resources. We accomplish this by close collaboration with individual crop curators from the National Plant Germplasm System to back-up and monitor their unique collections. We work to back-up world plant collections, collaborating with other national and international genebanks. Along with preserving crops for U.S. agriculture, we safeguard storage of threatened and endangered plants, crop wild relatives, plants for medicinal uses, and new crops being considered for future biofuel or bioproduct use. Linked to our mission, we propose to develop improved storage protocols of seed, clonally preserved crops, and microbes to become more efficient in our standard operating procedures. Our focus on germplasm preservation and will ensure that farmers have access to the most productive cropvarieties and help the U.S. remain as a world leader in genetic resources preservation.

Progress Report
Safeguarding key plant genetic resource collections for the long term was the primary objective during this period. From 2012 to 2017, the National Plant Germplasm System (NPGS) collection increased by 64,000 accessions, and the Plant Genetic Resources Preservation Program (PGRPP) security back up collection increased by 44,000 accessions. Safeguarding the Nation’s collection is the primary role played by PGRPP in the NPGS. Seed germination testing is an important part of safeguarding collections by obtaining baseline viability data on incoming seed samples and monitoring the seed collection over time. In the past five years, we conducted 40,463 germination tests. Another important role PGRPP plays within the NPGS is coordinating and shipping accessions to the Svalbard Global Seed Vault in Norway. In the past five years, we have sent 77,520 accessions to Norway. Although the NPGS base collection is of primary concern, safeguarding other national and global collections is also in the best interest of the United States for food safety and security. The project plays an important role in the seed genetic resource community by providing our storage service. From 2012 to 2017, germplasm received from 70 institutions increased from 310,603 to almost 450,000 accessions. The introduction of new improved germplasm into the NPGS is critical to ensure that researchers have the best material to work with. PGRPP plays an important role not only safeguarding cultivars certified by the Plant Variety Protection (PVP) Office and by the Journal of Plant Registration but by coordinating the transfer of this germplasm into the public domain upon expiration of intellectual property rights. From 2012 to 2017, PGRPP coordinated the transfer of 1543 expired certified cultivars into the NPGS collections from these two programs. In this same period, we processed incoming voucher specimens of 5260 accessions from these two programs, all of which will be transferred to the NPGS when intellectual property rights protections expire. The PGRPP microbe backup collection expanded from 4753 isolates in 2012 to over 111,000 isolates today representing 22 culture collections. Our lab has become an important resource to the microbial culture community, providing high quality back up storage for important and vulnerable collections. In collaboration with microbiologists, we completed and published a study that validated the use of liquid nitrogen to store a wide diversity of Fusarium species. We also converted an existing lab into a permitted microbial lab so we can conduct research in our own facilities and upgraded our Standard Operating Procedures so they are in-line with new biosafety regulations. Both the microbe curator and technician have received extensive formal and on the job training in biosafety and microbiology protocols. An important impact made during the last five years has been to assess the status of the base collection, in terms of viability of seed samples. We found that about 150,000 base samples have been tested two or more times (initial plus monitor test), and that 100,248 accessions were last tested more than a decade ago. Of this number, 12% had been tested over 20 years ago. In 2014 we prioritized our monitoring efforts, focusing on testing short-lived species. By 2017 we had conducted 22,000 monitor tests on species most likely to decline in storage. We found that for 71% of the samples, viability had changed (±) < 20% from initial viability. However, 23% had declined > 20%. The good news was that most of our short-lived species have had minimal decline, and we have initiated an NPGS-wide discussion to develop best practices for insuring high quality safety back-up samples through timely replenishment. Because of the range of diversity we manage, research has been needed to develop effective protocols for storing germplasm, especially in liquid nitrogen. In the last five years, we have made substantial progress in developing and validating protocols. To examine the relative effectiveness of seed storage in liquid nitrogen vapor compared to conventional storage at -18 degrees C we evaluated germination, seedling root characteristics, morphological attributes in a field environment, and DNA methylation of 20 rye accessions stored at -18 degrees C or in liquid nitrogen for 25 years. Our results published in Cryobiology indicated there was little difference between the two storage methods. We discovered that harvesting twigs at the right time leads to successful cryopreservation of blueberry. We found that fully dormant buds recover from storage at a much higher rate, making cryogenic storage of dormant buds an attractive option due to the lower costs and shorter processing times, compared to aseptic shoot tips. We also found that combinations of antioxidants and cryoprotectants increase cryogenic survival of apricot, peach, and plum. These new combinations set the stage for securing the USDA apricot, peach and plum germplasm collections in liquid nitrogen, safe from nature’s harm. Cryopreservation procedures were also established in pineapple, cacao, and sugarcane. Since 2015 our project plan has focused on conserving crop wild relatives. We have successfully refined spatial models to predict species distributions and have modeled over 1000 taxa. These models will be used in a crop wild relative gap analysis and were also incorporated into a two-volume book on crop wild relatives in North America that will be published in November 2018. Members of PGRPP were key leaders in producing the book. We have also been key leaders publicizing the importance of conserving and using North American crop wild relatives. We have developed a white paper outlining strategies and resources needed to secure U.S. crop wild relatives; organized two symposia, led efforts to produce a 17 paper Special Issue in Crop Science on crop wild relative use, and have given invited presentations to the Plant Conservation Alliance, National Genetic Resources Advisory Committee, National Native Seed Conference, USFS Regional Botanists, Dupont Plant Science Symposium, and Forest Tree Genetic Resource Conference.

1. Protocol for cryopreservation of quince. Fruit of the genus quince is used for fresh eating, jams, and paste, and contains levels of high vitamin C and other minerals. The USDA National Plant Germplasm System collection has 135 quince accessions preserved as single trees in a field, which exposes them to climate and biotic threats; therefore, these important genetic resources need to be securely backed up. Unfortunately, little work has been done to understand the most effective procedures to cryopreserve quince. To address this need, ARS scientists in Fort Collins, Colorado developed a cryopreservation technique for quince, which will allow for routine cryopreservation. The technique will be an asset to genebanks in safeguarding important germplasm so that it is available for distribution to support quince breeding efforts so that more fruit is available in the marketplace.

Review Publications
Da Silva Ledo, A., Jenderek, M.M., Da Silva Ledo, C.A., Ayala Silva, T. 2018. Antioxidants and phenolic secretion in sugarcane genotypes shoot culture. Journal of Agricultural Science. 10(5):1-13.
Norton, S.L., Khoury, C.K., Sosa, C.C., Castaneda-Alvarez, N.P., Achicanoy, H.A., Sotelo, S. 2017. Priorities for enhancing the ex situ conservation and use of Australian crop wild relatives. Australian Journal of Botany. 65(8):638-645.
Lu, J., Greene, S.L., Reid, S., Cruz, V.V., Dierig, D. 2018. Phenotypic changes and DNA methylation status in cryopreserved seeds of cereal rye (Secale cereale L.). Cryobiology. 82:8-14.
Webb, K.M., Holman, G.E., Duke, S.E., Greene, S.L., McCluskey, K. 2018. Frozen fungi: cryogenic storage is an effective method to store Fusarium cultures for the long-term. Annals of Applied Biology. 173:133-140.