1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter?
PGPRU is a fundamental component of the infrastructure of the National Plant Germplasm System (NPGS), with the primary responsibility of conducting research to support National Program 301 (Plant, Microbial and Insect Genetic Resources, Genomics and Genetic Improvement, Component 1 (Genetic Resource Management), section b (conserve genetic resources).

Objectives of PGPRU research are to.
1)develop analyses that assess, quantify and apportion genetic diversity so that it can be efficiently captured in germplasm that is amenable to preservation treatments,.
2)enhance survival of stored germplasm through better understanding of the mechanisms of damage and protection of cells during low temperature, desiccation and aging stresses, and.
3)improve tools that validate and predict viability, shelf life and genetic integrity through sensitive or non-invasive measures of health and genetic shifts.

Effective genebanking is predicated on the ability to efficiently capture and preserve genetic diversity. The Plant Germplasm Preservation Research Unit (PGPRU) conducts research to develop methods that measure genetic diversity and maintain the viability of diverse germplasm. The tools developed are applicable to the > 11,000 species represented in the NPGS collection. Without these tools, genetic diversity of plant species important to the US can be under-represented, over-represented, or lost entirely. Germplasm in genebanks varies widely in life history traits, reproductive biology, response to preservation stresses, and handling prior to arriving at the genebank. To manage the wide array of materials and to provide tools that are broadly applicable, PGPRU determines and examines key factors that affect distribution of genetic diversity and tolerance to stress.

In collaboration with PI Stations and Clonal Repositories, PGPRU also supports Component 1a, c and d activities (acquire, characterize and evaluate genetic resources). Research on crop domestication also supports National Program 301, Component 3 (Genome Databases). Research on plant growth and development leading to higher quality seeds and greater tolerance of low water and temperature stresses supports National Program 302 (Plant Biological and Molecular Processes (NP 302). PGPRU programs to promote conservation and seed quality of plant species native to the US supports National Program #205 (Rangeland, Pasture and Forages).

2.List by year the currently approved milestones (indicators of research progress)
Year 1 (2003) 1. Develop and validate assessments of genetic diversity and genetic change following cryopreservation using natural populations of Zizania texana, which produces recalcitrant seeds.

2. Initiate experiments to measure quantitative traits in Beta.

3. Evaluate genetic distances among accessions in NPGS garlic collection.

4. Determine relationships between phylogeny and cryopreservability among Malus species and M. domestica cultivars.

5. Establish in vitro cultures of grape, pear, rhizomatous peanut, and Jerusalem artichoke.

6. Initiate experiments to measure relationship between volatile emissions in seeds and aging kinetics.

7. Determine interaction between lipid composition, melting properties and seed storage behavior in Cuphea germplasm.

8. Complete experiments that measure optimum water content and shelf life in diverse desiccation-tolerant organisms.

9. Develop models of thermal conductivity as a function of propagule mass and water content for the eventual use in modeling heat transfer during cooling to liquid nitrogen temperatures.

10. Complete experiments correlating sugar accumulation and DTA profiles with cold hardiness in winter buds.

Year 2 (2004) 1. Develop and validate assessments of genetic diversity in natural populations of hops and initiate experiments to compare genetic erosion during seed banking and regeneration.

2. Optimize cryoprocedures for diverse garlics and transfer technology to PGPPP.

3. Quantify genetic diversity in wild-collected accessions of Malus.

4. Develop in vitro methods to recover cryopreserved buds of pear, cherry, and grape.

5. Develop protocols that measure water permeability in membranes of seed tissue and compare in bean embryos at different developmental stages.

6. Develop methods to quantify oxidative stress and antioxidant levels and apply to systems recovering from cryoexposure.

7. Evaluate cryoprotective behavior of solutions: isolate toxic component(s), evaluate water relations and biophysical properties.

8. Develop procedures for short-term storage of garlic.

9. Quantify aging rates of wheat, rye and intergeneric crosses.

10. Screen intergeneric crosses of Cuphea for sensitivity to -18C.

11. Evaluate feasibility of browning or electrolyte leakage techniques as indicators of viability in buds by comparing assays with budding success.

