APPLICATION OF SOMATIC EMBRYOGENESIS FOR ASH CONSERVATION AND RESTORATION

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: MCINTIRE-STENNIS
• Reporting Frequency: Annual
• Accession No.: 1016473
• Project Start Date: Jul 3, 2018
• Project End Date: Jun 30, 2023
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF GEORGIA
200 D.W. BROOKS DR
ATHENS,GA 30602-5016

Performing Department
School of Forestry & Natural Resources

Non Technical Summary
All North American ash species are under threat of extirpation from their native ranges by the emerald ash borer (EAB), an exotic wood-boring beetle introduced from Asia, first discovered in Michigan in 2002. The larvae feed on the inner bark of ash trees, disrupting the tree's ability to transport water and nutrients. Since its discovery, the insect has killed millions of ash trees in 31 U.S. states and Canada. The development of EAB-resistant ash trees will be critical for ash reforestation in both urban and natural forests. A tiny percentage of native white ash and green ash trees have been identified as potentially EAB-resistant by their persistence in populations where EAB-induced mortality exceeds 99%. These trees, commonly referred to as "lingering ash" by those researching the EAB infestation, constitute potentially valuable sources of genetic resistance that could be used in ash restoration programs. We propose to employ the in vitro clonal propagation system known as somatic embryogenesis (SE) for mass clonal propagation of EAB-resistant ash using seeds from these lingering ash parents as starting material. We have already developed SE systems for green ash and white ash and shown the potential of these systems to produce large numbers of clonal trees. We will apply this approach to initiate SE cultures from seeds collected from open-pollinated (and where available, control-pollinated) lingering ash parents of major North American ash species (green ash, white ash, black ash) as well as rarer ash species such as Texas ash and blue ash. We will produce populations of clonal trees (somatic seedlings) from these cultures and grow them in the greenhouse to the stage where they can be planted in clonal field tests to be screened for EAB resistance. We will also optimize a cryostorage protocol for the ash cultures, so that once elite EAB-resistant ash clones are identified based on their field performance, those clones can be recovered from cryostorage and scaled up to produce thousands of somatic seedlings for restoration plantings.

Animal Health Component

70%

Research Effort Categories

Basic

30%

Applied

70%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
123	0621	1060	40%
202	0621	1060	40%
211	0621	1081	20%

Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants; 202 - Plant Genetic Resources; 123 - Management and Sustainability of Forest Resources;

Subject Of Investigation
0621 - Broadleaf forests of the South;

Field Of Science
1060 - Biology (whole systems); 1081 - Breeding;

Keywords

forest health

ash

emerald ash borer

in vitro propagation

somatic embryogenesis

Goals / Objectives
To initiate embryogenic cultures from open-pollinated "lingering ash" trees representing the major North American ash species (white ash, green ash and black ash)To initiate embryogenic cultures from control-pollinated seeds derived from crosses between lingering ash parents provided by USDA Forest Service cooperatorsTo initiate embryogenic cultures from open-pollinated seeds of rare ash species, such as Texas ash (Fraxinus texensis) and blue ash (Fraxinus quadrangulata).To produce somatic embryos and somatic seedlings from the embryogenic ash cultures. (Note: Somatic seedlings from lingering ash-derived clones produced in this project will be provided to cooperators in a related project so that they can be screened for EAB tolerance in the field)To optimize cryostorage and recovery protocols for ash embryogenic cultures, and to cryostore copies of all embryogenic ash cultures for ash germplasm conservation. (Note: Although not part of this proposal, EAB-tolerant clones identified by field testing by cooperators in a related project will eventually be recovered from cryostorage for scaled-up somatic seedling production)

