2004 Annual Report 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? What does it matter? Genetic diversity is a natural resource, essential for sustainable agricultural landscapes. Genetic diversity is being lost or is increasingly unavailable due to environmental, political and demographic factors. US agriculture can be protected and enhanced by collecting and preserving germplasm (seeds or plant parts) from agriculturally important species in ex situ genebanks. PGPRU provides tools that enable genebanks to deliver the promise of preserving genetic diversity for more than 10,000 species of plants with different geographic origins, life histories, and physiologies. Tools that quantify and apportion genetic diversity, preserve it, and evaluate genetic erosion are needed for effective and economical operation of the genebank. PGPRU provides these tools by integrating disciplines of plant growth and development, preservation and population genetics. Without this research, genebanks are prone to becoming unwieldy collections that cannot be maintained with available resources and which inevitably fail to prevent inadvertent loss of valuable genetic resources. Genebanking would be easy if all germplasm behaved the same way. However, materials in the genebank vary widely (by definition), and PGPRU's research approach is designed to accommodate this variability while developing widely applicable genebanking procedures. 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. PGPRU is a fundamental component of the infrastructure of the National Plant Germplasm System, 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). 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 the milestones (indicators of progress) from your Project Plan. Year 1 (2003) Develop and validate assessments of genetic diversity and genetic change. following cryopreservation using natural populations of Zizania texana, which produces recalcitrant seeds. Initiate experiments to measure quantitative traits in Beta. Evaluate genetic distances among accessions in NPGS garlic collection. Determine relationships between phylogeny and cryopreservability among Malus species and M. domestica cultivars. Establish in vitro cultures of grape, pear, rhizomatous peanut, Jerusalem artichoke. Initiate experiment to measure relationship between volatile emissions in seeds and aging kinetics. Determine interaction between lipid composition, melting properties and seed storage behavior in Cuphea germplasm. Complete experiments that measure optimum water contents and shelf life in diverse desiccation-tolerant organisms. Develop models of thermal conductivity as a function of propagules mass and water content for the eventual use in modeling heat transfer during cooling to liquid nitrogen temperatures. Complete experiments correlating sugar accumulation and DTA profiles with cold hardiness in winter buds. Year 2 (2004) Develop and validate assessments of genetic diversity in natural populations of hops and initiate experiments to compare genetic erosion during seed banking and regeneration. Optimize cryoprocedures for diverse garlics and transfer technology to SVSRU. Quantify genetic diversity in wild-collected accessions of Malus. Develop in vitro methods to recover cryopreserved buds of pear, cherry and grape. Develop protocols that measure water permeability in membranes of seed tissue and compare in bean embryos at different developmental stages. Develop methods to quantify oxidative stress and antioxidant levels and apply to systems recoverying from cryoexposure. Evaluate cryoprotective behavior of solutions: isolate toxic component(s), evaluate water relations and biophysical properties. Develop procedures for short-term storage of garlic. Quantify aging rates of wheat, rye and intergeneric crosses. Screen intergeneric crosses of Cuphea for sensitivity to -18C. Evaluate feasibility of browning or electrolyte leakage techniques as indicators of viability in buds by comparing assays with budding success. Determine temperature coefficient for aging of lettuce seed. Identify NPGS accessions of Brassica to use in studies of genetic changes and linkage disequilibrium during genebanking. Year 3 (2005) Validate markers to measure DNA quality, genetic diversity and erosion in sorghum. Genetic assessments of ex situ collections of Beta based on neutral markers and genes that may be sensitive to selection. Using assessments of genetic diversity in wild-collected accessions of Malus, develop pollination strategy that minimizes loss of alleles in regenerated population of seeds. Comparison of ultrastructure in mint after cryoprotection procedures. Primers for