The following report summarizes research activities by the scientists in the Plant Germplasm Preservation Research Unit (PGPRU) at NSSL. Each project is briefly summarized and includes personnel assigned, the problem area, approach, and results for the past year. Following the reports is a list of publications appearing in print in 1998-99.
 

PERSONNEL: In March 1999, Dr. Eric Roos resigned as Research Leader of the PGPRU to become Assistant Area Director (NPA). He had been serving as Acting Assistant Area Director since June 1997. Dr. Christina Walters replaced Dr. Philip Stanwood as Acting Research Leader in October 1998, and she has recently been selected as the Research Leader. In November 1998, Ms. Jennifer Crane joined the research staff in a new Research Support Scientist position. Ms. Crane is initiating a pilot program to cryopreserve embryos of temperate trees and tropical fruit crops. Dr. Darren Touchell continues as a Postdoctoral Research Associate studying genetic conservation of wild and cultivated populations of the endangered species Zizania texana. A new Plant Physiologist/Molecular Biologist position was announced in February 1999. Research in this position will focus on enhancing our ability to cryopreserve vegetative tissues by determining environmental and developmental switches that enhance plant cell tolerance of desiccation and freezing stresses. Selection of the candidate will be made by July 1999.
 

VISITING SCIENTISTS: Ms. Paula Power (US Fish & Wildlife-Texas) visited for two weeks in July 1998 to study embryogenesis and desiccation tolerance of Texas wild rice, Zizania texana. Mr. James Wesley-Smith (University of Natal, Durban So. Africa) is on a year's sabbatical (beginning August 1998) to develop technology for ultra-rapid cooling rates. Mr. David Merrick (Kings Park Botanical Garden, Perth Australia) studied the physiology and water status of seeds from native Australian species at the NSSL in October 1998. Mr. Alvin Yoshinaga (Lyon Arboretum, Honolulu) visited for one week in February 1999 to discuss collaborative work on the physiology and storage behavior of seeds from endangered Hawaiian flora. Ms. Mirian Eira (EMBRAPA/CENARGEN Brasil) finished her doctoral research on the storage behavior of Coffea spp. seeds and will be defending her thesis in July 1999 in Brasilia. We received the usual large number of visitors from all over the world for periods of 1 to 2 days.
 

GRADUATE STUDENTS: Mr. Jian Fang, from the Peoples Republic of China, continues his doctoral thesis research on damage to seeds during ultra-dry storage (advisor: Dr. Roos). Ms. Kim Davidson continues her doctoral thesis research on the loss of desiccation tolerance during seed germination (advisor: Dr. Walters). Ms. Terri Christensen is writing her dissertation for a M.Sc. degree on the germination results of seeds from native California species stored for 50 years (advisor: Dr. Roos). Mr. Robert Cook is writing his dissertation for a M.Sc. degree on the effect of lipid composition on aging rates of soybean seeds (with Dr. Walters).
 

TECHNOLOGY TRANSFER: The research staff of the PGPRU routinely provides advice on the storage behavior of seeds, procedures to maximize seed longevity, cost-effective methods to process seeds for storage, and methods to germinate seeds. The PGPRU was represented at the annual meetings of the Regional Technical Advisory Committees, Plant Germplasm Operations Committee, American Society of Plant Physiologists, and American Society for Horticultural Science in 1998. Also this past year, research was presented at a Gordon Conference (Temperature Stress in Plants held 2/99) and international meetings in New Zealand (3/98), France (6/98), Japan (10/98), Malaysia (10/98), Mexico (1/99), and Denmark (3/99). Dr. Walters continues to consult with several groups (Leech Lake Reservation, US Fish & Wildlife, botanical gardens, and commercial growers) to conserve species endemic to the US in ex situ collections. She continues to serve on the Science Advisory Board of the Center for Plant Conservation. In cooperation with the International Plant Genetic Resources Institute (IPGRI, Rome, Italy), Dr. Walters worked to establish recommendations for seed genebanks in developing countries. Dr. Towill spent a month at the New Zealand Institute for Crops and Food to exchange information on cryopreservation techniques of plants grown in microculture. Dr. Walters spent a month at the Universite de Pierre et Marie Curie, Paris exchanging information on calorimetric techniques. Dr. Stanwood has now added about 4500 images to the GRIN database. PGPRU personnel received numerous inquiries from small companies preparing seed stuffs as a precaution against a possible Y2K disaster.