Project Methods
Objective 1: Embryogenic culture initiation from open-pollinated (OP) "lingering ash" trees representing the major North American ash species (white ash, green ash and black ash). Lingering ash parent trees will be identified by cooperators throughout the natural ranges of the major ash species, who will collect immature seeds from female lingering ash parents in August for the first two years of the project and ship them via next day courier to the Merkle Lab at UGA. There, cultures will be initiated from the immature seeds following the protocol described in Li et al. (2014). Briefly, samaras will be surface-disinfested and dissected under aseptic conditions to obtain immature zygotic embryos, which will be cultured on an induction-maintenance medium (IMM; Li et al. 2014) containing 2,4-D to obtain proembryogenic masses (PEMs).Objective 2: Embryogenic culture initiation from control-pollinated (CP) seeds derived from crosses between lingering ash parents provided by USFS cooperators. USDA Forest Service Cooperators in Delaware, OH will conduct controlled pollinations between lingering ash parents to produce full sib seeds. As with the open-pollinated material in Objective 1, immature seeds resulting from the crosses will be collected in August for the first two years of the project and shipped via next day courier to the Merkle Lab at UGA. Culture initiation from these seeds will proceed as described for OP seed under Objective 1.Objective 3. Embryogenic culture initiation from open-pollinated seeds of rare ash species, such as Texas ash and blue ash. We will make contact with USDA Forest Service and Camcore cooperators who know the locations of populations of rare ash species such as blue ash and Texas ash, and request that they collect immature OP seeds from these populations for us to culture. As with the open-pollinated material in Objective 1, seeds will be collected in August for the first two years of the project and shipped via next day courier to the Merkle Lab at UGA. Culture initiation from these seeds will proceed as described for OP seed under Objective 1.Objective 4: Somatic embryo and somatic seedling production. Ash somatic embryo and somatic seedling production will follow the protocol described in Li et al. (2014). Briefly, approximately 0.5 g of PEMs of each culture will be inoculated into 30 ml of liquid IMM in 125 ml Erlenmeyer flasks and grown on a rotary shaker at 90 rpm in the dark at 25° C. Suspension cultures will be fed after 3 weeks by decanting off the old medium and adding 30 ml of fresh IMM. At 6 weeks, suspensions will be collected on filter paper using a Büchner funnel with mild vacuum and washed with 200 ml of liquid basal WPM. Filters with PEMs will be placed on embryo development medium (EDM; Andrade and Merkle 2005), which is semi-solid basal WPM with 1 g/l filter sterilized L-glutamine, in 100 mm plastic Petri plates. Plates will be incubated in the dark at 25° C to allow somatic embryos to develop. When somatic embryos reach at least 3 mm in length with visible cotyledons, they will be picked from the masses of developing embryos, transferred individually to fresh plates of EDM and incubated for another 3-4 weeks in the dark at 25° C to mature before being given a pre-germination cold treatment at 4 C in the dark for 8 weeks. Then, they will be moved to a lighted incubator (100 µmol·m-2·sec-1) with 16 h day lengths at 25° C to encourage germination. Germinating embryos will be removed from in vitro conditions and potted in moistened peat:perlite:vermiculite (1:1:1) mix in plastic pots, which will be placed on water-saturated perlite in a clear plastic dome-covered tray under cool white fluorescent lights (80 µmol·m-2·sec-1) and 16 h day lengths to encourage somatic seedling growth. Following hardening off, somatic seedlings will be repotted in larger pots and transferred to the greenhouse to continue growth.Objective 5: Cryopreservation of embryogenic cultures. We will test cryopreservation protocols based on one that is currently used in our lab that has worked well for other hardwood trees, including yellow-poplar (Vendrame et al. 2001), sweetgum (Vendrame et al. 2001) and chestnut (Holliday and Merkle 2000). In this protocol, embryogenic cultures will be pretreated by suspending in liquid IMM supplemented with sorbitol for 24 hours, and protected from freezing damage with DMSO, prior to being transferred to Nalgene cryovials, which will be loaded into Nalgene Cryo 1° C freezing containers and placed into a -70° C freezer. After reaching -70° C, the cryovials will be immersed in liquid nitrogen where they will be held until recovery and regrowth is called for. Cryostored cultures will be removed from cryostorage, thawed in a 40° C water bath, collected on nylon mesh and washed with fresh IMM. The nylon mesh with embryogenic material will be transferred to plates of IMM medium, followed by transfer to fresh medium at 1, 8 and 24 hours to dilute residual DMSO.

