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


2014 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 this diversity for traits that convey resistance to adverse climate, devastating insects and disease. This past year a gene bank in Georgia narrowly missed being hit by a destructive tornado, an important reminder that individual gene banks are vulnerable to natural and manmade disasters. The Plant and Animal Genetic Resources Preservation Unit serves the role of providing safety duplication of a broad range of plant and microbe diversity, for government agencies, non-governmental organizations and the private sector; nationally and internationally. Building on our already substantial collection, in 2014, the Plant and Animal Genetic Resources Preservation Unit received 8,371 seed packets from National Plant Germplasm System sites. With the germplasm received this year, the Plant and Animal Genetic Resources Preservation Unit now provides safety back up for 82% of seed collections and 14% of vegetatively-propagated collections in the National Plant Germplasm System, amounting to 408,000 accessions. The Plant and Animal Genetic Resources Preservation Unit also provides safety back up for 314,000 germplasm accessions held by non-National Plant Germplasm System institutes, including 144,000 accessions from four international gene banks that are a part of the Consultative Group on International Agricultural Research. In 2014, we signed a new agreement to provide safety backup for the International Crops Research Institute for the Semi-Arid Tropics. Within the next five years, we will provide safe keeping for 120,000 additional accessions from the International Crops Research Institute for the Semi-Arid Tropics. We continued our partnership with the Bureau of Land Management Seeds of Success program, which has a mission to collect and make available native species. We received 1,099 Seeds of Success accessions in 2014, bringing our total to 8,389 accessions. Our institute stores seed and clonal propagules at -18 degrees C or -196 degrees C in liquid nitrogen. A critical activity conducted in the Unit is to assess initial viability of seed lots before they go into storage, and to monitor viability over time. In 2014, we conducted 7,448 germination tests of which 1,019 were monitor tests. Over 63% of the incoming seed from National Plant Germplasm System sites had over 85% germination and we can expect them to have maximum longevity in storage. Eleven percent had less than 60% viability and efforts are underway to get this information back to National Plant Germplasm System active sites so it can be used to refine seed production. The monitoring tests we performed on National Plant Germplasm System seed stored for 10 or more years indicated that 68% of the accessions had at least 85% viability, indicating they are storing well. We found that viability for native species was generally less; 48% of the accessions had viability greater than 80%. This reflects the greater inherent diversity in wild-collected seed lots, an issue we will be focusing on to ensure we can effectively store these types of accessions. A total of 170 clonally propagated plant accessions were also placed into long-term storage. These include: apples, banana, garlic, pineapple, potato, sugar cane, sweet potato, tart cherry and crop wild relatives of potato and strawberry. Additionally, 50 sugarcane accessions were established in tissue culture; this is the first in vitro sugarcane germplasm back-up in the National Plant Germplasm System. The 2014 activities described above contribute to the secure, long-term preservation of the National Plant Germplasm System collections and designated non- National Plant Germplasm System 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 2014, the Plant and Animal Genetic Resources Preservation Unit back-up microbe collection almost tripled in size with the addition of 18,160 new microbial isolates, representing Bradyrhizobium, E.coli, Neurospora and other fungi from five donors. This brings the microbial collection up to 28,827 microbial isolates representing bacteria, yeast, fungi and viruses from 18 collections donated by public and private scientists. We also coordinate the distribution of four International Seed Federation Differential Pathogen Sets, but had no distributions in 2014. 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 vapor compared to conventional storage at -18 degrees C. In 2014, we evaluated germination and seedling root characteristics of 32 rye accessions stored at -18 degrees C and in liquid nitrogen for 25 years. Our results indicated there was no difference in percent germination, seedling vigor index, and abnormal seedlings, between the two storage methods. We are conducting further work to evaluate rice, oats, wheat, and barley, and to examine if agronomic performance is negatively impacted by storage in liquid nitrogen. Implementing the long-term storage of cereals in liquid nitrogen can reduce storage and regeneration costs and provides yet another strategy gene banks around the world can use to effectively and efficiently store important cereal diversity. 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 or buds. Efficient procedures need to be developed to effectively store propagules cryogenically and still have acceptable viability upon thawing. In 2014, we continued to develop procedures for cryopreserving dormant buds. Selected oligosaccharides were extracted from dormant buds of almond, apricot and peach, harvested at different times during the winter. We are now analyzing the sugar content to determine the best time to harvest dormant buds for long term preservation. We established optimal harvest time for the cryopreservation of a blueberry variety’s dormant buds and began looking at nine additional varieties to determine if the procedures can be broadly applied to all blueberries in the collection. Cryopreservation procedures were also established for selected accessions of pineapple and we have begun to optimize protocols so a greater number of pineapple accessions can be preserved. We found that cryoprotectants and temperature regimes increased viability of a few selected accessions of currant and tart cherry dormant buds after being stored cryogenically. We are now determining if these procedures can be broadly applied to the currant and tart cherry collections. Currently, these collections are maintained at a single location, growing as shrubs or trees outdoors, where they are vulnerable to environmental stressors. If we can successfully cryogenically store dormant buds of these crops, the collections can be safely backed up at Fort Collins at minimal cost. We also successfully cryopreserved selected accessions of potato (10 accessions) and strawberry (14 accessions) crop wild relatives, indicating that the procedures we use for domesticated species of potato and strawberries also work on their wild relatives. This will allow us to backup these important wild relatives, insuring the collection is duplicated at more than one site. 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. Exploiting crop diversity relies upon developing effective strategies to identify a few useful accessions with the trait of interest from a large diverse collection. We have been examining the use of seedling root traits as a tool to help breeders. Accessions that can cope with climate change and reduced inputs of water and fertilizers may have root system characteristics that can be exploited to increase productivity and profitability. Genetic improvement of root traits requires initial screening for root growth and structure. If we can successfully identify important traits during the seedling stage we can save a significant amount of time. Using a wild and an improved breeding line of the industrial oil crop lesquerella, which grows in arid environments, root characteristics were measured in the field to verify whether traits previously noted in seedlings were consistent on mature plants. These traits were determined to significantly vary between field-grown plants of the wild line (PI 596434) and breeding line (WCL-LO4), and most importantly, were consistent with our observations of laboratory-grown seedlings of the two lines. This suggests that breeders can evaluate seedlings in the laboratory, which will allow them to screen more seedlings in a shorter period of time, thus speeding up the lengthy breeding process. The results will be presented at the Association for the Advancement of Industrial Crops and a manuscript is being prepared. Fine tuning of root characterization methods was also initiated, and roots characterized on other species such as field pennycress (Thlaspi), guayule (Parthenium), and corn (Zea). Devising and applying new methods for evaluating seedling roots in selected crops and their wild relatives is the fourth objective of our project plan, and will help shorten the pipeline for producing cultivars with root characteristics that support production under adverse conditions.

