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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
2010 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 methods to preserve citrus accessions. 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. Methods to cryopreserve citrus vegetative tissues were developed. Scientists at NCGRP in Fort Collins, CO developed a micrografting technique that enables recovery of meristem tissue after cryopreservation. Previously citrus could not be preserved by any method except seed. This new technique esures citrus can be preserved in long term storage in case diseases or other biotic or abiotic stresses occur that result in loss of valuable genetic resources important to industry, breeders and researchers, and the public.

2. Genetic erosion as a function of seed longevity modeled. Scientists at NCGRP in Fort Collins, CO developed a model to estimate the extent of random genetic shifts as a function of seed longevity and frequency of regeneration. The model shows the importance of maintaining original samples in the base collection and regenerating from these samples in parallel fashion. Currently the original sample is not kept at the NCGRP base collection and the model demonstrates an important recommended change in operations. The research could also be valuable in developing standards for storage for other genebanks internationally.

3. Seed longevity is detectable using non-invasive assays. Scientists at NCGRP in Fort Collins, CO developed assays that measure headspace above stored seeds and uses the identity of different molecular species and the rate of emission as a way to detect when seeds would begin to lose viability. Noninvasive assays detect deterioration sooner and do not deplete samples of valuable germplasm that are normally consumed during viability monitoring tests. Assays such as this could greatly impact genebanks and botanical gardens interested in managing collections of seeds for long term storage.


Review Publications
Volk, G.M. 2010. Application of functional genomics and proteomics to plant cryopreservation. Current Genomics. 11:24-29.

Volk, G.M., Richards, C.M., Henk, A.D., Reilley, A.A., Reeves, P.A., Forsline, P.L., Aldwinckle, H.S. 2009. Capturing The Diversity Of Wild Malus Orientalis From Georgia, Armenia, Russia And Turkey. Journal of the American Society for Horticultural Science. 134:453-459.

Volk, G.M., Bonnart, R.M., Waddell, J.W., Widrlechner, M.P. 2009. Cryopreservation Of Dormant Buds From Diverse Fraxinus Species. CryoLetters. 30:262-267.

Volk, G.M., Stern, D. 2009. Phenotypic Characteristics Of Ten Garlic Cultivars Grown At Different North American Locations. HortScience. 44:1238-1247.

Panella, L.W., Wheeler, L.J., Mcclintock, M.E. 2009. Long-term Survival of Cryopreserved Sugar Beet Pollen. Journal of Sugar Beet Research. Vol.46 No.1 pp 1-9.

Reeves, P.A., Richards, C.M. 2009. Accurate inference of subtle population structure (and other genetic discontinuities) using principal coordinates. Plos one. 4(1) e4269.

Davidson, R.M., Reeves, P.A., Manosalva, P.M., Leach, J.E. 2009. Germins: A Diverse Protein Family Important For Crop Improvement. Plant Science. 177:499-510.

Bonnart, R.M., Volk, G.M. 2010. Increased efficiency using the encapsulation-dehydration cryopreservation technique. CryoLetters. 31:95-100.

Richards, C.M., Volk, G.M. 2010. New Challenges for Data Managment in Genebanks. Acta Horticulturae. 859:333-336.

Volk, G.M., Richards, C.M., Forsline, P.L. 2010. A comprehensive approach toward conserving Malus germplasm. Acta Horticulturae. 859:177-182.

Niedzielski, M., Walters, C.T., Luczak, W., Hill, L.M., Wheeler, L.J., Puchalski, J. Assessment of Variation in Seed Longevity within Rye, Wheat and the Intergeneric Hybrid Triticale. Seed Science Research. 19:213-224.

Last Modified: 11/27/2014
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