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United States Department of Agriculture

Agricultural Research Service

Research Project: RESEARCH TO DEVELOP STRATEGIES AND TECHNOLOGIES FOR PRESERVING PLANT GENETIC DIVERSITY IN EX SITU GENEBANKS

Location: Plant Germplasm Preservation Research Unit

2009 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
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 allows curators to preserve viability of conservation targets and rationalize and validate the genetic diversity and integrity of those targets. The most significant research problems continue to be low survival after vegetatively propagated germplasm are exposed to liquid nitrogen; asymptomatic aging of seeds in storage, unidentified causes of varied response of plants to preservation stress, and representative sampling of genetic diversity for genebanks. Our research continues to quantify longevity of seeds, pollens and woody buds of diverse taxa in conventional and cryogenic storage. Research is expected to identify environmental, developmental and genetic factors that affect the ability of germplasm to survive in long term storage and structural or chemical changes to cells that correlate with changes in viability. Long-term storage results, initiated more than 20 years ago, continue to provide valuable information on the kinetics of deterioration under genebanking conditions. Factors such as geographic origin, phylogenetic relationships, winter hardiness, lipid composition, cell organization, presence of putative protectants, dry matter reserves, functional qualities of grains and water properties are correlated with duration of survival in diverse cell types. Large-scale collaborative studies have been initiated to link genomic data with preservation qualities of germplasm. Cryoprotectants are added to plant cells that do not naturally survive exposure to the low relative humidity or temperature needed to preserve germplasm. The mechanisms by which cryoprotectant solutions enable germplasm to survive and recover from these stresses and the reasons for cryoprotectant toxicity remain unknown. Research this year focused on recovery pathways of meristems following exposure to cryoprotectants and liquid nitrogen. We discovered that typical stress-recovery pathways are induced in cells after cryoexposure. Population genetic tools are an essential component of our research to link efficiency and accountability to preservation methods. We continue to use various statistical analyses to identify how genetic diversity is distributed among wild populations and to locate useful genes within collections. Ex situ preservation of genetic resources can result in inadvertent shifts in the genetic composition of accessions through drift or selection during collection, storage and regeneration. The risk of these potential bottlenecks are predicted through models that link preservation and genetic change. The models are validated with on-the-ground experiments comparing genetic composition of accessions during the genebanking process.


4.Accomplishments
1. Models developed to predict lifespans of cryopreserved plant germplasm. The lifespan of stored germplasm is difficult to measure empirically because expected timeframes are impractically long; however, this basic information is critical to all aspects of genebank management. ARS scientists at the National Center for Genetic Resources Preservation in Fort Collins, Colorado developed tools to understand temperature effects on reaction kinetics at extremely low temperatures and used these to model the time course of viability loss in plant germplasm. Models were validated with long-term storage experiments. The models demonstrate that despite the extreme cold of liquid nitrogen, cryopreserved germplasm is still likely to deteriorate over a period of a few thousand years. Liquid nitrogen storage is not effective for preserving viability in germplasm that is overdried or slightly deteriorated.

2. Models developed to maximize diversity of wild Malus collections with the fewest individuals. ARS Scientists at the National Center for Genetic Resources Preservation in Fort Collins, CO and the Plant Genetic Resources Unit in Geneva, NY used optimization models to develop a core subset of 112 individual wild apple trees that captured most of the genetic and phenotypic diversity of the M. sieversii collection containing over 1000 trees. Using microsatellite markers, a core collection of 27 individuals were identified to represent more than 700 Malus orientalis seedlings maintained at the Geneva repository. The reduction in numbers of trees without reducing the representative diversity will facilitate management of the collection while providing breeders valuable guidelines on where to look for novel traits.


Review Publications
Kothera, L., Zimmerman, E.M., Richards, C.M., Savage, H.M. 2009.Microsatellite characterization of subspecies and their hybrids in Culex pipiens complex mosquitoes along a north-south transect in the central United States of America. Journal of Medical Entomology 46:236-248.

Volk, G.M., Waddell, J.W., Towill, L., Grauke, L.J. 2009. Variation in low temperature exotherms of pecan cultivar dormant twigs. HortScience. 44:317-321.

Volk, G.M., Richards, C.M., Henk, A.D., Reilley, A., Miller, D.D., Forsline, P.L. 2009. Novel diversity identified in a wild apple population from Kyrgyz Republic. HortScience 44:516-519.

Richards, C.M., Volk, G.M., Reeves, P.A., Reilley, A., Henk, A.D., Forsline, P.L., Aldwinckle, H. 2009. Selection of stratified core sets representing wild apple (Malus sieversii). Journal of the American Society for Horticultural Science. 134:228-235.

Routson, K., Reilley, A., Henk, A.D., Volk, G.M. 2009. Identification of historic apple trees in the Southwestern United States and implications for conservation. HortScience 44:589-594.

Richards C.M., G. Volk, A.A. Reilley, A.D. Henk, D. Lockwood, P.A. Reeves, and P.L. Forsline. 2009. Genetic diversity and population structure in Malus sieversii, a wild progenitor species of domesticated apple. Tree Genetics and Genomics 5:339-347.

Last Modified: 8/22/2014
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