citrus giving polymorphic markers tested to distinguish between parents and progeny. Established procedure for somatic embryogenesis in sweet potato. Completed analysis of changes in desiccation tolerance of woody buds during winter. State diagrams of bean embryos developed. Analysis of ultrastructural, biochemical and biophysical changes in Rubus cultures differing in acclimation potential. Analysis of biochemical changes associated with aging during short term (hydrated) storage of various propagules. Models for cooling rate based on state diagrams (viscosity) and heat transfer mechanisms. Assessment of cryopreservability among Zizania seeds grown under greenhouse conditions. Correlate viability assays developed previously with ROS levels in propagules recovering after cryoexposure. Established assays to measure mechanical properties in sorghum seeds to predict longevity. Year 4 (2006) Develop core collections of Beta using quantitative traits and genetic markers and compare genetic diversity in both based on markers. Analysis of cryopreservation success between shoot tips and somatic embryos of sweet potato. Analysis of desiccation tolerance of Zizania seeds grown under greenhouse conditions. State diagrams of Zizania seeds. Development of optimum cooling rates for buds during 2 step cooling based on drying rates and desiccation sensitivity. Correlation of cold tolerance and cryopreservability. Established protocols to cryopreserve Jerusalem artichoke. Established protocols to cryopreserve grape. Improved protocols to cryopreserve recalcitrant seeds. List of genes expressed during acclimation of Rubus. Desiccation tolerance of Citrus and Coffea crosses. Assessment of viability assays using expression of cell cycle genes (such as knotted-1). Correlation of intracellular viscosity in hydrated germplasm with survival following cryoexposure. Determine extent of genetic shifts in sorghum seeds aged in NPGS collection. Regenerations of accessions for genetic erosion studies completed. Year 5 (2007) Pollination and seed collection strategies for wild-collected clonal crops extended to Pyrus, Prunus and Vitis. Techniques to measure DNA stability during storage developed. Assessment of DNA fragmentation patterns during recovery of apical shoot tips following cryoexposure. Assessment of slow ice formation in cryopreserved embryos (of hydrated seeds). Assessment of viability in apical shoot tips (2-4 years) and woody buds (> 10 year). Cryopreservation protocols established for sweet potato. Cryopreservation protocols established for rhizomatous peanut. Verification of genes important to stress tolerance. Analysis of genetic shifts in stored hops seed from natural populations and cultivated sites. Analysis of shifts in quantitative traits in serial regenerations of Brassica and correlation with changes in allelic frequencies. 3.Milestones: Milestones for 2004: met expectations · Develop and validate assessments of genetic diversity in natural populations of hops and initiate experiments to compare genetic erosion during seed banking and regeneration. · Optimize cryoprocedures for diverse garlics and transfer technology to SVSRU. · Quantify genetic diversity in wild-collected accessions of Malus. · Evaluate cryoprotective behavior of solutions: isolate toxic component(s), evaluate water relations and biophysical properties. · Develop procedures for short-term storage of garlic. · Quantify aging rates of wheat, rye and intergeneric crosses. · Determine temperature coefficient for aging of lettuce seed. Milestones for 2004: partially-met expectations · Develop in vitro methods to recover cryopreserved buds of pear, cherry and grape. Both untreated and cryoexposed buds show signs of viability in vitro, but growth is abnormal. Thus, the method is adequate to assess viability but must be improved to detect repairable damage imposed by cryoexposure. We are investigating whether additives to medium can stimulate normal growth or whether additional staining techniques can distinguish between healthy tissue and repairable damage. · Develop in vitro methods for recovery of shoot tips from grape materials. Grape explants (both ex vitro and in vitro) can be recovered in vitro, but growth is typically abnormal making it difficult to quantify levels of damage following cryo-treatments. We are investigating whether normal growth can be routinely obtained in control treatments using culture modifications. · Develop methods to quantify oxidative stress and antioxidant levels and apply to systems recovering from cryoexposure. Assays for measuring oxidative stress using TBA have been established for peach buds (large tissues), but need