A complete list of publications, excluding abstracts, from the National Seed Storage Laboratory dating from 1960 is available for distribution. Copies of papers can be requested from: Ms. Peggy Matti, National Seed Storage Laboratory, 1111 South Mason Street, Fort Collins, CO 80521-4500. We have set up a World Wide Web page on the INTERNET that can be accessed via the following: http://www.ars-grin.gov/nssl/nsslmain.htmlThe list of publications is also available through this web site.
 

FOREIGN TRAVEL: In January 1998, Dr Walters met in Reading, UK with colleagues from the University of Reading and from IPGRI to develop recommendations for seed storage in gene banks in developing countries. Dr. Towill was invited to spend six weeks at the New Zealand Institute for Crop and Food Research, Christchurch, New Zealand in February/March 1998. Dr. Walters spent six weeks in France in May/June 1998: 5 weeks at the Université de Pierre et Marie Curie, Paris as an invited professor to collaborate on calorimetric properties of seeds and one week visiting the germplasm repositories in Montpellier, France. In October 1998, Dr. Walters was invited to present a keynote address at a meeting sponsored by the International Union of Forestry Research Organizations in Malaysia. Also that month, Drs. Towill (declined) and Touchell were invited to present papers at a meeting held in Japan sponsored by Japan International Research Center for Agricultural Sciences and IPGRI. In January 1999, Dr. Touchell, Mr. Wesley-Smith, and Ms. Eira presented results of NSSL research at the VI International Workshop on Seeds held in Mexico. In March 1999, Dr. Walters was invited to participate in a Danish-sponsored symposium on seeds in Copenhagen. Dr. Walters plans to travel to Brazil to attend the thesis defense of Ms. Eira and to present a paper at the annual meetings of the Brazilian Plant Physiology Society.
 
 

CHRISTINA WALTERS (Plant Physiol), Jennifer Crane (Research Support Sci), Lisa Hill (Biol Sci Tech), N. Kameswara Rao (ICRISAT, India), Hu Xiaorong (ICGR, CAAS, China), Jan Engels (IPGRI, Rome), Julia Buitink (Agricultural U, the Netherlands), Mirian Eira (EMBRAPA, Brazil)
 

PROBLEM: Germplasm must be stored at precise water contents to maximize longevity. These water contents vary among species and with storage temperature. Clearly, there are insufficient resources to determine the optimum water content for all species represented in the NPGS. We are using thermodynamic principles as a tool to predict optimum water contents. This research is funded in part by the International Plant Genetic Resources Institute (IPGRI), Rome.
 

APPROACH: We have studied phylogenetically diverse organisms, pollen, and more than 30 species of seeds to determine the interaction of water content, temperature, relative humidity, and deterioration rates. Water properties in cells as a function of water content and temperature were also measured using sorption isotherms or differential scanning calorimetry. These thermodynamic properties are correlated with the optimum water content. Aging experiments are long-term, some with more than nine years of storage data.
 

RESULTS: We have identified two classes of desiccation tolerant organisms, and the optimum water content for storage varies among the classes. Many organisms survive the short-term effects of complete dehydration (examples are many crop seeds, Artemia cysts, some microflora). The optimum water contents for storage of these extremely desiccation tolerant organisms correspond to about 15-22% RH, regardless of the temperature, species, or tissue. A second class of organisms (seeds with this physiology are often called "intermediate") survive drying to as low as 20% RH, but the optimum moisture level for storage is about 55%. Above and below the optimum humidity, organisms deteriorate faster. This means that there is a limit to the beneficial effect of drying seeds, and once the water content is optimized, reducing the temperature is the only way of prolonging seed storage life. Drying protocols can be easily established to obtain optimum storage conditions for any storage temperature used. This research has enabled us to predict the best moisture content for seed storage and to expedite the preparation of seeds for storage. Storing seeds under optimum conditions will ultimately limit the frequency that samples need to be monitored and regenerated. These conclusions were published in a letter to NATURE (September 1998). In addition, Dr. Walters was guest editor of a special issue of SEED SCIENCE RESEARCH (September 1998) which summarizes the current knowledge of seed storage practices.
 
 

CHRISTINA WALTERS (Pl Physiol), Lisa Hill (Biol Sci Tech), Ming Zhang (post-doctoral research associate, China), Julia Buitink (Agricultural U, the Netherlands), Mirian Eira (EMBRAPA, Brazil), Robert Cook (M.Sc. student, CSU)
 

PROBLEM: Organisms that survive drying can be placed in "suspended animation" and remain viable for a long time. However, all organisms eventually die. The inevitable loss of viability presents a problem for genebanks since it necessitates monitoring germplasm and periodically regenerating it. Genebank operators need to predict which samples are more susceptible to deterioration and to know how to prevent deterioration. To address these needs, we must elucidate the mechanism(s) of deterioration during storage and the precise relationships among the kinetics of aging, the temperature and relative humidity of storage, and intrinsic properties of cells.
 