Progress 10/01/20 to 09/30/21

Outputs
Target Audience:The main target audience reached by our efforts during 2021 was other researchers working on ash species and emerald ash borer, and researchers working with forest health threats from other exotic pests and pathogens. The target audience also included stakeholders in the USDA Forest Service, state forestry agencies in states affected by emerald ash borer and NGOs interested in forest conservation and restoration, such as Trees Atlanta. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During this period, graduate student Mason Richins completed his cryostorage experiments as part of his M.S. thesis work on ash embryogenesis and cryostorage. He tested two different cryostorage approaches (slow-cooling and vitrification). He is writing up the results of his cryostorage research as a chapter of his M.S. thesis and as a manuscript to be submitted for publication. Mr. Richins presented the results of his ash cryopreservation research at the 36th Southern Forest Tree Improvement Conference in June 2021 and won the Van Buijtenen/Belle Baruch Institute Best Student Poster Award. He also regenerated somatic seedlings from his Texas ash and Mexican ash cultures, and his research with these species and Carolina ash will constitute another chapter of his thesis and another manuscript to be submitted for publication. How have the results been disseminated to communities of interest?During this period, results of our ash research were presented in the form of three presentations at national and regional conferences. A manuscript reporting a portion of this research has been accepted for publication in New Forests. What do you plan to do during the next reporting period to accomplish the goals?Currently, our plans call for additional production of ash somatic seedlings from cultures initiated 2018 - 2020 and transfer of the somatic seedlings of these clones to Forest Service and other collaborators for eventual testing for resistance to EAB. We also plan to initiate additional ash cultures from seeds from controlled pollinations between lingering ash parents, and hope to identify more collaborators who will breed among lingering ash individuals of different ash species (white ash, black ash) in different parts of the ranges of these two species and share the seeds with us for culture initiation. A specific goal for 2022 is to expand our efforts to initiate embryogenic cultures of Oregon ash with the help of Richard Sniezko of the USFS Dorena Genetic Resource Center in Oregon.

Impacts
What was accomplished under these goals? 1. To initiate embryogenic cultures from open-pollinated "lingering ash" trees representing the major North American ash species (white ash, green ash and black ash). In 2021, although we attempted to initiate cultures from lingering green and white ash trees from the Atlanta area and the Blue Ridge region of Georgia, our cooperators at Trees Atlanta and the Georgia Forestry Commission could only provide seeds from a single source tree and none of the explants we cultured from that tree produced embryogenic cultures, so no new culture lines were generated in 2021. 2. To initiate embryogenic cultures from control-pollinated seeds derived from crosses between lingering ash parents provided by USDA Forest Service cooperators. Our collaborators at the USDA Forest Service Northern Research Station in Delaware, OH provided seeds from a single cross between lingering green ash parents and from two open-pollinated lingering green ash parents in 2020. By the end of the project year (9/30/20), it was still too early to tell if any embryogenic cultures were produced from these explants, but during 2021, we ended up capturing one new embryogenic culture from the control-pollinated family and 4 new cultures from the two open-pollinated families. No new material was supplied by USDA Forest Service collaborators for culturing in 2021. 3. To initiate embryogenic cultures from open-pollinated seeds of rare ash species, such as Texas ash (Fraxinus albicans) and blue ash (Fraxinus quadrangulata). Cultures were initiated from seed explants of Texas ash, Mexican ash (F. berlandieriana) and blue ash trees in 2020. By the end of the project year (9/30/20), it was still too early to tell if any embryogenic cultures were produced from these explants, but during 2021, we ended up capturing 17 embryogenic cultures from seeds of two Texas trees and 6 embryogenic cultures from seeds of a single Mexican ash source tree. No blue ash cultures were produced. In 2021, we initiated cultures from seeds of 4 blue ash trees and 2 Oregon ash (F. latifolia) trees, but by the end of the project year (9/30/21), it was too early to tell if any embryogenic cultures were produced. 4. To produce somatic embryos and somatic seedlings from the embryogenic ash cultures. (Note: Somatic seedlings from lingering ash-derived clones produced in this project will be provided to cooperators in a related project so that they can be screened for EAB tolerance in the field). We have continued to produce somatic seedlings of lingering white ash and green ash from cultures started in previous years and at last count, we had over 100 green ash and white ash plantlets, representing 17 potentially EAB-resistant clones, in the greenhouse. We are preparing to ship these to our collaborators in Pennsylvania and Ohio to grow out for field testing. In 2021, we produced the first somatic seedlings from the Texas ash and Mexican ash cultures and these have been acclimatized and transferred to the greenhouse. 5. To optimize cryostorage and recovery protocols for ash embryogenic cultures, and to cryostore copies of all embryogenic ash cultures for ash germplasm conservation. (Note: Although not part of this proposal, EAB-tolerant clones identified by field testing by cooperators in a related project will eventually be recovered from cryostorage for scaled-up somatic seedling production). Final cryostorage experiments were completed in 2021 and data were analyzed. Results indicate that using the vitrification protocol results in more consistent and more rapid regrowth of green ash and white ash embryogenic culture material following recovery from cryostorage than the slow-cooling protocol, which previously had been our standard protocol. A manuscript reporting these results is in preparation. We have proceeded to apply vitrification operationally to get copies of all of our embryogenic ash cultures into cryostorage.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Merkle, S.A. 2021. Somatic embryogenesis: a multifunctional tool for conservation and restoration. Society and Policy Influences on Biotechnology Risk Assessment for Restoration of Threatened Forest Tree Species, April 21-22, 2021.
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Merkle, S.A. 2021. Propagating rare and threatened North American trees using tissue culture. International Society of Arboriculture Southern Chapter Virtual Conference, April 27, 2021
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Tull, A.R., H.J. Gladfelter, F. Pampolini, S. Fan, L. Rieske-Kinney, C.D. Nelson, A.G. Abbott and S.A. Merkle. 2021. Development of a new genetic transformation system for white and green ash using embryogenic cultures. 36th Southern Forest Tree Improvement Conference, June 7-9, 2021
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Richins, M.W.M., A.R. Tull and S.A. Merkle. 2021. Propagation and conservation of three rare North American ash species using somatic embryogenesis. 36th Southern Forest Tree Improvement Conference, June 7-9, 2021