to be adapted for smaller tissues such as in vitro shoot tips. Other assays that measure ROS such as hydrogen peroxide production fluorescence or colorimetric assays will be tested by the end of 2004. A fluorimetric assay to measure total antioxidant capacity will be adapted to a multi-welled system by the end of 2004. We will look at total antioxidant capacity in recovering tissues before we develop assays for specific antioxidants. · Evaluate feasibility of browning or electrolyte leakage techniques as indicators of viability in buds by comparing assays with budding success. Browning accurately indicates dead buds. Non-browning predicts ability to grow in cold (0 to -40C) treated buds but not in cryoexposed buds. Correlations of browning with desiccation stress will be tested in apple in winter 2004/2005. The feasibility of combining browning with other viability assessments (such as electrolyte leakage) to more accurately predict growth potential needs to be developed. · Complete experiments correlating sugar accumulation and DTA profiles with cold hardiness in winter buds (2003 milestone). The test of whether sugar composition or low freezing temperatures correlates with cold hardiness in diverse germplasm (correlation is well established by other labs for buds during winter) is delayed because of the unavailability of cold tolerance data for various genotypes and species. Milestones for 2004: expectations not met · Develop protocols that measure water permeability in membranes of seed tissue and compare in bean embryos at different developmental stages. This project was designed to study critical stages during embryogenesis that affect seed storage behavior. At the time the project was planned, the PGPRU was confident that base funds would be added to support complimentary research on the molecular biology of seed quality. The funds were not added, so research efforts were redirected to evaluate storage behavior of seeds currently in the genebank. That assessment has been completed and the paper submitted to the journal. Until SY capacity in the PGPRU is expanded, studies linking seed maturation events with seed quality will be de-emphasized in favor of more general studies comparing properties among species. · Screen intergeneric crosses of Cuphea for sensitivity to -18C. The purpose of the study was to understand how lipids within seeds contribute to their storage behavior. Upon re-evaluation of the proposed project with the curator, it was decided that the effort to make intergeneric crosses of Cuphea would require too much effort for the amount of information gained. Hence, the project has been refocused from a genetic study to a biophysical one to determine how the physical behavior of the lipids affects storage behavior in seeds of diverse genera. One manuscript has been submitted. · Identify NPGS accessions of Brassica to use in studies of genetic changes and linkage disequilibrium during genebanking. This research was designed as a collaborative project between the PGPRU and the PI Station in Ames, IA. The curator for Brassica was hired in FY2004, making it prudent to delay selection of accessions for one year until she became more knowledgeable about the collection. In the meantime, the project was extended to include a grass species, rye, and a Specific Cooperative Agreement with the Polish Academy of Sciences Botanical Garden to collaborate on the project was established. For rye, the milestone is partially met since the range of genetic improvement within the collection has been ascertained, but notes on the number of regeneration cycles and parental lines used to regenerate still need to be searched. Selection of specific accessions is expected to be completed by October. The extra time gained by delaying the project allowed for greater progress on genetic assessments of Beta, and a 2005 milestone to determine genes sensitive to selection during regeneration was marked by the identification of a flowering gene that regulates biennial growth habit. B. List the milestones (from the list in Question #2) that you expect to address over the next 3 years (FY 2005, 2006, & 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? Year 3 (2005) · Validate markers to measure DNA quality, genetic diversity and erosion in sorghum. We expect to determine if DNA extracted from herbarium samples is sufficiently intact to retrieve PCR product. We expect to identify sorghum accessions in the NPGS collection ranging in heterogeneity and viability for future tests on genetic changes during seed storage. · Genetic assessments of ex situ collections of Beta based on neutral markers and genes that may be sensitive