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


2015 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. We will characterize germplasm in our collection to identify plant traits that increase crop productivity under water shortages. Our focus on germplasm preservation and characterization 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
The crop diversity housed in gene banks is foundational for plentiful food supplies. Breeders mine such collections for genetic diversity traits that convey resistance to adverse climate, devastating insects and disease or provide superior nutrition. Since individual genebanks are vulnerable to natural and manmade disasters, germplasm collections need to be backed up at multiple locations to protect against loss. The Plant and Animal Genetic Resources Preservation Unit (PAGRP) in Fort Collins serves the role of providing safety duplication, free of charge, for a broad range of plant and microbe diversity, not only for the USDA and other U.S. agencies, but for non-governmental organizations and the private sector, both here and abroad. The USDA National Plant Germplasm System is one of the largest crop germplasm repositories in the world, and currently houses over 572,000 accessions. In 2015, the PAGRP received 11,689 packets, and assessed viability, packaged and stored 8285 seed packets in conventional storage and 982 packets in cryo storage. 362 in vitro samples and 193 shoot meristem samples were processed and stored in cryo. Overall, the PAGRP provides safety back up for 82% of NPGS seed collections and 16% of vegetatively-propagated collections. Currently 431,000 NPGS accessions are securely backed up at the Fort Collins repository. The PAGRP also provides safety back up for 315,000 germplasm accessions held by non-NPGS institutes, including germplasm from four international genebanks that are a part of the Consultative Group on International Agricultural Research. In 2015, we signed a new MTA with AfricaRice and 2500 rice accessions are currently enroute from Africa. We continued our partnership with the Bureau of Land Management (BLM) Seeds of Success (SOS) program, which has a mission to collect and make available native species for revegetating land impacted by fire and other natural disasters. From July 1, 2014 -June 30, 2015 we received 1961 new samples, bringing our total SOS samples to 9230 accessions. We also reported back to the BLM on seed quality issues to help improve seed handling procedures throughout the production chain, to improve seed quality. The PAGRP stores seed and clonal propagules at -18° F or -196° F in liquid nitrogen. A critical activity in our Unit is to monitor viability of stored seed lots. In 2015, we reprioritized our monitor tests to focus on testing short lived species that have not been tested in 10-20 years. Our goal of 3000 accessions was reached by establishing a new specific cooperative agreement with Colorado Seed Testing Laboratory. We also developed a prototype software application that prioritizes monitor testing based on estimates of species longevity and last viability test date. Over 58% of the incoming seed from the National Plant Germplasm System (NPGS) sites had over 85% germination and we expect them to have maximum longevity in storage. Eighteen percent had less than 65% viability and NPGS active sites were contacted to provide feedback on the possibility of subpar seed production methods. The monitoring tests we performed indicated that 67% of our high priority, short longevity accessions had at least 85% viability, and only 8% had less than 65% germination, indicating that viability was being maintained for most accessions tested. The 2015 activities described above contribute to the secure, long-term preservation of the NPGS collections and associated information and of safety back-up storage for designated non-NPGS plant genetic resources; objective one of our current five-year project plan. A recent government report on public microbe collections stated that many are vulnerable to loss as scientists retire. The report suggested that microbe collections be backed-up to protect the investment made in establishing and maintaining these collections. In 2015, the PAGRP back up microbe collection increased by 2,893 samples, for a total of 31,721 isolates. We also coordinate the distribution of four International Seed Federation Differential Pathogen Sets, and had one distribution in 2015. These activities support the secure, long-term preservation of agriculturally important microbial genetic resources by backing-up collections from ARS and other institutions; objective two of our current five-year project plan. Preservation protocols for orthodox seeds are straight forward, however, questions remain regarding the relative effectiveness of seed storage in liquid nitrogen vapors compared to conventional storage at -18°C. In 2014, we completed the germination and seedling root characteristics of 40 rye accessions stored at -18°C and in liquid nitrogen for 25 years. We also planted a trial out in the field to evaluate the morphological variation between seed lots stored in conventional and cryo storage. Although many crops can be stored as seed, a large number of fruit and nut crops are preserved as clonal propagules, either as small shoots plantlets or buds. Efficient procedures need to be developed to effectively store propagules cryogenically and still have acceptable viability upon thawing. In 2015, 120 clonally maintained accessions were placed in long-term storage according to the established; 90 accessions were cryopreserved via meristem shoots and 30 accessions via dormant buds (DB). The successful cryopreservation of dormant buds for 30 genotypes of the same species supported the adequacy of using the DB technology for cryopreservation of that large germplasm collection. Over 100 sugarcane accessions were established in vitro and were placed in slow-growth conditions until a reliable cryopreservation protocol is established. The projected cryopreservation number was not fully achieved due to critical vacancies and staffing changes. These efforts support Objective 1 in our project plan. Progress was also made in several sub objectives in Objective 3. Samples for oligosaccharide analysis have been collected; however, due to vacancies, the analysis has not been completed and oligosaccharide dynamics pattern have yet to be established. Pretreatment of dormant buds with sucrose combined with selected anti-oxidants as vitamin C, L-cysteine, L-proline and ABA increased post cryopreservation viability in tested genotypes of apricot, hazelnuts, peach, pear and plum to a level required in routine long-term preservation of germplasm. The data supports the possibility of cryopreserving germplasm of these collections by dormant buds that is faster and less expensive than using meristem shoots. We also found that using a shoot etiolating step promoted the physiological uniformity of shoots (a critical phase in preparation of shoots for cryopreservation) and reduced culture transfer time. Our activities to devise, adapt, and/or apply optimal methods for secure long-term preservation of plant genetic resources support objective three of our project plan. We changed our project plan in 2015 due to the departure of two scientists. Objective 4 was dropped, and Object 3 was expanded to include a gap analysis of crop wild relatives in the United States. To this end we hired a new postdoc who will begin work on the project in September 2015.