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:The main target audience reached by our efforts during 2020 was other researchers working on ash species and emerald ash borer, and researchers working with forest health threats from other exotic pests and pathogens. The target audience also included stakeholders in the USDA Forest Service and state forestry agencies in states affected by emerald ash borer. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During this period, graduate student Mason Richins began new cryostorage experiments as part of his M.S. thesis work on ash embryogenesis and cryostorage. He is testing two different cryostorage approaches (slow-cooling and vitrification) and has completed some of the replications of his vitrification experiments during 2020. How have the results been disseminated to communities of interest?During this period, results of our ash research was presented in the form of two published abstracts in the proceedings of a regional tree genetics conference, and a poster presented at a national invasive species conference. What do you plan to do during the next reporting period to accomplish the goals?Currently, our plans call for additional production of ash somatic seedlings from cultures initiated 2018 - 2020 and transfer of the somatic seedlings of these clones to Forest Service collaborators for eventual testing for resistance to EAB. We also plan to initiate additional green ash cultures from seeds from controlled pollinations between lingering ash parents, and hope to identify more collaborators who will breed among lingering ash individuals of different ash species (white ash, black ash) and share the seeds with us for culture initiation.

Impacts
What was accomplished under these goals? 1. To initiate embryogenic cultures from open-pollinated "lingering ash" trees representing the major North American ash species (white ash, green ash and black ash). Cultures were initiated in August 2020 from 96 seed explants collected from two open-pollinated lingering green ash parents by cooperators at the USDA Forest Service Northern Research Station on Delaware, OH. By the end of the project year (9/30/20), it was still too early to tell if any embryogenic cultures were produced from these explants. 2. To initiate embryogenic cultures from control-pollinated seeds derived from crosses between lingering ash parents provided by USDA Forest Service cooperators. Our collaborators at the USDA Forest Service Northern Research Station in Delaware, OH could only provide seeds from a single cross between lingering green ash parents for our culture initiations in 2020, and most of these seeds were empty or damaged, so only 10 explants were cultured. By the end of the project year (9/30/20), it was still too early to tell if any embryogenic cultures were produced from these explants. 3. To initiate embryogenic cultures from open-pollinated seeds of rare ash species, such as Texas ash (Fraxinus albicans) and blue ash (Fraxinus quadrangulata). Cultures were also initiated from 135 seed explants collected from two Texas ash trees and from 119 seed explants collected from 2 Mexican ash trees by a cooperator at the Ladybird Johnson Wildflower Center in Austin, TX. We also initiated cultures from 93 blue ash seed explants collected by cooperators at the University of Kentucky in Lexington, KY. By the end of the project year (9/30/20), it was still too early to tell if any embryogenic cultures were produced from these explants. 