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


2014 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
Activities contributing toward Objective 1 (understanding how genetic diversity in collections and in nature is quantified and apportioned) involved studies with apple, pear, beet and sunflower. In previous years, apple and pear accessions were genotyped using microsatellite markers and FY2014 efforts were applied towards analyzing these data to a) identify gaps in the National Plant Germplasm System (NPGS) coverage of wild Malus species native to North America, b) determine extent of lost genetic diversity in domesticated apple over centuries from comparisons of old and modern cultivars, and c) assess the genetic relationship between wild Pyrus species native to Georgia and domesticated pear varieties. In addition, estimates of Malus genetic diversity using chloroplast DNA was used to identify relationships among diverse Malus species. Objective 1 goals are also to develop tools that use latitude/longitude coordinates, included as passport data for accessions, to predict where to find additional useful genetic variation that can further augment collections. We have found that genetic variation modeled from spatial data alone do not predict the location of unique ecotypes. More sophisticated models that account for geneflow are needed. An example using Beta maritima, collected from populations along the Atlantic coast of Europe, suggested that some geneflow may arise from ships. Another example using Helianthus humilis, dwarf sunflower, showed that barriers to geneflow near Pikes Peak, rather than climate, determined how genetic diversity of this species was distributed along the front range of Colorado and Wyoming. Activities contributing to Objective 2 (enhancing survival of genebanked germplasm) used diverse propagule forms including shoot tips, dormant buds, orthodox and recalcitrant seeds and pollen. Significant progress has been made to refine the Citrus shoot tip cryopreservation protocols developed by PGPRU. Over 50 diverse species of Citrus relatives from the NPGS Riverside collection have been placed in backup cryostorage and survival of materials placed in storage 2-3 years ago have shown no change in viability, providing excellent validation of the procedure. Work to develop techniques to cryopreserve grape and sugarcane shoot tips is proceeding and giving acceptable survival and recovery. Cryotherapy work is progressing and an opposite and alternative method to maintain pathogen strains using diseased plants is under investigation. Cryopreservation methods for recalcitrant embryos of tropical and temperate plants continue. Major efforts in FY2014 focused on surveying the desiccation tolerance of seeds from diverse oak, chestnut, cottonwood/willow and magnolia species – all believed to produce recalcitrant seeds. We continue work to understand development and germination in culture of seeds from tropical species, with particular focus on avocado, cocoa, palms and citrus relatives. We investigated the feasibility of a new technique, which involves treating the plumule tip of large embryos like clonally produced shoot tips. We initiated studies to examine viability of fresh pollen from avocado and cotton to test the feasibility of using those propagules. Longevity studies of orthodox seeds involved comparisons of aging rates under diverse temperature (including freezer and liquid nitrogen storage) and moisture conditions (including accelerated aging, ambient and ultra-dry conditions). Several large experiments were tested for viability this year, including a cross section of seeds from Brassicaceae, Solanaceae, rice and rye accessions, and the 65 year old Went-Munz experiment on the longevity of seeds from California native species. We report changes in after-ripening and seed coat permeability with storage time in some seeds which make these traits good candidates to study additional changes besides lost viability under seed storage conditions. We have been investigating the use of dynamic mechanical analyses (DMA) to predict seed longevity. Activities contributing to Objective 3 (assessing changes in germplasm during genebanking) involve studies that link physical and chemical assays to viability loss and studies that compare genetic changes of wild-collected germplasm stored in the genebank with similar populations in situ. The former studies volatile emission or lipid crystallization kinetics in seeds. A new technique of measuring RNA integrity as a function of storage time and degradation was initiated using seeds stored for 20 to 30 years. Species studied included lettuce, soybean, onion and Brassica relatives. All genotyping and phenotyping were completed this past year for the study comparing genetic changes of barley during genebanking ex situ and in situ. We are now poised to perform analyses to measure the magnitude and direction of change of accessions and natural populations over a 35 year period. Activities related to Objective 4 (finding genes with agronomic benefit using geo-spatial and genomic data) involved working with partners to find common and mutually beneficial approaches. Gene evolution approaches for fatty acid synthesis are of interest to partners’ investigation of mechanisms for lipid composition and PGPRU interests in genetic diversity. PGPRU has also been working with partners on the apple genotyping-by-sequencing (GBS) project. We have made a preliminary assessment of the dataset to determine its suitability to address genetic relationships, domestication and gene evolution questions.

