Progress 09/01/08 to 08/31/12
Outputs
OUTPUTS: A variety of transgenic American chestnut tree events were compared to non-transgenic control trees produced by conventional breeding methods. The transgenic events contained two or more of the following transgenes: GFP, NPT2, BAR, OxO, and/or ESF39 AMP. The events varied by insert copy number (2 to 5 inserts) and transgene expressions levels. For example in two of these events, OxO expression was over 400 fold higher in the Darling 4 event, which had confirmed blight resistance approaching levels measured in Chinese chestnut, compared to the Wirsig event, in which blight resistance was not significantly different than the American chestnut control. Two transgenic events were produced by a single vector, and the remaining four events were produced by co-transformation with two vectors. Most events had plant defense transgenes; one had only selectable and scorable marker transgenes. The control plants included a tissue culture-produced, non-transformed clonal line to the transgenic events, seedling American and Chinese chestnuts, and three conventionally bred hybrids. This combination of transgenic event trees and non-transgenic control trees were planted in a randomized block design in one open field plot and one shelterwood plot at each of two study sites, for a total of four separate plantings in central New York state. Growth and survival, ectomycorrhizal colonization, insect feeding, and vascular plant colonization around the trees were measured and analyzed statistically. The first year of the grant was used to produce the transgenic trees from tissue culture and get them (along with seedling controls) established in the field. General weed control (mechanical treatment) was used only during the first year to help establish the trees. Afterwards, only small (1ft sq) ground cloths were used to reduce competition around the base of the trees. Insert copy number and transgene expression were analyzed throughout the study. The second and third years were used to collect and analyze field data. Insect feeding studies were limited by the small size of the trees and the resulting scarcity of available leaves, yet interesting results were obtained. All the tissue culture-derived plants (transgenic and non-transgenic) started out smaller than the seedlings because of the lack of the large nut that provides additional nutrients and energy to the seedlings. Trees that did not survive through a given winter were replaced and tagged so that the data could be adjusted accordingly. PARTICIPANTS: In addition to the co-PIs, below are additional researchers: Andrew Newhouse (technician) Katherine D'Amico (graduate student) Samual Tourtellot (graduate student) James Johnson (graduate student) Keith Post (graduate student) Partner organizations: The American Chestnut Foundation The Forest Health Initiative ArborGen LLC TARGET AUDIENCES: Regulators, researchers and the general public are the target audiences. Presentations have been presented to: 1. Update on American chestnut research. 10/29/10, Annual meeting of the New York chapter of The American Chestnut Foundation, Syracuse, NY 2. Progress on transgenic chestnut research. 9/16 - 9/18/10, Annual NE1033 chestnut researchers meeting, Cataloochee Ranch in Maggie Valley, NC 3. American Chestnut Research and Restoration Project. 1/28/11. Invited speaker: Clemson University Seminar, Clemson, SC 4. Restoration of the American chestnut: Old problem, New solutions. 2/17/11. Fayetteville Free Library, Fayetteville, NY 5. Developing Blight Resistance in Transgenic American Chestnut for Agroforestry and Restoration. 3/1-3/2/11. Consortium for Plant Biotechnology Research (CPBR) symposium, Washington, DC 6. Restoration of the American chestnut: Old problem, New solutions. 3/15/11. Council on Environmental Health meeting. Layfayette, NY 7. Update on American chestnut research. 4/7 - 4/8/11. Forest Health Initiative, Science Advisory Committee's reverse site visit, Raleigh, NC 8. Update on American chestnut research. 4/15-4/16/11. The American Chestnut Foundation's annual Board and Cabinet meetings, Abingdon, VA 9. American Chestnut Research and Restoration Project. 4/28/11. Invited speaker. Missouri University of Science and Technology, MO 10. American Chestnut Research and Restoration Project. 4/29/11. Danforth Plant Science Center, St Louis, MO 11. American Chestnut Research and Restoration Project. 4/29/11. Monsanto, St Louis, MO 12. Restoration of the American chestnut: Old problem, New solutions. 4/30/11. Missouri Botanical Gardens, St Louis, MO 13. Update on American chestnut research. The American Chestnut Science Cabinet planning meeting. 