4. To produce somatic embryos and somatic seedlings from the embryogenic ash cultures. (Note: Somatic seedlings from lingering ash-derived clones produced in this project will be provided to cooperators in a related project so that they can be screened for EAB tolerance in the field). We ultimately obtained 18 new embryogenic cultures from the open-pollinated seeds collected from three lingering green ash parents in Delaware, OH, and 11 embryogenic cultures from the OP seed explants collected from three Carolina ash (F. caroliniana) trees at Savannah River Ecology Lab, Aiken, SC in 2019 (see 2019 progress report for details on source trees and culture initiations). Somatic embryos were produced and somatic seedlings were produced from multiple Carolina ash cultures, hardened-off and moved to the greenhouse to continue growth. We have also continued to produce somatic seedlings of white ash and green ash from cultures started in previous years. 5. To optimize cryostorage and recovery protocols for ash embryogenic cultures, and to cryostore copies of all embryogenic ash cultures for ash germplasm conservation. (Note: Although not part of this proposal, EAB-tolerant clones identified by field testing by cooperators in a related project will eventually be recovered from cryostorage for scaled-up somatic seedling production). New cryostorage experiments were conducted testing a vitrification protocol. So far, regrowth of cultures seems to be more consistent and rapid than regrowth following our standard slow-cooling protocol, but experiments comparing both treatments in the same experiment have yet to be conducted.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Merkle, S.A., J. Koch, R. Tull, D. Carey, P. Montello, B. Barnes, L. House, K. Eidle, D. Herms and K. Gandhi. 2020. Combining breeding, somatic embryogenesis and cryostorage to create emerald ash borer-resistant ash varietals. In Proceedings: Booklet of Abstracts - 35th Southern Forest Tree Improvement Conference, June 3-6, 2019, Lexington KY, p. 32 [abstract]
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: House, L., C.R. Montes, P.M. Montello, A. R. Tull and S.A. Merkle. 2020. Development and optimization of a cryostorage protocol for embryogenic ash cultures. In Proceedings: Booklet of Abstracts - 35th Southern Forest Tree Improvement Conference, June 3-6, 2019, Lexington KY, p. 51 [abstract]
• Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2020 Citation: Merkle, S.A., J. Koch, R. Tull, D. Carey, P. Montello, B. Barnes, D. Herms, and K. Gandhi. 2020. A new hope: ash embryogenic research for creating emerald ash borer-resistant ash clones. 30th USDA Interagency Research Forum on Invasive Species, January 14-17, 2020, Annapolis, MD [poster]

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:The main target audience reached by our efforts during 2019 was other researchers working on ash species and emerald ash borer, as well as researchers working with forest health threats from exotic pests and pathogens. The target audience also included stakeholders in the USDA Forest Service and state forestry agencies in states affected by emerald ash borer. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During this period, an undergraduate in our program, Mr. Logan House, received training in ash embryogenic culture initiation and cryostorage as part of his Senior Thesis project, which he completed in May 2019. A new graduate student, Mr. Mason Richins, began his M.S. thesis work on ash embryogenesis and cryostorage in August 2019. How have the results been disseminated to communities of interest?During this period, results of our ash research was presented in the form of two oral presentations and two poster presentations at a regional tree genetics conference, a regional forest insect work conference and a national conference on innovations in forestry. What do you plan to do during the next reporting period to accomplish the goals?Currently, our plans call for production of ash somatic seedlings from cultures started during 2019 and the initiation of more white ash and green ash cultures from seeds from controlled pollinations between lingering ash parents, as well as for initiations from open pollinated seeds collected from additional rare ash species, specifically blue ash and Texas ash.