to selection. We expect to pursue characterization of the flc gene. · Using assessments of genetic diversity in wild-collected accessions of Malus, develop pollination strategy that minimizes loss of alleles in regenerated population of seeds. This milestone was met in 2004. Studies are now being applied to other genera. · Comparison of ultrastructure in mint after cryoprotection procedures. We expect to characterize level of plasmolysis and impact on plasmodesmata. · Primers for citrus giving polymorphic markers tested to distinguish between parents and progeny. This project relies on the availability of published primers. This project was designed to determine whether citrus clones could be cryopreserved using nucellar embryos rather than vegetative tissues. While still a promising approach, priority for this project has been reduced because of new budgetary constraints leading to reduced PGPRU staff and because of weakened support for preservation of citrus germplasm. · Established procedure for somatic embryogenesis in sweet potato. Initial attempts to induce somatic embryos using a published method were not successful. Modifications of the technique will include applying thidiazuron, using immature inflorescences, inducing embryogenic suspensions and screening for more amenable genotypes. · Completed analysis of changes in desiccation tolerance of woody buds during winter. 2005 marks the 3rd year of the measurements of critical water contents during the winter months in Chardonnay grapes. · Develop state diagrams of bean embryos. DSC will be used to determine melting temperature and energies of water and glass transitions temperature and infer glass relaxation rates as a function of water content. State diagrams will be completed for mature pea embryos rather than bean. · Analysis of ultrastructural, biochemical and biophysical changes in Rubus cultures differing in acclimation potential. Research on sugar composition and vitrification response as a result of acclimation will be completed. Other aspects of the project will be conducted using Arabidopsis and are delayed by one year due to the shift in model systems. · Analysis of biochemical changes associated with aging during short term (hydrated) of various propagules. Changes in sugar content, anti-oxidant activity, ROS content will be completed in trifoliate orange, grape buds and garlic cloves. · Models for cooling rate based on state diagrams (viscosity) and heat transfer mechanisms. Models for heat transfer have been developed and need to be integrated with measurements of viscosity for high moisture materials. Finalized viscosity measurements are not expected until the following year. · Assessment of cryopreservability among Zizania seeds grown under greenhouse conditions. Culture of seed-bearing Zizania plants has been established in the greenhouse. Yields are low, and the produced seed differs considerably from those produced in raceways in Texas. Study is to be redesigned to ensure greenhouse grown seed is representative for the species. · Correlate viability assays developed previously with ROS levels in propagules recovering after cryoexposure. This project cannot proceed until 2004 milestones are met: assays for ROS levels (2004 milestones) and improved viability assessments. · Established assays to measure mechanical properties in sorghum seeds to predict longevity. Training on DMA equipment was completed in 2004. In 2005, experiments will be initiated to determine if seed water content affects mechanical properties. Year 4 (2006) · Develop core collections of Beta using quantitative traits and genetic markers and compare genetic diversity in both based on markers. Analyses are on schedule and will be completed by 2006. · Analysis of cryopreservation success between shoot tips and somatic embryos of sweet potato. Success is dependent on a 2005 milestone to develop a procedure to produce somatic embryos. Meeting this milestone depends heavily on maintaining technical assistance. · Analysis of desiccation tolerance of Zizania seeds grown under greenhouse conditions. Pending satisfactory results that Zizania texana seeds are viable (see 2005), we will continue with this project to compare water relations during development of Z. texana and Z. palustris. · State diagrams of Zizania seeds. Pending satisfactory results that Zizania texana seeds are viable (see 2005), we will perform relaxation studies using DSC to estimate viability in recalcitrant seeds and compare with earlier determinations using orthodox seed. · Development of optimum cooling rates for buds during 2-step cooling based on drying rates and desiccation sensitivity. Desiccation