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United States Department of Agriculture

Agricultural Research Service


Location: Plant Germplasm Preservation Research Unit

2012 Annual Report

1a.Objectives (from AD-416):
Objective 1: Develop physiological and biophysical approaches and tools to assess changes in plant germplasm viability and the potential causes during genebank preservation. Objective 2: Develop statistical genetic strategies and tools to sample and preserve plant genetic diversity in genebank collections and in situ reserves.

1b.Approach (from AD-416):
The Preservation of Plant Genetic Diversity in Ex Situ Genebank program scientists conduct research to improve the biological and genetic integrity of genebanked germplasm and to standardize procedures for handling accessions and reporting associated data. Interrelated research goals will allow curators to preserve viability of conservation targets (Objective.
1)and rationalize and validate the genetic diversity and integrity of those targets (Objective 2). Using taxa that are empirically tractable systems, we will • define tolerances to preservation stresses of selected propagules, • develop methods to improve survival or reliably predict loss of viability over time, • model the effects of mortality and regeneration on genetic composition, and • develop sampling strategies for wild-collected germplasm that maximize genetic diversity while minimizing curator inputs for storage and regeneration.

A central theme is identifying appropriate conservation targets that capture desired genetic diversity, remain viable during storage and are available to the user when needed. A conservation target is a group of propagules (such as seeds or pollens) or an individual propagule (such as an explant) that comprises an accession valued for specific genes, genetic richness (number and frequency of alleles) or an allelic combination (genotype). PGPRU scientists and their collaborators will investigate major conceptual issues of repository biology and standardization using within-unit expertise in biophysics, plant physiology, cell and molecular biology and population genetics and National Plant Germplasm System (NPGS) curators’ expertise on reproductive biology, phenotypic diversity, history and cultivation of their assigned collections. Our central position within NPGS allows us to develop protocols and predictive tools that are applicable to a wide variety of species and propagules.

3.Progress Report:
Research on preservation of vegetatively-propagated germplasm provides new insights on ways to increase efficiency in handling materials and survival of cryo-exposed materials. The major time bottleneck for backing-up clonally propagated germplasm is obtaining in vitro cultures with sufficient plant sizes. Research has shown that the initial in vitro step may be obviated by using shoot tips harvested from greenhouse grown plants. Oxidative stress appears to be a major limitation to obtaining explants that can be recovered and grown in culture. Cryo-exposed shoot tips show up-regulation of antioxidants during recovery and application of antioxidants increases survival and health of shoot tips after excision and cryoexposure. Cryotherapy appears to be a feasible method to eliminate graft transmissible pathogens from Citrus and citrus relatives without inducing juvenility. Pathogens reside in large, differentiated cells of the meristem which are killed during cryoexposure. This enables grafters to use much larger shoot tip pieces which increases the efficiency of grafting.

Research on seed quality and storage behavior suggests that desiccation damage in developing embryos is related to the amount of mechanical disruption when cells shrink during drying. Damage decreases as embryos mature and this can be explained by accumulation of dry matter into cells. The results lead to a quantitative description of seed storage physiology that replaces existing categories of orthodox and recalcitrant behavior. Assays that measure molecular mobility within seeds are providing promising tools for predicting seed longevity or describing the early effects of aging continue to expand. Dynamic mechanical analysis (DMA) detects interactions of moisture and temperature on the tendency for molecular mobility in seeds.

Assessments of the effectiveness of genebanking are critical to support the high priority of wild-collected accessions. Tools that quantify genetic erosion within genebanked samples are being developed using barley. Collections of original wild populations were made and genetic diversity of these will be compared with accessions that have been stored and regenerated for more than 20 years in different genebanks using different genebanking strategies. This experiment complements additional research projects that integrate geospatial modeling and population genetic in order to estimate local adaptation. Advanced statistical methods to estimate genetic structure and Geographic Information Systems mapping have been used to detect patterns of diversity and help locate potential gaps in collections. The results from these projects have particular application to finding gaps in the representation of genetic diversity of species within a genebank.

1. Accurate identification of collection structure. It is often hard to tell the identity of individuals within a collection of wild plant germplasm. Duplicate samples and interspecific hybrids are less useful components of the collection and identifying them can save resources. ARS researchers in Fort Collins, CO developed a set of procedures that can be used to assess whether a sample within the collection is an accurate representative of the species or is the result of inadvertent cross-pollination between two distant relatives or species. The procedure uses genetic marker data to accurately define variation captured within a particular species and remove accessions having genes from different genepools. This procedure allows curators within the National Plant Germplasm System to cross-compare holdings among global genebanks using calibrated datasets.