1. Safeguarding plant and microbial genetic resources. Plant and microbial genetic diversity forms the foundation upon which successful production systems are built that secure our food, fiber, forage and environment, not only in the United States, but around the world. Gene banks house this diversity, but are vulnerable to human and environmental disasters unless their holdings are duplicated elsewhere. The Fort Collins, CO genebank safeguards a wealth of the world’s plant and microbial diversity. ARS researchers in Fort Collins grew the collection by adding 11,689 new seed collections and performing germination tests on 9,267 samples, plus 3,000 monitor tests on samples already in storage. ARS researchers added 2,894 microbial isolates to the Agricultural Microbe Back-up collection. ARS Fort Collins researchers also sent the largest single shipment from any country (18,000 accessions), to the Svalbard Seed Vault in Norway. We also provided 61 tours of our facilities resulting in over 800 people learning about the importance of conserving agricultural biodiversity and USDA efforts to ensure our germplasm is available to be used by future generations. With more than 941,760 accessions housed in its vaults, the plant and microbe gene bank at Fort Collins, CO truly serves the nation, and the world, as the Fort Knox of plant and microbial diversity, ensuring that germplasm is safeguarded so it remains available for use, and is not lost.

2. Overcoming germination barriers in native wild species used for revegetation. Wild fires in the western United States have catalyzed the use of native species in revegetation efforts, however understanding basic species biology such as germination are creating a bottleneck in using native seeds. ARS researchers in Fort Collins reviewed and collated National Center for Genetic Resource Preservation Seeds of Success germination test data for over 700 different wild species and reported the germination protocols that worked best. They also identified which species have dormancy challenges. This data will help natural resource managers working with native species to better understand the conditions needed to germinate specific species.

3. Effective cryopreservation of black current. Although the United States does not consume many currents, it is an important fruit in the northern hemisphere and is being used more frequently as an additive to juice products because of its high antioxidant properties. The National Plant Germplasm system has 249 accessions of black currant germplasm; however, only 38 accessions have been cryopreserved as meristem shoots. The remainder of the living collection is vulnerable since it is planted at a single location. In 2015, ARS scientists at the Fort Collins Plant and Animal Genetic Resource Preservation Unit were successful in doubling the number of cryopreserved genotypes using dormant buds. Most importantly this two year study identified a set of protocols that is highly effective for a diverse range of black current germplasm. These protocols can be adapted by other gene banks and breeding programs for long term storage of these species.

Review Publications
Cruz, V., Comas, L.H., Dierig, D.A. 2014. Root phenotypic characterization of lesquerella genetic resources. Industrial Crops and Products. 62:130-139.
Smýkal, P., Coyne, C.J., Ambrose, M.J., Maxted, N., Schaefer, H., Blair, M.W., Berger, J., Greene, S.L., Nelson, M.N., Besharat, N., Vymyslický, T., Toker, C., Saxena, R.K., Roorkiwal, M., Pandey, M.K., Hu, J., Li, Y.H., Wang, L.X., Guo, Y., Qiu, L.J., Redden, R.J., Varshney, R.K. 2014. Legume crops phylogeny and genetic diversity for science and breeding. Critical Reviews in Plant Sciences. 34:43-104. doi: 10.1080/07352689.2014.897904.
Greene, S.L., Kisha, T.J., Yu, L., Parra-Quijano, M. 2014. Conserving plants in gene banks and nature: Investigating complementarity with Trifolium thompsonii Morton. PLoS One. 9(8):e105145.