tolerances have been partially determined for pear, sweet cherry and peach nodal sections. More detailed studies using excised buds are delayed until normal shoot growth can be obtained in vitro (a 2004 milestone). · Correlation of cold tolerance and cryopreservability. This project was originally designed for Rubus species (2005 milestone). However, we have determined that a model system with sufficient expression libraries and genomic information will hasten accomplishments in this area. Hence we have switched to Arabidopsis. Preliminary results indicate that cold acclimation is not required for survival of cryoexposed Arabidopsis, so we have switched approaches and are evaluating mutant Arabidopsis types (altered lipids, altered cold acclimation responses) to determine key components needed for survival in cryoexposed materials. · Established protocols to cryopreserve Jerusalem artichoke. In vitro cultures of J. artichoke have been established and cryopreservation attempts have been successful. A medium term cold storage experiment will be started to determine how long tissue cultured accessions can be stored in vitro at reduced temperatures. · Established protocols to cryopreserve grape. Survival following cryoexposure of shoot tips using both vitrification and encapsulation/dehydration methods is variable depending on greenhouse conditions and genotype. Further progress is dependent on better understanding of the source of experimental variability and the assays for recovery metabolism (a 2004 milestone). · Improved protocols to cryopreserve recalcitrant seeds. Using models of heat transfer and intracellular viscosity and measurements of critical water content for desiccation damage that were investigated in previous years, we will determine the moisture content parameters in Zizania that allow fast cooling without causing desiccation damage. Meeting this milestone depends heavily on maintaining technical assistance. · Identify genes expressed during acclimation of Rubus. This project was originally designed for Rubus species (see 2005 milestone), but we have switched to focus on Arabidopsis. · Desiccation tolerance of Citrus and Coffea crosses. This work was designed as a preliminary test of whether seed storage behavior was inherited, with the future intention of approaching the genetics of seed storage behavior using QTL analysis. A recent publication using interspecific crosses of Coffea measures heritability of recalcitrance, obviating the need for our study at this time. · Assessment of viability assays using expression of cell cycle genes (such as knotted-1). This study will be consolidated with the study of gene expression during acclimation that leads to greater survival during cryoexposure. Sequences for these genes will be included on the microarray and expression will be probed in surviving shoot tips. Micro-array studies were postponed 1 year to hasten progress in other areas. We believe this milestone will be met in 2007 pending current levels of technical assistance. · Correlation of intracellular viscosity in hydrated germplasm with survival following cryoexposure. These studies will test predictions made from heat transfer models and viscosity measurements. · Determine extent of genetic shifts in sorghum seeds aged in NPGS collection. Using primers verified in 2005, accessions of sorghum that vary in heterogeneity and viability during storage will be genotyped to determine extent of changes in allelic frequencies. Meeting this milestone depends heavily on maintaining technical assistance. · Regenerations of accessions for genetic erosion studies completed. Regeneration schemes of Brassica are delayed 1 year. Regeneration of rye accessions will be underway. Year 5 (2007) · Pollination and seed collection strategies for wild-collected clonal crops extended to Pyrus, Prunus and Vitis. Studies for Pyrus and Prunus are underway with Pyrus accessions mostly genotyped in 2004-5. Work with Vitis will depend on curator interest. Fewest numbers of individuals to capture 90% of allelic diversity will be determined and used to develop pollination schemes. · Techniques to measure DNA stability during storage developed. Studies depend on the ability to get PCR product from deteriorated samples. These experiments are underway currently. The feasibility of using pulsed electrophoresis to determine DNA fragment size will be tested. Meeting this milestone depends heavily on maintaining technical assistance. · Assessment of DNA fragmentation patterns during recovery of apical shoot tips following cryoexposure. Studies will depend on feasibility of using pulsed electrophoresis to determine DNA fragment