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Research Project: Innovations that Improve the Efficiency and Effectiveness of Managing and Preserving Ex Situ Plant Germplasm Collections


2015 Annual Report

Genebanks have an urgent mandate to increase efficiency and number of germplasm forms and species within collections. This mandate will be achieved through technologies that prolong germplasm shelf-life, tools that nondestructively detect early changes in viability and genetic integrity, and methods that quantify and compare wild and collected diversity. Through time, genebanked materials must stay fit-for-purpose. PGPRU’s goals are to provide state-of-art genebanking methods that address current needs of genebank operators to manage collections cost-effectively and future needs of users for access to well characterized, diverse collections of living germplasm. To meet this challenge, PGPRU will perform research in the next 5 years that will: OBJECTIVE 1: DETECT GAPS AND REDUNDANCIES IN GENEBANK COLLECTIONS Develop and validate methods that integrate statistical genetics and spatial analyses to detect gaps and redundancies in genebank collections, and to estimate and compare the genetic diversity among genebank collections and in situ populations, especially for crop landraces and wild relatives. • Subobjective 1a. Partition genetic diversity within NPGS collections into subsets and provide metrics to relate genetic distance among accessions. • Subobjective 1b. Confirm whether geospatial and climatic data can identify collection gaps or ecotypes. OBJECTIVE 2: IMPROVE INITIAL AND LONG-TERM SURVIVAL OF STORED GERMPLASM Devise or refine tools that enhance the long-term viability of stored germplasm, including clonal propagules, and provide the means for curators to assess and predict the response of germplasm to conventional and cryogenic storage treatments. • Subobjective 2a: Develop methods to recover vigorous plants from shoot tips. • Subobjective 2b: Quantify variation of shoot tip response to established preservation methods of desiccation and liquid nitrogen exposure. • Subobjective 2c: Quantify variation of dormant bud response to preservation methods of desiccation and liquid nitrogen exposure. • Subobjective 2d: Quantify variation of seed response to desiccation and cooling. • Subobjective 2e: Quantify interactions between temperature, moisture and seed longevity. OBJECTIVE 3. EVALUATE CHANGES IN QUALITY AND GENETIC IDENTITY Design metrics for monitoring and validating biological quality (viability, health, etc.) of stored and regenerated plant germplasm and assess genetic integrity of germplasm and the genetic shifts that occur during germplasm management. • Subobjective 3a: Develop new tools to measure initial vigor and detect aging. • Subobjective 3b: Assess risks of genetic change during genebanking. OBJECTIVE 4: LOCATE MASKED DESIRABLE GENES IN CROP WILD RELATIVES Develop and apply genome annotation methods to evaluate collections of crop land races and wild relatives for genetic diversity in key agricultural traits, so as to enable more effective germplasm curation and to improve access to that diversity for marker-assisted breeding.

The Plant Germplasm Preservation Research Unit (PGPRU) has the unique mission of troubleshooting plant genebanking methods to solve the most critical problems of genetic resource collections: keeping germplasm alive, healthy and representative of the source population; describing collection composition; and ensuring stored germplasm meets the needs of diverse users. This 5 year plan describes PGPRU’s strategy to apply creative, multidisciplinary approaches that balance the special requirements of diverse living germplasm (seeds, pollen and explants) with the practical needs of curators and users. Research will provide tools that compare genetic diversity of collections with species diversity extant in the wild; broaden the array and longevity of propagules in storage; assess germplasm health with minimum sample depletion; and account for genebanking effects on the biological and genetic integrity of the sample. Our research efforts will be vital to the overall goal of creating relevant scientific collections to understand, protect and use plant diversity in a changing world. PGPRU will approach the task of improving genebanking as the challenge of achieving apparently contradictory goals, such as maximizing genetic diversity while minimizing collection size; standardizing preservation treatments for diverse propagules that respond differently; monitoring for signs of deterioration during early storage when few changes are known to occur; maintaining genetic heterogeneity in an agricultural context where quality and uniformity are highly valued; and finding specific alleles of interest that may be masked by the genetic background. In a real sense, these contradictions underscore the complex mission of genebanking. PGPRU will remain at the forefront of plant repository biology and will continue to play a global role in technology transfer for plant genebank management.

Progress Report
(Objectives 1 and 4) Efforts continue to combine geospatial and genetic data to understand diversity (number and frequency of alleles for a gene) and differentiation (physical appearance or function) of crop wild relatives that already exist or should be included in NPGS collections. Research in this unit continues to show that diversity and differentiation do not correlate with geographic distance and warns against strategies that only use spatial data to guide collections. Research primarily focused on wild apple (Central Asia) and barley (Jordan) and shows complex interrelationships among species or populations. Therefore, analyses that use networks to uncover genetic distances or suites of genes are most informative and will be most useful in locating ‘genes of interest.’ On the international scale, there is increased interest in acquiring genetic data for crop wild relatives and scientists at NCGRP are actively involved in this initiative. (Objective 2) Big strides were made in understanding the stress of germplasm preservation and the process of recovery in both seed and vegetative tissues. Despite the extremely low temperatures of liquid nitrogen and the high moisture level of many cells, ice formation is not the primary stress. Surviving tissues may form very small, nonlethal ice crystals intracellularly. Cryoprotectants reduce ice formation and induce formation of a glassy matrix. The kinetics of cryoprotectant influx into cells to induce glass formation can now be measured. Recovering tissues show symptoms of oxidative stress and, perhaps, programmed cell death (PCD) cascades that are manifested differently than PCD observed in animal cells. This information is important to guide the next steps in preventing initiation of stress cascades before they reach “the point of no return.” Behavior (chemical reactions and physical change) within the glassy matrix of preserved cells is a brand new area of research, called “solid-state” biology that presents many logistical challenges for exploration because most current assays rely on fluid systems. (Objective 3) Research on changes in biological integrity and genetic identity in stored germplasm continues. The major challenges are that initially there are no symptoms of change and that our current assays consume large amounts of germplasm and only provide a snapshot of germplasm health. Hence, we have focused on developing assays that provide early indications of deterioration or that increase statistical rigor of germination or growth assays. Development of several tests is progressing: mechanical tests show changes in structure of aged germplasm (e.g., increased brittleness); calorimetric tests indicate admixture of impurities within liposomes of seeds; lost RNA integrity indicate seed aging while only using a few seeds; volatile profiles change in deteriorated seeds and can be detected completely non-invasively. Work on genetic changes in seed accessions during gene banking continues – most of this is related to changes that occur as a consequence of regeneration. Assuming minor changes in genetic identity of wild-collected samples, gene banked materials are being used to evaluate changes in natural populations over time, especially in response to climate change.