Impacts
What was accomplished under these goals? Impact statement The major expected impact of the project will be the production of hundreds of somatic seedlings derived from "lingering ash" parents of different ash species that will be made available to collaborators for use in clonal screens for EAB resistance. Ultimately, once testing of these clones is completed we believe these clones will provide the basis for new, EAB-resistant ash varieties that can be planted by landowners and communities in restoration efforts. The approach will not only generate clonal material for replicated testing to establish a strong genetic basis for EAB resistance, but will also provide a vehicle for scaled-up production of millions of EAB-resistant ash trees to use as planting stock. Accomplishments (by goal) 1. To initiate embryogenic cultures from open-pollinated "lingering ash" trees representing the major North American ash species (white ash, green ash and black ash). Cultures were initiated in August 2019 from 240 seed explants collected from three open-pollinated lingering green ash ash parents by cooperators at the USDA Forest Service Northern Research Station on Delaware, OH. By the end of the project year (9/30/19), it was still too early to tell if any embryogenic cultures were produced from these explants. 2. To initiate embryogenic cultures from control-pollinated seeds derived from crosses between lingering ash parents provided by USDA Forest Service cooperators. Unfortunately, our collaborators at the USDA Forest Service Northern Research Station in Delaware, OH, who provided CP material in 2018, could not provide any CP material for our experiments in 2019, so they provided OP material (see above). 3. To initiate embryogenic cultures from open-pollinated seeds of rare ash species, such as Texas ash (Fraxinus texensis) and blue ash (Fraxinus quadrangulata). Cultures were initiated in July and August 2019 from 300 seed explants collected from three open-pollinated Carolina ash (Fraxinus caroliniana) trees at Savannah River Ecology Lab, near Aiken, SC. Cultures were also initiated from 82 seed explants collected from a single Texas ash tree (state champion tree; Pioneer's Rest: Lat: 32.765931; Long: -97.329060; Tarrant County, Texas). By the end of the project year (9/30/19), it was still too early to tell if any embryogenic cultures were produced from these explants. 4. To produce somatic embryos and somatic seedlings from the embryogenic ash cultures. (Note: Somatic seedlings from lingering ash-derived clones produced in this project will be provided to cooperators in a related project so that they can be screened for EAB tolerance in the field). We obtained 4 embryogenic cultures from seeds derived from controlled pollinations between lingering green ash parents in 2018, and 19 embryogenic cultures from OP white ash and green ash seeds collected in Mercer County, PA that may have been lingering ash. We began producing somatic embryos and somatic seedlings from cultures initiated in 2018 during the spring of 2019 and this continued over the rest of the year. Somatic embryos were produced by all of the cultures and tested for germination, which ranged from 0 - 75%, depending on clone. Somatic seedlings representing many of these clones have already been produced, hardened off and moved to the greenhouse to continue growth. We have also continued to produce somatic seedlings of white ash and green ash from cultures started in previous years. 5. To optimize cryostorage and recovery protocols for ash embryogenic cultures, and to cryostore copies of all embryogenic ash cultures for ash germplasm conservation. (Note: Although not part of this proposal, EAB-tolerant clones identified by field testing by cooperators in a related project will eventually be recovered from cryostorage for scaled-up somatic seedling production). Experiments to test cryostorage protocols were conducted by an undergraduate researcher, results remained inconsistent, with some genotypes re-growing well following recovery from cryostorage and others not re-growing at all in one experiment, and behaving differently in the next experiment. However, the student's experiments did at least demonstrate the feasibility of applying cryostorage of the embryogenic cultures for conservation of ash germplasm.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Merkle, S.A., J. Koch, R. Tull, D. Carey, P. Montello, B. Barnes, L. House, K. Eidle, D. Herms and K. Gandhi. 2019. Combining breeding, somatic embryogenesis and cryostorage to create emerald ash borer-resistant ash varietals. 35th Southern Forest Tree Improvement Conference, June 3-6, 2019, Lexington, KY
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: House, L., C.R. Montes, P.M. Montello, A. R. Tull and S.A. Merkle. 2019. Development and optimization of a cryostorage protocol for embryogenic ash cultures. 35th Southern Forest Tree Improvement Conference, June 3-6, 2019, Lexington, KY
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Merkle, S.A. 2019. A partnership to restore threatened trees. 2019 Forest Innovation Reviews (FIRz) event, September 18, 2019, University of Georgia, Athens, GA
• Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2020 Citation: Merkle, S.A., J. Koch, R. Tull, D. Carey, P. Montello, B. Barnes, D. Herms, and K. Gandhi. 2019. Combining selection, breeding and in vitro mass propagation to generate emerald ash borer-resistant ash clones. 60th Southern Forest Insect Work Conference, July 23-26, 2019, Savannah, GA