APPROACH: Thermodynamic properties of water in seeds are measured since they appear to correlate with the nature and kinetics of deterioration reactions. We are currently trying to evaluate the use of volatile emission from seeds as a non-destructive assay of seed aging rates. Once reliable estimates of aging rates under a variety of storage conditions are known, we can produce predictive models for storage longevity and begin studies of the environmental and genetic components of seed quality that affect seed aging rates. Ultimately, this will enable us to find the underlying chemical or physical properties of seeds that give rise to the quality factors.
 

RESULTS: We have developed phase diagrams for seeds based on calorimetric measurements of water. We have established that the optimum moisture content for seed storage corresponds to a change in the heat capacity of water, but not to so-called glass transitions. At water contents below the optimum, lipid peroxidation reactions appear to dominate, and the kinetics of these are presently being described. At water contents above the optimum, reactions involve glycolysis and unregulated respiration. The effect of temperature on these reactions can be described predominantly by Arrhenius behavior with apparent activation energy similar among tissue types. This information gives us powerful tools to predict aging rates for different seed species and lots.
 
 

CHRISTINA WALTERS (Pl Physiol), J. Crane (Biol Sci Tech), L. Hill (Biol Sci Tech), K. Davidson (Ph.D. student, CSU), P. Berjak (U of Natal, So. Africa), N.W. Pammenter (U of Natal, So. Africa) J. Farrant (U of Cape Town, So. Africa), F. Corbineau (U P.M. Curie, Paris), D. Come (U P.M. Curie, Paris), C. Bailley (U P.M. Curie, Paris), J. Wesley-Smith (U of Natal, So. Africa)
 

PROBLEM: Embryos acquire, to varying degrees, the ability to survive the immediate (desiccation damage) and long-term (aging damage) effects of desiccation during development. They lose this ability when they germinate. Many economically-important species from tropical areas produce seeds that have limited abilities to survive drying and storage. Our task is to understand the basis of the limitations, first at a physiological level and then at a genetic level. We believe that this information will allow us to successfully cryopreserve all embryos--somatic or zygotic--and perhaps to artificially enhance the desiccation tolerance of other plant propagules that we wish to cryopreserve.
 

APPROACH: We have started to "map" out the developmental changes in seeds of diverse phylogenetic backgrounds on a biophysical, chemical, and ultrastructural basis and to determine which changes lead to greater tolerance of desiccation.
 

RESULTS: We have shown that as embryos mature there is almost a continuous decrease in the critical water content at which desiccation damage occurs. This has led to the idea that desiccation tolerance is a purely quantitative feature. However, we have shown that there are only a few critical water potentials (-1, -3.5, -12, -50 MPa), and developing embryos approach these in discrete steps. We believe this is an important finding as it allows us to search for developmental switches and the genetic basis of desiccation tolerance.
 
 

CHRISTINA WALTERS (Pl Physiol), J. Wesley-Smith (U of Natal, So. Africa)
 

PROBLEM: Developing embryos and fully mature embryos of some species are not tolerant of desiccation and must be preserved in the hydrated state. Cryopreservation in liquid nitrogen is the only alternative. To prevent ice crystals during cooling to liquid nitrogen temperatures, cryobiologists either add cryoprotectants (or exploit the natural cryoprotectants present in cells) or cool extremely rapidly to prevent the formation and growth of ice crystals. Because most of the propagules we work with are large (>500,000 cells), they cannot be cooled using methods developed for other organisms.
 

APPROACH: Our goal is to develop technology to cool larger propagules (500,000 cells) sufficiently rapidly to prevent lethal ice damage. There are several factors that determine the necessary cooling rate: the thermal mass of the propagule, the intrinsic level of cryoprotectants in the cell, the level of cell differentiation, and how large ice crystals can be without causing damage. We will consider all of these factors in a multidisciplinary study using electron microscopy, differential scanning calorimetry, seed physiology, and tissue culture. Cooling rates are controlled by a spring loaded-plunging device that shoots propagules at different speeds into various depths of different cryogens.
 

RESULTS: Cooling rates of up to 1000⁰C/sec have been achieved. Embryonic axes that are slightly dried have a larger window of allowable cooling rates, and close to 100% survival has been achieved for numerous species. Embryos that are fully hydrated have a small window of allowable cooling rates. We have shown that ice crystals can form in cells with no apparent damage and are currently evaluating the size of those crystals.
 
