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


2016 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
The crop diversity housed in this ARS gene bank is foundational for food security. Breeders mine this collection for traits that convey resistance to adverse climate, devastating insects and disease or for improved nutrition. The Fort Collins collection also supports basic and applied biological research. The USDA-ARS National Plant and Genetic Resources is the largest agricultural gene banking system in the world and distributes over 250,000 orders annually to users around the globe. Our first objective is to ensure secure, long-term preservation of the National Plant Germplasm System base collections and associated information and to provide safety back-up storage for designated non-NPGS plant genetic resources. Our vaults hold the single largest collection of crop genetic resources in the world. We store both seed and clonal propagules (i.e. dormant buds, shoot meristems). We have 1.7 million seed packets representing 550,000 NPGS accessions, and store an additional 362,000 accessions for non NPGS organizations such as the USDA Forest Service, U.S. Tribal agencies, botanical gardens, seed saving organizations, and international seed collections. We have over 14,000 clonal propagules in cryogenic storage. In 2016, a total of 18,201 samples were received. Thirty-five percent came from National Plant Germplasm System sites. In addition to seed samples, we now have 743 National Plant Germplasm System clonal samples stored cryogenically. This exceeds our 48 month cryo preservation milestone by 118 accessions. Twenty-eight percent of the samples we received in 2016 came from non ARS agencies (i.e. AfricaRice). Four percent were seed voucher samples came from the Plant Variety Protection Office and Journal of Plant Registration. The final 33% were National Plant Germplasm System samples destined for storage at the Svalbard Global Seed Bank in Norway. In September, 19,375 samples will be shipped to Svalbard, bringing our total backup level at Svalbard to 20% of the U.S. seed collection. In the course of a year, we tested seed viability, packaged and stored 5,682 packets in conventional storage and 584 seed packets in cryogenic storage. Since 2013, 80 in vitro samples, 369 shoot meristem and 294 dormant bud samples were processed and stored cryogenically. In 2016, we completed an inventory of our non NPGS collections (Obj. 1) and identified 42 collections that required new Material Transfer Agreements. We received approval from the ARS Tech Transfer office to extend our MTAs from 5 to 10 years. We continued our partnership with the Bureau of Land Management, Seeds of Success program. From July 1, 2015 to June 30, 2016 we received 1,239 new samples of native species, bringing the Seeds of Success samples total to 10,469. We have established a new five year agreement with the Bureau of Land Management, Seeds of Success program for processing and storage of wild species. Our second objective is to facilitate and promote the secure, long-term preservation of plant-associated microbial genetic resources by backing-up collections from ARS and other public-sector. In 2016 we received collections of algae (1,095 samples), bacteria (882) and fungi (885). We will also be receiving an entire copy of the ARS National Regional Research Laboratory culture collection, which consists of almost 80,000 isolated. One of our sub objectives is to aid in the development of the United States Culture Collections Network. In October of 2015 we hosted the annual United States Culture Collections Network Workshop at the National Laboratory for Genetic Resources Preservation. Culture collection curators discussed collections that are vulnerable because of changes in funding programs, or are at risk of loss because of retirement or lack of funding. We reported on our efforts to provide security back up and contributed to a peer-reviewed paper that reported on the meeting outcomes. A second sub objective is developing improved long term storage systems for microbe storage. In 2016 we collaborated with other ARS units to evaluate the viability of Fusarium species stored in cryogenically. We began evaluating isolates in August, and should be done evaluating the entire collection by spring of 2017. This should provide insight into the effectiveness of cryogenic storage to preserve Fusarium species. Further research will be facilitated by the setup of a microbial laboratory in our Unit which is occurring at this time. Our third objective is to devise, adapt, and/or apply optimal methods for securing long-term preservation of plant genetic resources, and promote research, training, and domestic and international technology transfer of the preceding approaches. An important sub objective is to set priorities to monitor viability of our seed collection. In 2015, we reprioritized our monitor tests to focus on testing short lived species that have not been tested in 10-20 years and monitor tested 3000 accessions. In 2016 we increased our monitor tests to 5000. In FY 2016 ARS Fort Collins examined the effects of cryogenic storage on plant germplasm (Objective 3b). We conducted an analysis of morphological data gathered from field grown plants, root growth and seedling viability studies on 20 accessions of rye. We compared cryogenically and at -18 C samples that were stored for 25 years. DNA methylation differences between accessions were found. There was also minor difference observed in spike length. Significant differences were not detected for other traits. Obj. 3b milestones were met with the following clonal crops: pear, potato, strawberry and sweet potato. Obj. 3c involves the establishment of optimal harvest time for dormant winter buds to be used in cryopreservation of selected tree species based on carbohydrate concentration. A comparison of pre-harvest twig temperature with post cryopreservation viability was conducted on blueberry in FY 15/16. This led to a better understanding of how harvest temperature supports high dormant bud survival and suggests a strategy for optimizing timing of dormant bud harvest. Research continued to develop cryopreservation protocols for various species (Obj. 3d1). In cacao, research on somatic embryos and cold-hardened axillary buds were conducted. Somatic embryos were successfully generated for 15 accessions as well as cold-hardened cacao plants (2 accessions). Micrografting of axillary buds and shoots were successful (>50%) and in vitro rootstocks were established. In sugarcane, literature-reported cryopreservation procedures were tested on a variety of germplasm types. Several factors in medium composition, osmoprotection time and regeneration techniques were improved and a protocol was established. We also evaluated the applicability of the Malus dormant bud cryopreservation method on apricot, peach and plum species. The method was significantly improved upon by introducing cryoprotectans and antioxidants. Sub-objective 3d2 focused on determining gene banking protocols for crop wild relatives including the use of GIS and gap analysis to identify and prioritize U.S. Crop Wild Relatives. This ARS unit has been active in increasing awareness among plant breeders and other Federal agencies regarding the importance of conserving and using native crop wild relatives. For example we organized a symposium at the crop society meeting held in Nov. 2015, put out a call for papers to publish a special issue of Crop Science on crop wild relative use, initiated publication of a book on the crop wild relatives and wild utilized species of North America, gave an invited presentation on U.S. Crop Wild Relatives activities to the Plant Conservation Alliance in Washington D.C and organized a workshop at the Forest Tree Genetic Resource conference held at the Chicago Botanical Garden.