Progress 07/03/18 to 09/30/18

Outputs
Target Audience:The main target audience reached by our efforts during 2018 was other researchers working on ash species and emerald ash borer, as well as researchers working with forest health threats from exotic pests and pathogens. My target audience also included stakeholders in the USDA Forest Service and state forestry agencies in states affected by emerald ash borer. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During this period, an undergraduate in our program, Mr. Logan House, received training in ash embryogenic culture initiation and cryostorage as part of his Senior Thesis project. How have the results been disseminated to communities of interest?During this period, results of our ash research was presented in poster format at one forest health conference. Also, paper for a presentation made at a 2016 tree conservation conference that included information on our ash work was published in the conference proceedings in 2018. What do you plan to do during the next reporting period to accomplish the goals?Currently, our plans call for production of ash somatic seedlings from cultures started during 2018 and the initiation of more white ash and green ash cultures from seeds from controlled pollinations between lingering ash parents, as well as for initiations from open pollinated seeds collected from rare ash species.

Impacts
What was accomplished under these goals? 1. To initiate embryogenic cultures from open-pollinated "lingering ash" trees representing the major North American ash species (white ash, green ash and black ash). Cultures were initiated in August 2018 from over 600 seeds collected from multiple lingering ash parents by cooperators in Minnesota, Pennsylvania and North Carolina. By the end of the project year (9/30/18), it was still too early to tell if any embryogenic cultures were produced from these explants. 2. To initiate embryogenic cultures from control-pollinated seeds derived from crosses between lingering ash parents provided by USDA Forest Service cooperators. Cultures were initiated in August 2018 from over 70 seeds from two crosses between lingering green ash parents made by collaborators at the USDA Forest Service Northern Research Station on Delaware, OH. By the end of the project year (9/30/18), it was still too early to tell if any embryogenic cultures were produced from these explants. 3. To initiate embryogenic cultures from open-pollinated seeds of rare ash species, such as Texas ash (Fraxinus texensis) and blue ash (Fraxinus quadrangulata). We were unable to get immature seeds of any of the rare ash species for culturing, but we did receive stored seed samples of F. texensis, F. quadrangulata and F. caroliniana from cooperator Dr. Robert Jetton (CAMCORE). The seeds of F. texensis supplied were all empty, but we cultured seeds of F. quadrangulata and F. caroliniana. By the end of the project year (9/30/18), it was still too early to tell if any embryogenic cultures were produced from these explants. 4. To produce somatic embryos and somatic seedlings from the embryogenic ash cultures. (Note: Somatic seedlings from lingering ash-derived clones produced in this project will be provided to cooperators in a related project so that they can be screened for EAB tolerance in the field). We continue to produce somatic seedlings of white ash and green ash from cultures started in previous years. The trees are acclimatized and moved to the greenhouse to continue growth. We should be able to start producing somatic seedlings from the new cultures during summer 2019. 5. To optimize cryostorage and recovery protocols for ash embryogenic cultures, and to cryostore copies of all embryogenic ash cultures for ash germplasm conservation. Experiments to test cryostorage protocols started in 2018, but to date, results have been inconsistent, with some genotypes re-growing well following recovery from cryostorage and others not re-growing at all.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Merkle, S.A., P.M. Montello, A. R. Tull, D.A, Herms and K.J.K. Gandhi. 2018. Somatic embryogenesis and cryostorage for restoration of ash forests devastated by emerald ash borer. Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry, August 5-10, 2018, Mt. Sterling, OH.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Merkle, S., Tull, A., Gladfelter, H., Montello, P., Mitchell, J., Ahn, C., & McNeill, R. (2018). Somatic embryogenesis and cryostorage for conservation and restoration of threatened forest trees. In R. A. Sniezko, G. Man, V. Hipkins, K. Woeste, D. Gwaze, J. T. Kliejunas, & B. A. McTeague (Eds.), Proceedings of Workshop on Gene Conservation of Tree SpeciesBanking on the Future. Gen. Tech. Rep. PNW-GTR-963 (pp. 113-116). Portland, OR: USDA Forest Service, Pacific Northwest Research Station.