 

PHILLIP C. STANWOOD, (Res Agron); Lana Wheeler, (Biol Sci Tech)
 

PROBLEM: Seeds are used as the primary means of preserving plant diversity for future generations. From a practical point of view, seed moisture content and storage temperature are the two primary factors that one can modify to lengthen the time that seeds can be preserved. The use of ultra-cold temperatures for storage, cryopreservation (LN_2, -196⁰C), has been suggested as a means of greatly extending the storage life of seeds and other biological materials. Short-term studies (< two years) have demonstrated the efficacy of seed cryopreservation on more than 130 species. However, longer-term responses are needed to evaluate the full potential of this technology. A significant problem is how one evaluates and monitors the deterioration of seeds over time. Seed germination has been, and is currently, used as the evaluation technique. There are certain limitations to this technique. Early detection of deterioration before loss of germination would be highly desirable, reducing the likelihood of loss of genetic diversity from reduced seed viability.
 

APPROACH: Current research is directed at: 1) developing technologies and understanding principles of cryopreservation of seed and pollen using liquid nitrogen (LN₂, -196⁰C) as a storage medium; 2) developing and using digital imaging to measure vigor (deterioration) of seed germplasm; and 3) evaluating the concept of image oriented databases as a means of data archiving and distribution.

A robotic system based on digital image analysis is being developed to simultaneously conduct 100 seedling root-growth vigor tests. The Slant Growth Robotic (SGR2) system is temperature controlled and continuously slewing, which provides a similar microenvironment to each sample being tested. The slewing activity is critical for a significant reduction of experimental variation. This reduced error allows for more reliable results, use of a smaller number of seeds per test, reduced labor and material cost, and enhanced evaluation of the seed germplasm. The output of the SGR2 system is a series of time course root-growth curves for individual germinating seeds. From these curves, analyses can be done to determine the relative vigor (deterioration) of a sample. Reduction of vigor precedes loss of seed germination; thus, identification of vigor loss provides an extremely sensitive and valuable tool in accessing the storability of a sample and thus expected longevity. This information greatly enhances the management of genetic resources, ultimately improving the preservation of the material while reducing costs and labor inputs.

A digital image oriented database concept is being investigated to enhance the collection, storage, and dissemination of information concerning our preserved plant genetic resources. Chickpea, lettuce, and sugar beet image sets are being used as test species for this part of the project. Information from these studies is consolidated and provided on photo CD-ROM. Information and images from these image databases are also placed on the USDA-ARS, Germplasm Resources Information Network (GRIN) in Beltsville, Maryland and are available through the Internet and World Wide Web (www.ars-grin.gov).

Cryopreservation of plant genetic resources using liquid nitrogen (LN₂, -196^oC, -320^oF), offers the opportunity to enhance the longevity and quality of stored materials such as seeds, embryos, cell suspension, pollen, and vegetative buds. This technique can also improve the reliability of the storage system and reduce costs and other resources needed per sample. Seeds from more than 130 species have been successfully exposed and stored in liquid nitrogen. Long-term preservation studies are underway to determine the practical and biological feasibility of the cryopreservation technique for seed germplasm.

RESULTS: Much of the effort in 1998 was directed at the development of the SGR2 vigor evaluation system. Several mechanical design problems in the robotic slewing system were corrected. A new "structured" light fiber optic device was installed on the SGR2. This greatly improved the distribution of light over the test area, which is needed by the digital imaging analysis software. New software routines were developed to address root cross-over detection problems. Several new species have been successfully grown on the SGR2 including bromegrass, timothy, red clover, alfalfa, orchard grass, and sorghum seed. A large experiment (150,000 images) was conducted on lettuce and onion seed to evaluate root-growth-rate standard error vs. number of seedlings.

Seed germination testing was started for 30 species stored for 20 years at 5⁰C, -18⁰C and -196⁰C (in liquid nitrogen). Preliminary results indicate that highest deterioration is occurring at 5⁰C (as expected). There are a few species where loss of germination is occurring at -18⁰C; however, most species do not show a loss of germination at this temperature. Most species stored at -196⁰C have shown no significant loss of germination. Upon completion of the germination testing, we will start to evaluate the seed materials for loss of vigor. This will give us a more sensitive measure of seed deterioration. Ten-year storage studies on lettuce and onion seed indicated no loss of germination at -18 and -196⁰C storage; however, there were significant reductions in seed vigor for seed stored at -18⁰C compared to -196⁰C. The 20-year result will further clarify seed deterioration differences at the three storage temperatures.