1. Scientists collaborating within the NPGS completed the most comprehensive phylogenetic analysis to date of the genus Malus (apple). The analysis reveals a complex domestication history with repeated mixing among several distinct species. Understanding how cultivated apple relates to its wild progenitors helps us target novel diversity for collection and find useful genes that are hidden by a wild, weedy phenotype.

2. Cryopreserved germplasm is subjected to extremely low temperatures and recovery is dependent on “jumpstarting” metabolism upon thawing. We examined gene expression in recovering shoot tips of Arabidopsis thaliana and discovered that surviving cells “turned on” genes that are typically expressed following stress – in particular, genes for antioxidant enzymes. Our next step is to test whether addition of these gene products can aid recovery of severely damaged cells.

3. Scientists at NCGRP provide an explanation for orthodox, recalcitrant and intermediate storage categories of seeds. The explanation is based on mechanical principles, not unlike principles used in materials sciences that explain performance of plastics. The unified concepts provide a rational way to predict seed longevity in gene banks.

Review Publications
Xia, K., Hill, L.M., Li, D., Walters, C.T. 2014. Factors affecting stress tolerance in recalcitrant embryonic axes from four Quercus (Fagaceae) species native to the US or China. Annals Of Botany. 114(8):1747-1759. DOI: 10.1093/aob/mcu193.
Gross, B., Henk, A.D., Richards, C.M., Fazio, G., Volk, G.M. 2014. Genetic diversity in Malus × domestica (Rosaceae) through time in response to domestication. American Journal of Botany. 101(10):1770-1779. DOI: 10.3732/ajb.1400297.
Bonnart, R.M., Waddell, J.W., Haiby, K., Widrlechner, M., Volk, G.M. 2014. Cryopreservation of Populus trichocarpa and Salix using dormant buds with recovery by grafting or direct rooting. CryoLetters. 35(6):507-515.
Walters, C.T. 2015. Genebanking seeds from natural populations. Natural Areas Journal. 35(1):98-105. DOI: 10.3375/043.035.0114.
Volk, G.M., Bonnart, R.M., Shepherd, A., Krueger, R., Lee, R.F. 2015. Cryopreservation of citrus for long-term conservation. Acta Horticulturae. 1065:187-191.
Asanidze, Z., Akhalkatsi, M., Henk, A.D., Richards, C.M., Volk, G.M. 2014. Genetic relationships between wild progenitor pear (Pyrus L.) species and local cultivars native to Georgia, South Caucasus. Flora. 209(9):504-512. DOI: 10.1016/j.flora.2014.06.013.
Fazio, G., Chao, C.T., Forsline, P., Richards, C.M., Volk, G.M. 2014. Tree and root architecture of Malus sieversii seedlings for rootstock breeding. Acta Horticulturae. 1058:585-594.
Wesley-Smith, J., Berjak, P., Pammenter, N., Walters, C.T. 2014. Intracellular ice and cell survival in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum: an ultrastructural study of factors affecting cell and ice structures. Annals Of Botany. 113(4):695-709. DOI: 10.1093/aob/mct284.
Volk, G.M., Chao, C.T., Norelli, J.L., Brown, S.K., Fazio, G., Peace, C., McFerson, J., Zhong, G., Bretting, P.K. 2015. The vulnerability of US apple (Malus) genetic resources. Genetic Resources and Crop Evolution. 62(5):765-794. doi:10.1007/s10722-014-0194-2.
Zhang, J., Huang, B., Lu, X., Volk, G.M., Xin, X., Yin, G., He, J., Chen, X. 2015. Cryopreservation of in vitro-grown shoot tips of Chinese medicinal plant Atractylodes macrocephala Koidz. using a droplet-vitrification method. CryoLetters. 36(3):195-204.
Wesley-Smith, J., Walters, C.T., Pammenter, N., Berjak, P. 2015. Why is intracellular ice lethal? A microscopical study showing evidence of programmed cell death in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum. Annals of Botany. 115(6):991-1000. DOI:10.1093/aob/mcv009.
Walters, C.T. 2015. Orthodoxy, recalcitrance and in-between: describing variation in seed storage characteristics using threshold responses to water loss. Planta. 242(2):397-406.
Volk, G.M., Henk, A.D., Baldo, A.M., Fazio, G., Chao, C.T., Richards, C.M. 2015. Chloroplast heterogeneity and historical admixture within the genus Malus. American Journal of Botany. 102(7):1198-1208. DOI: 10.3732/ajb.1500095.