12. Determine temperature coefficient for aging of lettuce seed.

13. Identify NPGS accessions of Brassica to use in studies of genetic changes and linkage disequilibrium during genebanking.

Year 3 (2005) 1. Validate markers to measure DNA quality, genetic diversity, and erosion in sorghum. 2. Complete genetic assessments of ex situ collections of Beta based on neutral markers and genes that may be sensitive to selection.

3. Develop pollination strategy that minimizes loss of alleles in regenerated population of seeds using assessments of genetic diversity in wild-collected accessions of Malus. 4. Compare ultrastructure in Mentha after cryoprotection procedures.

5. Test primers for citrus giving polymorphic markers to distinguish between parents and progeny.

6. Establish procedure for somatic embryogenesis in sweet potato.

7. Complete analysis of changes in desiccation tolerance of woody buds during winter. 8. Develop state diagrams of bean embryos.

9. Analyze ultrastructural, biochemical and biophysical changes in Rubus cultures differing in acclimation potential.

10. Analyze biochemical changes associated with aging during short term (hydrated) storage of various propagules.

11. Model cooling rate based on state diagrams (viscosity) and heat transfer mechanisms.

12. Assess cryopreservability among Zizania seeds grown under greenhouse conditions.

13. Correlate viability assays developed previously with ROS levels in propagules recovering after cryoexposure.

14. Establish assays to measure mechanical properties in sorghum seeds to predict longevity.

Year 4 (2006) 1. Develop core collections of Beta using quantitative traits and genetic markers and compare genetic diversity in both based on markers.

2. Analyze cryopreservation success between shoot tips and somatic embryos of sweet potato.

3. Analyze desiccation tolerance of Zizania seeds grown under greenhouse conditions.

4. Develop state diagrams of Zizania seeds.

5. Develop optimum cooling rates for buds during 2 step cooling based on drying rates and desiccation sensitivity.

6. Correlate cold tolerance and cryopreservability in vegetative tissue.

7. Establish protocols to cryopreserve Jerusalem artichoke.

8. Establish protocols to cryopreserve grape.

9. Improve protocols to cryopreserve recalcitrant seeds.

10. List genes expressed during acclimation of Rubus.

11. Assay desiccation tolerance of Citrus and Coffea crosses.

12. Assess viability assays using expression of cell cycle genes (such as knotted-1).

13. Correlate intracellular viscosity in hydrated germplasm with survival following cryoexposure.

14. Determine extent of genetic shifts in sorghum seeds aged in NPGS collection.

15. Regenerate accessions for genetic erosion studies.

Year 5 (2007)

1. Extend pollination and seed collection strategies for wild-collected clonal crops to Pyrus, Prunus, and Vitis.

2. Develop techniques to measure DNA stability during storage.

3. Assess DNA fragmentation patterns during recovery of apical shoot tips following cryoexposure.

4. Assess long term viability (after 10 years of storage) of preserved apical shoot tips and woody buds.

5. Verify genes important to stress tolerance. This study will be conducted using Arabidopsis and a microarray approach.

6. Analyze genetic shifts in stored hops seed from natural populations and cultivated sites. This study also includes Limnathes (in collaboration with Parlier) and sage (in collaboration with Western growers).

7. Analyze shifts in quantitative traits in serial regenerations of Brassica and Secale correlate with changes in allelic frequencies. This project depends on the selection of appropriate Brassica accessions (a 2004 milestone that was delayed to 2005). Studies with rye germplasm are well underway. Milestone has been delayed because of limited personnel at NCGRP and recruitment of a new curator for oilseeds at NC-7. Brassica accessions remain the ideal genus to work in on this project and we anticipate getting this project underway after the Secale genotyping is completed.