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 and the USDA-ARS Fort Collins, CO genebank safeguards a wealth of the world’s plant and microbial diversity. Plant and Animal Genetic Resources Preservation Unit researchers grew the collection by adding 8,371 new seed collections and performing germination tests on 7,448 samples, of which 1,019 were monitor tests on samples already in storage. ARS researchers placed 170 clonal accessions in liquid nitrogen storage. ARS researchers almost tripled the size of the Agricultural Microbe collection, with the addition of 18,160 new microbial isolates. With more than 700,000 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.

2. Effectiveness of liquid nitrogen storage on rye seeds. Long term preservation of seed in liquid nitrogen is cost effective but long term studies examining the impact on seeds are rare. ARS researchers in Fort Collins, CO compared rye seed that had been kept for 25 years in conventional freezer storage and in liquid nitrogen. The results did not show difference in seedling germination and vigor. This provides assurance that cost efficient liquid nitrogen can be used to store rye seed, and suggests this method might be an effective and cost-efficient method for storing other cereals.

3. Optimizing protocols for cryopreservation success in blueberry. Until now, blueberries were cryopreserved as tissue culture; this is several times more costly than preservation of dormant buds in liquid nitrogen. The success rate for cryopreserving blueberries from dormant buds has been variable; therefore, it has been difficult to advance the cryopreservation of this important crop. Previous research suspected that temperatures and day length before bud harvest might be related to cryopreservation success. Plant and Animal Genetic Resources Preservation Unit researchers in Fort Collins, CO examined day length and temperature data and determined an optimal combination of these variables at blueberry bud harvest would ensure successful post-thaw viability of buds during the cryopreservation process. Optimizing our protocols allows us to safeguard blueberry diversity using a more cost effective procedure then was previously used, which contributes to genetic security and reduces the cost of collection, processing and storage of this crop.

4. Root growth and development in lesquerella oilseed. Lesquerella is a new oilseed crop, native to the arid regions of the US Southwest, and breeders are working to develop varieties that can be used as a source of lubricants and biodiesel additives. The breeding pipeline is lengthened when mature plants, as opposed to seedlings, need to be evaluated for desirable traits. ARS researchers at Fort Collins, CO in collaboration with staff of the University of Arizona and University of California compared root traits on seedlings grown in the laboratory to root traits on mature plants grown in the field and found they were positively correlated. Screening young seedlings will speed the process of breeding effective varieties that can support the development of a new oil seed industry in the US Southwest.

Review Publications
Alves, A.A., Manthey, L.K., Isbell, T., Ellis, D., Jenderek, M.M. 2014. Diversity in oil content and fatty acid profile in seeds of wild cassava germplasm. Industrial Crops and Products. 60:310-315.
Comas, L.H., Becker, S., Cruz, V.V., Byrne, P.F., Dierig, D.A. 2013. Root traits contributing to plant productivity under drought. Frontiers in Plant Science. 4:1-16. doi: 10.3389/fpls.2013.00442.
Kaya, E., Alves, A., Rodrigues, L., Jenderek, M.M., Hernandez-Ellis, M., Ozudigru, A., Ellis, D.D. 2013. Cryopreservation of eucalyptus genetic resources. CryoLetters. 34:608-618.
Jenderek, M.M., Holman, G.E., De Noma, J.S., Reed, B.M. 2013. Medium- and long-term storage of the Pycnanthemum (Mountain mint) germplasm collection. CryoLetters. 34:490-496.