Seed images can be evaluated to determine variations in seed size, shape, and color. Characterization of seed color has been problematic in that 16.7 million color variations are possible in most models. The use of this number of color variations in describing seed color is impractical. A modified color model using luminescence as a basis was developed for seed. Thirty-six "basic" color (luminescence) standards were selected with appropriate standard errors. Using this model, seed color from 505 chickpea genetic selections were characterized.
 
 

LEIGH E. TOWILL (Pl. Physiol.), John W. Waddell (Biol. Sci. Techn), Philip Forsline and Warren Lamboy (National Clonal Germplasm Repository, Geneva, NY), L.J. Grauke (National Clonal Germplasm Repository, Brownwood, TX), Charles Simon (National Clonal Germplasm Repository, Davis, CA.
 

PROBLEM: Long-term preservation of species that are vegetatively propagated is needed to avoid potential loss of germplasm and is a priority area for NPGS. Cryopreservation allows for safe, long-term storage which then gives clonal repositories options for minimizing costs with field or greenhouse maintenance. Apple (Malus spp.) was the first clonal species to be routinely placed into cryogenic storage at NSSL using dormant vegetative buds and the so-called two-step cooling method. Other cold hardy, woody species were also shown to survive such a method, but often in lower percentages. Certain aspects of the method still need examination. What alternatives can be used with particularly cold-tender lines? Some of these studies are ongoing and are summations from 2-3 years of work.
 

APPROACH: Several parameters of the dormant vegetative bud method for cryopreservation are expected to be species specific. Grape lines from the Davis, CA repository were determined to be a priority to the NPGS and were examined in winter 1998-9. Cut nodal sections and isolated, whole buds were examined using modifications of a dormant, vegetative bud method. Initial studies for the eventual cryopreservation of pecan were begun. Hardiness levels of three lines of pecan were determined at three dates during the dormant season using an oxidative browning test for samples slowly cooled to subzero temperatures. Differential thermal analysis was used to determine the freezing temperature in buds which may correlate with relative extent of cold hardiness among 100 pecan lines from the Brownwood/Somerville TX locations.
 

RESULTS: Hardiness levels were determined by cooling nodal sections from three lines of grapes harvested from Davis, CA to -30⁰C. Buds survived amongst the three lines to about -18 to -21⁰C. Cambial and wood parenchyma hardiness was greater than bud hardiness. Storage at -3⁰C for several weeks increased bud hardiness about 3⁰C. Buds removed from desiccating nodal sections (ca 12 mm in length) were about 2-5% lower in moisture content (FW basis) than that of the stem. Differential thermal analysis (DTA) using both thermocouples and thermoelectric modules showed that undried buds exhibited low temperature exotherms in the range of -15 to -25⁰C. This confirms that grape vegetative buds supercool and do not freeze-dehydrate extensively during slow cooling. Desiccation below about 24% moisture for the whole nodal section was damaging to the bud, but the large and variable diameters of the canes and somewhat lower bud moisture content from these sections precluded a precise determination. Samples of undried or partially desiccated (ca. 20-24%) whole nodal sections from Davis materials were cooled to LN vapor from either -3, -18, -21, -24⁰C. Survival after ca. -160⁰C treatment was very low. Because retrieval of cryo-treated samples will probably be via culture, isolated bud complexes were used for tests. Desiccation alone (ca. 20% moisture content, FW basis) gave some survival from hardier hybrid lines (from Fort Collins, CO). Exposure to sugar followed by desiccation improved survival after low temperature exposure in lines from Davis, CA.

Three pecan lines from Texas, selected to represent tender, moderate, and cold hardy lines, could be distinguished using a slow cooling procedure with nodal sections and an oxidative browning viability assay. DTAs of 100 lines from Texas showed distinct low temperature exotherms, probably due to supercooling of xylem ray parenchyma. The position of the exotherm and its pattern are being used to classify cold hardiness of the 100 lines. Correlations with field observations of hardiness will then be done. Similar studies are planned for the winter of 1999-2000. This information will be useful in determining which cryopreservation procedure might be most successful in obtaining survival after LN exposure.
 
 

LEIGH E. TOWILL (Pl Physiol)
 

PROBLEM: Vitrification, a method to cryopreserve diverse cells, tissues, and organs, has been shown by us and others to be effective for a range of species. This still is a relatively new method and is, as yet, not used as a routine method for cryopreservation. Vitrification is a process containing a series of steps which must be optimized. Modifications of the vitrification process usually need to be explored to develop an efficient, effective procedure. When incorporated, cryopreservation will allow clonal repositories options for cost savings in managing their field, greenhouse, or in vitro collections. We continue to investigate aspects of vitrification for several species.
 