4a.List the single most significant research accomplishment during FY 2006.
Discovery of a flower regulatory gene in Beta that functions broadly in Angiospermae

This research was conducted under National Program 301 Plant Genetic Resources, Genomics, and Genetic Improvement Action Plan, and directly contributes to research Component 1: Plant and Microbial Genetic Resource Management, ARS Strategic Plan Goal 1: Enhance Economic Opportunities for Agricultural Producers, namely 1.2.8: Maintain, characterize, and use genetic resources to optimize, safeguard, and enhance genetic diversity and promote viable and vigorous plant production systems.

Many plant species require a lengthy cold treatment in order to flower and, as induced by winter cold, is termed "vernalization." In Arabidopsis thaliana FLOWERING LOCUS C (FLC), a MADS-box gene, functions as a repressor of flowering. Using EST databases of tomato, poplar, and sugar beet, species which are distant relatives of Arabidopsis, we provide the first report of FLC homologs outside the Brassicaceae. The FLC homolog that we identified in sugar beet (named BvFL1) behaves in a manner similar to Arabidopsis FLC--acting as a repressor of flowering downregulated by cold. Vernalization response in wheat operates using a different genetic mechanism so it appears that the capacity to flower in response to cold has evolved at least two times during the diversification of the angiosperms. Based on known flowering plant phylogenetic relationships, we predict that it will be possible to find FLC-like genes in the caryophyllid and rosid lineages, collectively about half of eudicot species.

Flowering time is a trait of critical agronomic importance and considerable ecological interest. Conservation of the basic genetic mechanism underlying the vernalization response implies an opportunity to study (and control) flowering time in the caryophyllid and rosid lineages, which collectively include about half of eudicot species.

4b.List other significant research accomplishment(s), if any.
This research was conducted under National Program 301 Plant Genetic Resources, Genomics, and Genetic Improvement Action Plan, and directly contributes to research Component 1: Plant and Microbial Genetic Resource Management, ARS Strategic Plan Goal 1: Enhance Economic Opportunities for Agricultural Producers, namely 1.2.8: Maintain, characterize, and use genetic resources to optimize, safeguard, and enhance genetic diversity and promote viable and vigorous plant production systems.

Established a physiological basis for the syndrome of damage in seeds with “intermediate” storage behavior.

Developed a framework to study the feasibility of DNA banking.

Completed the "Global Experiment" commissioned by IPGRI to determine optimum moisture contents for seed storage and the interaction of water content and storage temperature.

Developed protocols to cryopreserve oak germplasm (Quercus seeds are recalcitrant).

Cryopreservation protocols for Jerusalem artichokes have been established.

Cellular effects (permeability, plasmolysis) of PVS2 as a cryoprotectant for shoot tip systems have been determined.

Diversity and representation of wild Pyrus communis at National Clonal Germplasm Repository (Corvallis) were published.

Salix cryopreservation results were confirmed for multiple species, and technology was transferred to service units.

Critical analyses of published collection methods for wild germplasm were completed.

4c.List significant activities that support special target populations.
PGPRU along with other units in NCGRP presented an overview of plant preservation research to the 2005 National Conference of SACNAS, the Society for Advancement of Chicanos and Native Americans in Science held in Denver, CO. The presentations were monitored and scored highly with the conference organizers. However, there was no follow-up by student attendees despite our efforts to encourage their engagement with us for future career options.

4d.Progress report.
None.

5.Describe the major accomplishments to date and their predicted or actual impact.
This Research Unit contributes to the accomplishment of ARS Strategic Plan Goal (Goal # 1.2.8: Maintain, characterize, and use genetic resources to optimize, safeguard, and enhance genetic diversity and promote viable and vigorous plant production systems). This research unit is responsible for investigating and conducting research to develop strategies and technologies for preserving plant genetic diversity in ex situ genebanks (ARS National Program #301: Plant, Microbial & Insect Genetic Resources, Genomics & Genetics Improvement). We have integrated preservation technology with population genetic techniques to enable systematic approaches to curatorial decisions. Some examples of successful research are.
1)development of cryopreservation techniques that are applicable to genetically diverse samples of a species (e.g., garlic, wildrice),.
2)maximization of genetic diversity with minimum sample sizes (apple, hops, garlic, wildrice),.
3)identification of gaps in genetic diversity (pear),.
4)development of analytical approaches to identify under-sampling in genetic variation, .
5)more efficient genebanking by creating seed collections from wild germplasm that has been maintained clonally. Curators now have information based on species characteristics and genetic variability to guide genebanking such as sampling strategies, preservation protocols, viability testing, development of core collections, and effort per incremental increase in genetic diversity. The scientific and agricultural communities now have information on tolerance of species to preservation stresses that can be used for mechanistic studies or crop improvement to water and low temperature stresses.