size and on maintaining level of technical assistance. · Assessment of slow ice formation in cryopreserved embryos (of hydrated seeds). This study depends on successful initial survival of recalcitrant seeds (Zizania) and their consequent storage under cryogenic temperatures. Samples will be tested for whether a water melting endotherm increases in size with storage time. Similar studies can be conducted with stored buds or shoot tips if samples are available. · Assessment of viability in apical shoot tips (2-4 years) and woody buds (> 10 year). Viability after cryogenic storage will be evaluated. · Cryopreservation protocols established for sweet potato. Meeting this milestone depends heavily on ability to measure recovery following cryotreatments as well as keeping the current level of technical assistance. · Cryopreservation protocols established for rhizomatous peanut. This project has been postponed indefinitely because of the high level of bacterial contamination in microculture. PGPRU does not have the mission or resources to develop axenic cultures of germplasm. When these cultures exist, research toward this milestone will resume. · Verification of genes important to stress tolerance. This study will be conducted using Arabidopsis and a microarray approach. This milestone is heavily dependent on maintaining current levels of technical assistance. · Analysis of genetic shifts in stored hops seed from natural populations and cultivated sites. Allelic frequencies of unaged seeds collected from the wild will be compared with seedlings surviving following storage. Allelic frequencies of unaged wild seeds will also be compared with progeny of this population from a regeneration cycle. · Analysis of shifts in quantitative traits in serial regenerations of Brassica and correlation with changes in allelic frequencies. This project depends on the selection of appropriate Brassica accessions (a 2004 milestone that was delayed to 2005). 4.What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY 2004 (one per Research (OOD) Project): In collaboration with curators, PGPRU led the way towards developing a new tool to quantify genetic diversity in wild accessions. The work uses new population genetic approaches to quantify allelic diversity in wild populations. Application of this tool makes it feasible to convert accessions that are typically maintained as planted trees (costly, risky and difficult to back up) into seed accessions with minimum risk of genetic erosion. Between 25 and 30% of the NPGS clonal collection (about 7500 accessions) will benefit from this tool, which will lead to safer, more cost-effective maintenance of wild-collected accessions that are typically maintained clonally. Application of this work also enables curators attempting to evaluate germplasm to select the smallest group of individuals that represent the greatest level genetic diversity. B. Other significant accomplishment(s), if any. Storage performance of seeds at NCGRP was quantified for the first time. Description of the average time for seeds to remain viable for about 280 species provide the first concrete data on which to base regeneration and monitoring frequencies of seed banked materials and comparisons of intraspecific variation of seed shelf life. In addition to providing critical information necessary to any genebanking operation, these analyses also led to suggestions for improvements in NCGRP procedures and additional GRIN fields to record harvest date and anomalies in harvest and postharvest treatments. Studies of genes that are susceptible to selection during genebanking led to the discovery a flowering transcription factor flc in Beta. This gene, which shares sequence homology with one reported in Arabidopsis, appears to regulate annual/biennial growth habit. Studies of the variation of flc alleles can contribute to better understanding of how genetic diversity is structured in natural populations and how it changes in captive populations. C. Significant activities that support special target populations. None D. Progress Report opportunity to submit additional programmatic information to your Area Office and NPS (optional for all in-house ("D") projects and the projects listed in Appendix A; mandatory for all other subordinate projects). None. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. Population genetic approaches that quantify and apportion genetic diversity are new to NPGS and have had immediate impact. The first application showed key points of the genebanking process for wild species that pose risks to genetic erosion (acquisition, field collections, preservation). Later applications, which incorporated preservation research of seed and clonal materials, have allowed researchers to select accessions that provide the broadest range of genetic diversity for evaluation or preservation purposes. Future analyses will enable us to increase the accountability of genebanking procedures in maintaining genetic integrity and provide a prioritizing system for maintaining accessions. Documentation of germplasm longevity under cryogenic conditions has profound impacts on the understanding of cryogenics and cell biology in many disciplines. The finding that cryogenic temperatures do not limit aging reactions to the extent once believed has immediate impacts on the future of the seed cryogenic program at NCGRP and could save hundreds of thousands of dollars in operating expenses each year. 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 transferred information on genetic diversity of garlic, fruit crops and hops germplasm to curators and user groups. PGPRU transferred cryopreservation protocols for garlic to the SVSRU. PGPRU transferred seed performance analyses of conventional and cryogenic storage to SVSRU and NPS. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. Cover article for July issue of Journal of American Society of Horticultural Science, vol 129. Channel 9 News Denver (KUSA) "Where in the World is Greg Moss" October 2003. Cross K, G. Volk and W. Lyons. 2004. Spring planting of garlic. The Garlic Press 43:17. Cross K, G. Volk and W. Lyons. 2004. Fresh garlic in the spring? Cold storage may be the answer. The Garlic Press 43:16. Walters, C. 2004. Principles for Preserving Germplasm in Genebanks. pp.113-138. In E. Guerrant, K. Havens and M. Maunder (eds) Ex Situ Plant Conservation: Supporting species survival in the wild. Island Press, Covela, CA. Walters, C. 2004. Guidelines for Seed Storage. pp.442-453. In E. Guerrant, K. Havens and M. Maunder (eds) Ex Situ Plant Conservation: Supporting species survival in the wild. Island Press, Covela, CA. Review Publications Ashfield, T., A. Bocian, D. Held, A.D. Henk, L.F. Marek, D. Danesh, S. Penuela, K. Meksem, D.A. Lightfoot, N.D. Young, R.C. Shoemaker and R.W. Innes. 2003. Genetic and physical localization of the soybean Rpg1-b disease resistance gene reveals a complex locus containing several tightly linked families of NBS-LRR genes. Molecular Plant-Microbe Interactions 16:817-826. Reeves, P.A. and R. Olmstead. 2003. Evolution of the TCP gene family in Asteridae: cladistic and network approaches to understanding regulatory gene family diversification and its impact on morphological evolution. Molecular Biology and Evolution 20:1997-2009. Volk G.M., L.J. Goss, V.R. Franceschi. 2004. Calcium channels are involved in calcium oxalate crystal formation in specialized cells of Pistia stratiotes L. Annals of Botany 93:741-753. Wheeler, R., C. Kenneth, G.M. Volk, C. Mackowiak, N.C. Yorio, and J.C. Sager. Soybean canopy gas exchange rates: Effects of lighting. Ecological Engineering 16(3):209-214. Volk, G.M., A.D. Henk and C.M. Richards. 2004. Genetic Diversity among US garlic clones as detected using AFLP methods. Journal of the American Society for Horticultural Science 129(4):559-569. Volk G.M., N. Maness, and K. Rotindo. 2004. Crypreservation of garlic (Allium sativum L.) using plant vitrification solution 2. CryoLetters 25:219-226. Volk G.M., K. Rotindo and W. Lyons. 2004. Low-temperature storage of garlic for spring planting. HortScience (39)3:571-573. Walters, C. 2003 Optimizing seed banking procedures. 36:723-743. In R.D. Smith, J.B. Dickic, S.H. Linington, H.W. Pritchard and R.J. Probert (eds) Seed Conservation: turning science into practice. The Royal Botanic Gardens, Kew, London. Walters, C. 2004. Temperature-dependency of molecular mobility in preserved seeds. Biophysical Journal 86:1253-1258. Walters C., L.J. Wheeler and P.C. Stanwood. 2004. Longevity of cryogenically-stored seeds. Cryobiology 48:229-244. Richards, C.M., M. Brownson, S. Mitchell, S. Kresovich and L. Panella. 2004. Polymorphic microsatellite markers for inferring diversity in wild and domesticated sugar beet (Beta vulgaris). Molecular Ecology Notes 4:243-245. Towill, L.E., Bonnart, R.M. 2003. Cracking in a vitrification solution during cooling or warming does not effect growth of cryopreserved mint shoot tips. CryoLetters. 24:341-346. Towill, L.E., Widrlechner, M.P. 2004. Cryopreservation of salix species using sections from winter vegetative scions. CryoLetters 25:71-80. Towill, L.E. 2004. Pollen storage as a conservation tool. pp.180-188. In E. Guerrent, K. Havens and M. Maunder (eds) Ex Situ Plant Conservation: Supporting species survival in the wild. Island Press, Covela, CA.