APPROACH: Most studies used axillary buds excised from plants maintained in vitro. Such systems are axenic and minimize contamination when the treated axillary buds are cultured to produce the shoots. Both in vitro stock plants and the buds isolated from them may be treated prior to cryopreservation. Both liquid-based and encapsulation-based vitrification procedures are being examined. Most studies have been with sweet potato, given our previous work and observations of considerable variation in survival after cryopreservation.
 

RESULTS: Personnel changes during the year delayed progress in approaching some studies. Crop priorities were reevaluated for materials that would use in vitro culture, either as in vitro stock plants and in vitro retrieval or greenhouse/field plants with in vitro culture. Sweet potato (Ipomoea spp.), potato (Solanum spp.), grape, garlic and pineapple will be emphasized. Culture systems are being tested to provide better stock materials for shoot tip isolation.
 
 

LEIGH E. TOWILL (Pl Physiol), Hyla Schreurs (federal, temporary appointment)
 

PROBLEM: The cryopreservation of clonally propagated lines provides a backup should stock plants be lost. The mint industry in the US is based on a few lines with known production potential and oil quantity/quality. The development of a backup system for the foundation stock program would be beneficial should loss occur. This backup is desired for micropropagated (test tube) and greenhouse stocks. The greenhouse stock presents challenges because of endogenous bacteria. The desire is to maintain the endobionts and not alter the stock plants since it may be argued that the presence of these endogenous bacteria contribute to the field performance characteristics. This project is partially funded by the Mint Industry Research Council.
 

APPROACH: Initial tests will determine whether we can use an antibiotic to suppress external growth of microbes from shoot tips or small nodal sections when samples are cultured on normal culture media. We will examine more recent methods of cryopreservation using PVS2 as a vitrification solution and glycerol preculture. Mint plants readily over-winter if acclimated, and we will examine whether acclimation can be induced in in vitro plants for achieving higher levels of survival after cryogenic treatment. We will also examine aspects of rate of cooling and warming on survival.
 

RESULTS: Antibiotic tests have centered on the use of PPM, but have been disappointing since levels that suppress (but not eliminate the bacteria) also retard growth. Several modifications of vitrification procedures have been tested. Preculture with 2M glycerol and 0.4M sucrose for 1-3 hours was beneficial. With this system, rapid cooling with shoot tips on foil strips gave better survival than cooling within vials. Several methods seem to give approximately similar levels of survival.
 
 

LEIGH E. TOWILL (Pl. Physiol.), H.G. Hughes (Prof. of Horticulture, Colorado State Univ.)
 

PROBLEM: Bermudagrass, zoysiagrass, saltgrass, and buffalograss are clonally propagated and methods are desired to avoid loss of selected species. Cryopreservation offers the potential for backup but no studies have utilized shoot tips from grass lines.
 

APPROACH: First we will initiate microbe free lines of each species and then develop a system of micropropagation to provide plant material for cryopreservation. This research is funded by the US Golf Association Turfgrass and Environmental Research Program. H.G. Hughes, Colorado State is responsible for the culture aspects and L.E. Towill for the cryopreservation aspects.
 

RESULTS: Plants free from microbes have been isolated from all four lines. These have been rather slow in growth and proliferation has only occurred in buffalograss and saltgrass. As an initial step in cryopreservation, alternative viability tests (tetrazolium salts, FDA) to regrowth are being examined from greenhouse grown materials. Preliminary tests suggest that tetrazolium should provide a semiquantitative estimate of bud survival and should allow use of buds from greenhouse plants to define some aspects of cryopreservation protocols until in vitro plant materials are available.
 
 

JENNIFER CRANE (Pl Physiol), C. Walters (Pl Physiol), L. Hill (Biol Res Tech), R. Freeman (student), R. Krueger (ARS, Riverside, CA), F. Zee (ARS, Hilo, HI), J. Perscell (Leech Lake Tribal Council, MN)
 

PROBLEM: The physiology of seeds dictates the most appropriate storage strategy. Some species produce seeds that are non-orthodox, meaning that they do not survive desiccation and so can not be stored dry. Many tropical fruit crops, tree species, and plants growing in threatened habitats produce seeds of this category. The physiology of seeds from vegetatively-propagated crops, wild relatives of commercially produced crops, or plants growing in threatened habitats may be unknown and so appropriate storage conditions are also unknown. In order to preserve the genetic diversity of these valuable resources, the storage physiology of the seeds must be established and appropriate storage protocols determined. In addition, appropriate culture methods need to be established for recovery of these materials following storage.