6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products?
PGPRU transfers preservation protocols to curators and seed companies on a routine basis. Genetic diversity profiles of garlic, fruit crops, hops, beet, and wildrice have been transferred to curators and land managers. Adoption of preservation protocols to backup germplasm is constrained by the labor required for producing and preparing explants for preservation. A change in genebanking culture is needed to accept that collection sizes cannot grow indiscriminately.

A thorough inventory of liquid nitrogen purchase and usage was conducted to support future analyses of cost/benefit of cryopreservation versus conventional storage and to guide emergency plans in case of national emergencies that interrupt liquid nitrogen supply. In 2005, NCGRP consumed an average of 777 liters of LN each day (PGRPP: 369 l/d, NAGP: 217 l/d, PGPRU: 118 l/d, unaccounted: 73 l/d). Most of the LN (about 62%) is used to cool cryovats and the average evaporation rate for LN from a cryovat is 9.8 l/day. As of June 2005, there were 43 cryovats online and another 17 cryovats empty. Cryovats have a 15 to 30 year projected lifespan and need to be reconditioned about every 10 years to maintain a warranty (for 10 years). About 22% of the LN is used for processing samples or research in laboratories using low pressure tanks. About 16% of the LN is consumed when pipes are cooled. LN is purchased through a DOD contract with AirLiquide for $0.84/liter. More information on LN use is available upon request.

Over the years, some historical collections, old samples or original accessions received a GRIN status of “research,“ which effectively removed them from the base collection. An accession-by-accession inventory identified these accessions as valuable genetic resources and we alerted curators of their existence. Among these accessions are 4356 accessions of genera ranging from Agropyron to Zea dating back to the 1960s, the Oaks-Leffel-Hyland collection of forage crop seeds dating from 1963-1973, 400 accessions from the B. Berrier Collection of heirloom varieties of corn, soybean, cherry, bean, etc. from Mexico, India, and Japan dating from the early 1900s, 26 accessions of guayule dating from 1955, 5 accessions of hemp and kenaf dating from 1947 and an accession of wild jojoba. Inventory assessments are underway for about 2000 accessions dating from the 1930s from the Cheyenne (WY) Experiment Station of vegetables and alpine plants expected to do well in dry climates and 160 accessions from a potato seed collection from Greeley, CO dating from the early 1960s.

PGPRU SYs devised schema to allow entry of molecular genetic data into GRIN. They developed areas, tables, and drop down menus to handle an array of genetic data. The proposed schema implementation is underway in collaboration with the DBMU. With a user-friendly interface and search engine, genetic data collected on NPGS accessions will be publicly available.

SY presented garlic genetic diversity data by invitation at Hudson Valley (New York) Garlic Festival in 2006 (attendance >50,000).

SY invited to present Malus diversity data at departmental seminars and at Rosaceae Genomics International Symposium (New Zealand) that led to the development of collaborations with European Malus community to ensure genetic marker data can be compared across labs and continents. Those collaborations established a set of international control samples and markers for Malus diversity studies with European collaborators.

7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below).
Volk, G.M. 2005. Genetically similar garlic varieties. The Garlic Press 45:7.

Anon. 2006. Warming up to Cuphea: Seeds get special treatment after cold storage. Agricultural Research p.11-13.

Review Publications
Walters, C., P. Landre, L.M. Hill, F. Corbineau and C. Bailly. 2005. Organization of lipid reserves in cotyledons of primed and aged sunflower seeds. Planta 222:397-407.