1. Crop Vulnerability Statement (CVS) for Apple. ARS researchers took the lead in reviewing and updating a risk analysis for apple production that reflects the urgency and extent of abiotic and biotic threats and the availability of genetic resources to mitigate these threats. The apple CVS extensively uses products of National Plant Germplasm System (NPGS) research on apple genetic diversity within NPGS collections and extant in the wild. This CVS serves as a template for other crops and represents how information technology for genetic and geo-spatial data can be integrated to identify needs for genetic resource collections and research.

2. Successful cryopreservation of recalcitrant seeds from oak. Oak trees are iconic to many North American habitats and yet their genetic diversity is threatened by numerous pressures. In the past, oak germplasm has been difficult to store because trees produce “recalcitrant” seeds that cannot be preserved using conventional methods. ARS scientists in Fort Collins, CO, working in collaboration with US Agencies and international groups, have developed methods to cryopreserve embryonic axes dissected out of acorns. The methods provide high recovery, especially for diverse species which are adapted to temperate winters or dry habitats. Over twenty species of oak have been tested so far and found to be amenable to the developed protocols. ARS is now poised to initiate a cryopreserved collection of oak genetic resources.

Review Publications
Volk, G.M., Henk, A.D., Bonnart, R.M., Shepherd, A., Gross, B.L. 2014. Plant shoot tip response to treatment with plant vitrification solution 2. Acta Horticulturae. 1039:81-84.
Reeves, P.A., Richards, C.M. 2014. Effect of a geographic barrier on adaptation in the dwarf sunflower (Helianthus pumilus Nutt.). International Journal of Plant Sciences. 175:688-701.
Richards, C.M., Reeves, P.A., Fenwick, A.L., Panella, L.W. 2014. Genetic structure and gene flow in Beta vulgaris subspecies maritima along the Atlantic coast of France. Genetic Resources and Crop Evolution. 61(3):651-662. DOI 10.1007/s10722-013-0066-1.
Volk, G.M., Henk, A.D., Richards, C.M., Forsline, P.L., Chao, C.T. 2013. Malus sieversii: a diverse Central Asian apple species in the USDA-ARS National Plant Germplasm System. HortScience. 48(12):1440-1444.
Volk, G.M., Shepherd, A., Bonnart, R.M. 2014. Strategies for improved efficiency when implementing plant vitrification techniques. Acta Horticulturae. 1039:85-89.
Yin, Z., Chen, L., Bi, W., Volk, G.M., Wang, Q. 2014. Somatic embryogenesis and organogenesis from cryopreserved shoot tips of Lilium Oriental hybrid ‘Siberia’. Acta Horticulturae. 1039:193-200.
Walters, C.T. 2014. Extreme biology: probing life at low water contents and temperatures. Acta Horticulturae. 1039:49-56.
Wang, B., Wang, R., Cui, Z., Li, J., Bi, W., Li, B., Ozudogru, E., Volk, G.M., Wang, Q. 2014. Potential applications of cryogenic technologies to plant genetic improvement and pathogen eradication. Biotechnology Advances. 32(3):583-595. DOI: 10.1016/j.biotechadv.2014.03.003.
Yin, Z., Bi, W., Chen, L., Zhao, B., Volk, G.M., Wang, Q. 2014. An efficient, widely applicable cryopreservation of Lilium shoot tips by droplet vitrification. Acta Physiologiae Plantarum. 36(7):1683-1692. DOI: 10.1007/s11738-014-1543-7.