2. Quantification of genetic diversity. The value of a collection is often expressed by how representative it is of extant diversity. The diversity of plant genetic resource collections can be assessed by diversity in parts of the genome that are not expressed but give information about genetic relatedness (neutral variation). However, when making collections for crop improvement, we want to have diversity in the parts of the genome that are expressed and contribute to phenotype (functional variation). ARS researchers in Fort Collins, CO developed a model to test whether neutral (i.e., unexpressed or having no adaptive function) genetic markers can serve as surrogates for markers of agriculturally useful traits when structuring genetic diversity of a genetic resource collection and developing subsets to improve crops. The simulation demonstrated that neutral markers failed to reliably predict gene variation that regulated desired traits. This finding has major impacts on how core subsets within genetic resource collections should be formed. Alternative methods that use gene genealogies or ecotypes may help breeders locate valuable genes that cannot be detected by phenotype.

3. Lipid crystallization properties detect seed aging. The problem with seed aging is that it can’t be detected until the seed has aged so much that it dies. This means that a seed lot can go from seemingly high to very low quality in a short time. ARS researchers in Fort Collins, CO showed that aging of lettuce seeds could be detected without a germination test. We use a similar test as used to detect heat abuse in frying oils. The test measures the tendency of seed storage lipids to crystallize. As seeds age, less lipids crystallize; we can measure this very accurately and without damaging the seed.

Review Publications
Volk, G.M., Richards, C.M. 2011. Integration of georeferencing, habitat, sampling, and genetic data for documentation of wild plant genetic resources. HortScience. 46(11): 1446-1449.

Dierig, D.A., Wang, G., Crafts-Brandner, S.J. 2012. Dynamics of reproductive growth of Lesquerella (Physaria fendleri) over different planting dates. Industrial Crops and Products. 35:146-153.

Wegrzyn, J.L., Main, D., Figueroa, B., Choi, M., Neale, D.B., Jung, S., Stanton, M., Zheng, P., Ficklin, S., Cho, I., Peace, C., Evans, K., Volk, G.M., Oraguzie, N., Chen, C., Gmitter, F.G., Abbott, A.G. 2012. Uniform standards for genome databases in forest and fruit trees. Tree Genetics and Genomes. 8:549-557.

Ballesteros, D., Estrelles, E., Walters, C.T., Ibars, A. 2012. Effects of temperature and desiccation on ex situ conservation of nongreen fern spores. American Journal of Botany. 99(4): 721-729.

Gross, B.L., Henk, A.D., Forsline, P.L., Richards, C.M., Volk, G.M. 2012. Identification of interspecific hybrids among domesticated apple and its wild relatives. Tree Genetics and Genomes. (2012)8:1223-1235. Available: DOI 10.1007/s11295-012-0509-4.

Perez, H.E., Hill, L.M., Walters, C.T. 2012. An analysis of embryo development in palm: interactions between dry matter accumulation and water relations in Pritchardia remota (Arecaceae). Seed Science Research. 22:97-111.

Gross, B.L., Volk, G.M., Richards, C.M., Forsline, P.L., Fazio, G., Chao, C.T. 2012. Identification of “duplicate” accessions within the USDA-ARS National Plant Germplasm System malus collection. Journal of the American Society for Horticultural Science. 137(5):333-342.

Routson, K.J., Volk, G.M., Richards, C.M., Smith, S.E., Nabhan, G.P., De Echeverria, V.W. 2012. Genetic variation and distribution of Pacific crabapple. Journal of the American Society for Horticultural Science. 137(5):325-332.

Volk, G.M. 2011. Chapter 25: Collecting pollen for genetic resources conservation. In: Guarino, L., Ramanatha Rao, V., Goldberg, E., editors. Collecting Plant Genetic Diversity: Technical Guidelines. 2011 Update. Rome, Italy: Bioversity International. Available:

Reeves, P.A., Panella, L.W., Richards, C.M. 2012. Retention of agronomically important variation in germplasm core collections: implications for allele mining. Theoretical and Applied Genetics. 124(6) 1155-1171. DOI: 10.1007/S00122-011-1776-4.

Last Modified: 4/17/2014
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