APPROACH: The storage physiology of seeds is defined in terms of a mature seed's tolerance to freezing and desiccation stress. With this information, the appropriate storage procedure become apparent: seeds that appear orthodox can be stored using conventional procedures (optimizing water content and storing at -18⁰C), and seeds that are non-orthodox must be cryopreserved in liquid nitrogen. Experiments consist of measuring the viability of seeds and excised embryonic axes following exposure to a range of moisture contents and low temperatures. Water sorption isotherms are used to predict the optimum water content for storage at subzero temperatures. Freezing characteristics of water in the seeds are measured using differential scanning calorimetry and provide insights into the feasibility of cryopreserving a species for which there is no published information.
 

Using rapid cooling procedures, embryonic axes of several species of non-orthodox seeds survive exposure to liquid nitrogen. Thus, cryopreservation protocols only require fine-tuning to maximize survival and increase handling efficiency. Efforts are now underway to monitor survival in liquid nitrogen in a pilot project using species of Rutaceae (citrus family), papaya, macadamia, and wild rice.
 

RESULTS: This research program officially began in November 1998. Survival of embryonic axes exposed to liquid nitrogen range from 70 to 100%. Next year's harvest of seeds will be used to initiate the pilot project. Curators of plants that produce seeds with non-orthodox or unknown physiologies are encouraged to contact

Ms. Crane (jcrane@lamar.colostate.edu) or Dr. Walters (chrisv@lamar.colostate.edu)
 
 

DARREN TOUCHELL (post doctoral research associate), Christina Walters (Pl Physiol), M Antolin (CSU), J. Wesley-Smith (U of Natal, So Africa), Paula Power (US Fish and Wildlife Texas), Kathryn Kennedy (US Fish and Wildlife Texas).
 

PROBLEM: Zizania texana is a critically endangered wild rice species found only in a four km stretch of the upper San Marcos river in southern Texas. It is closely related to the more common and often cultivated wild rice species, Zizania palustris, and as such may have agronomic importance. Ex situ germplasm strategies should facilitate conservation of the genetic diversity of this species. However, Zizania texana does not often produce seeds in the wild, and the seeds that are produced cannot withstand desiccation to water contents that are amenable to conventional seed storage protocols. Furthermore, genetic assessment of extant populations of Zizania texana will aid in maximizing the genetic diversity of the ex situ collection.
 

APPROACH: This study has two components, the development of cryostorage procedures for Zizania texana and the assessment of genetic diversity of populations of the species. Because of the limited biodiversity of Z. texana and the difficulties in cryopreserving propagules, two strategies will be used to obtain an ex situ collection that is representative of the genetic diversity of plants growing in the wild. In one strategy, seeds, collected from plants in the wild (if there are sufficient supplies) or from plants grown in "captive" collections, will be cryopreserved. In the second strategy, clones of plants that give the greatest level of genetic diversity will be cryopreserved as shoot tips. At each step of the conservation effort, the genetic heterogeneity of the accession will be compared to that existing in wild populations to determine how much of the genetic diversity of the species can be preserved. In order to reduce impact on Zizania texana populations, initial studies on the development of cryostorage procedures used the closely related common wild rice, Zizania palustris, with successful procedures then being tested for Zizania texana. The use of a combination of rapid drying, ultra rapid cooling, and tissue culture procedures were used to optimize cryostorage protocols. Microsatellites were chosen to assess the genetic diversity of Zizania texana. To develop markers, a combination of screening genomic DNA with markers developed for closely related species and screening a genomic library for microsatellites is required. This should provide high number of polymorphic markers suitable for assessing genetic variability within a species.
 

RESULTS: Successful cryostorage was achieved for the cultivated wild rice, Zizania palustris. The optimal procedure (89% post-thaw survival) involved rapidly drying embryos to a water content of 0.3 g/g dry weight followed by cooling in a sub cooled liquid nitrogen (-208⁰C). This same procedure did not result in high survival of embryos of Zizania texana (11% post-thaw survival). This suggested that, although Z. texana and Z. palustris are closely related, their seed physiology differs significantly in terms of their ability to survive cryostorage. Microsatellite markers developed for Oryza sativa, the closest agronomically related species to Zizania, were applied to Zizana texana without success. Preliminary results suggest that the screening of a genomic library for microsatellites is a more reliable procedure for developing polymorphic markers for this species.
 

Becker, H. 1998. Saving Seeds for the long term. Agricultural Research September 1998: 12-13 (documentary).
 

Buitink, J, C WALTERS, FA Hoekstra and J CRANE. 1998. Storage behavior of Typha latifolia pollen at low water contents: Interpretation on the basis of water activity and glass concepts. Physiologia Plantarum 103:145-153.
 