1. Harvesting twigs at the right time leads to successful cryopreservation of blueberry. Blueberries are a nutritional super star, with a market value of $824.9 million. Breeders rely on blueberry germplasm collections to develop productive varieties but field collections are vulnerable to disease, insects and inclement weather. Storing living tissues cryogenically allows us to safeguard these important genetic resources. Cryopreserving dormant buds is less expensive than processing aseptic shoot tips; however, dormant buds often do not have high viability after being stored cryogenically. In a three-year study, ARS scientists at Fort Collins, Colorado in collaboration with Corvallis, Oregon colleagues have discovered they can track daily air temperature to time twig harvest during the winter to ensure buds are completely dormant. 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. Tracking daily air temperature to optimize twig harvest may hold promise for cryopreserving other fruit species using dormant buds. Being able to successfully store dormant buds in liquid nitrogen provides a cost effective way to safe guard important fruit collections, which are otherwise vulnerable to the vagaries of nature.

2. Combinations of antioxidants and cryoprotectants increase cryogenic survival of apricot, peach and plum. Field collections of apricots, plums and peaches are important resources used by breeders to develop new varieties and the USDA maintains field grown collections just for this purpose. However, field collections are vulnerable to extreme weather, pests and disease. Therefore the USDA-ARS has been working to develop protocols that allow for the safety backup of our germplasm collections. Preserving living tissue in the extremely cold temperatures of liquid nitrogen, called cryopreservation, is an important protocol and cryopreservation of dormant buds has been successfully worked out for apples, and used to ensure the security of our USDA apple collection. But field collections of apricot, peach, and plum remain vulnerable. A combined market value of $1 billion (2014), USDA has a strong incentive to develop cryopreservation protocols for these fruits and combinations of antioxidants and sugars significantly increased the survival of dormant buds in liquid nitrogen. These new combination set the stage for securing the USDA apricot, peach and plum germplasm collections in liquid nitrogen, safe from nature’s harm. Successful cryopreservation using dormant buds allows our fruit collections to be secured at a lower cost and more quickly, compared to cryopreserving living tissue via tissue culture.

Established new Material Transfer Agreements to provide long term seed storage for imperiled Ash forests with the following Tribal organizations: Leech Lake Band of Objiwe, Forest County Potawatami Community, Grand Traverse Band of Ottawa, Chippewa Mohawk Nation, St Regis Mohawk Tribe, Mohican Nation Stockbridge-Munsee Community.

Review Publications
Souza, F.V., Ergun, K., Vieria De Jesus, L., De Souza, E.H., Amorim, V., Skogerboe, D.M., Matsumoto Brower, T.K., Alves, A.A., Ledo, C., Jenderek, M.M. 2015. Droplet-vitrification and morphohistological studies of cryopreserved shoot tips of cultivated and wild pineapple genotypes. Plant Cell Tissue and Organ Culture. doi:10.1007/s11240-015-0899-8.
Khoury, C.K., Achicanoy, H.A., Bjorkman, A.D., Navarro-Racines, C., Guarino, L., Flores-Palacios, X. 2016. Origins of food crops connect countries worldwide. Proceedings of the Royal Society of London B. 283: 20160792.
Mcclusky, K., Alvarez, A., Bennett, R., Bokati, D., Boundy-Mills, K., Brown, D., Bull, C., Coffey, M., Dreaden, T., Duke, C., Dye, G., Ehmke, E., Eversole, K., Fentstermacher, K., Geiser, D., Glaeser, J.A., Greene, S.L., Gribble, L., Griffith, M.P., Hanser, K., Humber, R.A., Johnson, B.W., Kermode, A., Krichevsky, M., Laudon, M., Leach, J., Leslie, J., May, M., Melcher, U., Nobles, D., Fonseca, N., Robinson, S., Ryan, M., Scott, J., Silflow, C., Vidaver, A., Webb, K.M., Wertz, J., Yentsch, S., Zehr, S. 2016. United States Culture Collection Network: 2015 Meeting report and call to action. Phytopathology. Phytopathology 106(6): 532-540. doi:10.1094/PHYTO-02-16-0074-RVW.