Christensen, TL, EE ROOS, DA DAVIDSON and RJ Reinsvold. 1998. Germination of seed after fifty years in vacuum storage. American Journal of Botany 85: 87-88. (Abstract).
 

Christensen, TL, EE ROOS, and RJ Reinsvold. 1998. Seeds of the past for the future. American Journal of Botany 85: 69-70 (Abstract).
 

Davidson, KGV and C WALTERS. 1998. Thresholds for desiccation tolerance during pea axis germination. Plant Physiology 114 (suppl) (Abstract).
 

EBERHART, SA and LE WIESNER. 1998. Long-term storage of rye at the National Seed Storage Laboratory. Pp27- 36. In T Gass, W Podyma, J Puchalski and SA Eberhart (eds) Challenges in rye germplasm conservation: Proceedings of an international conference on crop germplasm conservation with special emphasis on Rye. IPGRI, Rome
 

Eira, MTS, C WALTERS and LS Caldas. 1998. Avoiding desiccation and freezing damage in Coffea germplasm. Plant Physiology 114 (suppl) (Abstract).
 

Fang, J, F Moore, E ROOS and C WALTERS. 1998. Three-dimensional models represent seed moisture content as a function of relative humidity and temperature. HortScience 33: 1207-1209.
 

Farrant, JM and C WALTERS. 1998. Ultrastructural and biophysical changes in developing embryos of Aesculus hippocastanum L. in relation to the acquisition of tolerance to drying. Physiologia Plantarum 104:513-524.
 

Franco J, J Crossa, J Villasenor, S Taba and SA EBERHART. 1998. Classifying genetic resources by catergorical and continuous variables. Crop Science 38: 1688-1696.
 

Forsline, PL, LE TOWILL, JW WADDELL, C Stushnoff, WF Lamboy and JR McFerson. 1998. Recovery and longevity of cryopreserved dormant apple buds. J. Amer. Soc. Hort. Sci. 123:365-370.
 

LAUFMANN, JE and LE WIESNER. 1998. Rapid Germination of Western Dogwood Using Embryo Extraction, Cut Cotyledons and Gibberellic Acid. Seed Technology 20: 99-105.
 

Reed, BM, J. DeNoma, J. Luo, Y. Chang and L TOWILL. 1998. Cryopreservation and long-term storage of pear germplasm. In Vitro Cell. Dev. Biol.-Plant 34:256-260.
 

WALTERS, C. 1998. Understanding the mechanisms and kinetics of seed aging. Seed Science Research 8:223-244 (Invited review).
 

WALTERS, C. 1998. Ultra-dry technology: perspective from the National Seed Storage Laboratory, USA. Seed Science Research 8 (suppl):11-14 (Invited commentary).
 

WALTERS, C (invited guest editor). 1998. Recent Advances on Ultra-dry Research. Seed Science Research 8(suppl)
 

WALTERS, C. "Water Activity: Bad Habits Die Hard": A response. CryoLetters 19: 265-266 (editorial)
 

WALTERS, C and J Engels. 1998. The effects of storing seeds under extremely dry conditions. Seed Science Research 8 (suppl): 3-8 (Invited commentary).
 

WALTERS, C and LM HILL. 1998. Water sorption isotherms of seeds from ultradry experiments. Seed Science Research 8 (suppl): 69-73.
 

WALTERS, C, NK Rao, and X Hu. 1998. Optimizing seed water content to improve longevity in ex situ genebanks. Seed Science Research 8 (suppl): 15-22.
 

WALTERS, C, EE ROOS, D TOUCHELL, PC STANWOOD, LE TOWILL, L WIESNER and SA EBERHART. 1998. Ultradry seed storage cuts cost of genebank: Response. Nature 395: 758 (Peer-reviewed commentary).
 

WALTERS C, EE ROOS and SA EBERHART. 1998. An analysis of seed storage practices. Pp12-23. In T Gass, W Podyma, J Puchalski and SA Eberhart (eds) Challenges in rye germplasm conservation: Proceedings of an international conference on crop germplasm conservation with special emphasis on Rye. IPGRI, Rome
 

WIESNER, LE. 1998. Important Biological Factors for Utilizing Native Plant Species. In: Proceedings Revegetation with native species. USDA Forest Service.
 

Zhang M, C WALTERS and EE ROOS. 1998. Analysis of seed aging using moisture sorption isotherms, glass transition and volatile detection. Plant Physiology 114 (suppl) (Abstract).
 

Note: Names in caps are present or former ARS-NSSL employees