5/4/11. Penn State, State College, PA 14. Restoration of the American chestnut. 5/18/11. Cary Institute of Ecosystem Studies, Millbrook, NY 15. New York Botanical Garden transgenic American chestnut tree planting and seminar, 4/18/12, Bronx, NY 16. The 5th International Chestnut Summit, 9/6/12. Shepherdstown, WV 17. The Chestnut Symposium, 10/20/12, Asheville, NC 18. The Annual Meeting of the New York Chapter of The American Chestnut Foundation, 10/26/12, Syracuse NY PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The primary outcome of this study was that no significant differences could be detected between any of the various American chestnut transgenic events and the non-transgenic controls. This lack of differences between the transgenic trees and the conventionally bred trees was not due to the inability to detect differences. For example, in the growth and survival study, significant differences were detected between tissue culture trees and seedling trees, with the seedlings having higher survival rates, growing faster the first year and staying ahead in subsequent years. There were also site differences, in which the lower-elevation open field site promoted the largest growth, and the higher-elevation open field site showed the lowest growth and survival rates. Likewise, both site type and plant size had an effect on ectomycorrhizal colonization, with shelterwood sites providing more root colonization than open field sites, and larger plants showing more root colonization than smaller ones. Despite limitations due to plant size, the insect studies detected feeding differences with gypsy moth larva but not fall webworms. The differences were attributed to genotype and not to transgene presence, because two transgenic events and one traditionally-bred backcross tree all showed marginally higher feeding by gypsy moth and were not significantly different from one another. The plant colonization studies found significant site differences, but no differences associated with transgene presence. So, differences were detected in all these studies, but none were due to the tree being transgenic. The impact of this study for regulators is to be aware that planting sites and characteristics of the plant, i.e., plant production methods, base genotypes, and plant size, can all influence environmental impact studies, and therefore proper controls and replications are necessary to distinguish these effects from any caused by the transgenes. This study examined the impacts of small transgenic trees, as they would appear in initial plantings. A follow-up study is underway to track larger trees as they reach sexual maturity, and observe whether similar results are obtained. In conclusion, differences were detected for various reasons but none were due to transgene presence, copy number, expression level, or the number of different transgenes in a given event using the American chestnut transgenic events in this study.
Publications
- Post, K. H. and D. Parry. 2011. Non-Target Effects of Transgenic Blight-Resistant American Chestnut (Fagales: Fagaceae) on Insect Herbivores. Transgenic Plants & Insects. 40:955-963
- D'Amico, T. Horton, C. Maynard, and W. Powell. 2011. Assessing ectomycorrhizal associations and transgene expression in transgenic Castanea dentata. (Extended abstract for the IUFRO meeting in 2011) BioMed Central (BMC) Proceedings 2011, 5(Suppl 7):O54
- D'Amico, K.M. T.R. Horton, C.A. Maynard, S.V. Stehman, and W.A. Powell. 2012. Assessing Ectomycorrhizal Associations on Transgenic American Chestnut Compared to the Wild-Type, a Conventionally-Bred Hybrid, and Related Fagaceae Species (in review)
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: A variety of transgenic American chestnut tree events were compared to non-transgenic control trees produced by conventional breeding methods. The transgenic events contained two or more of the following transgenes: GFP, NPT2, BAR, OxO, and/or ESF39 AMP. The events varied by insert copy number (2 to 5 inserts) and transgene expressions levels (for example OxO expression was over 400 fold higher in Darling4 event compared to the Wirsig event). Two transgenic events were produced by a single vector, and the remaining four events were produced by co-transformation with two vectors. Most events had plant defense transgenes; one had only selectable and scorable marker transgenes. The control plants included a tissue culture-produced, non-transformed clonal line to the transgenic events, seedling American and Chinese chestnuts, and three conventionally bred hybrids. This combination of transgenic event trees and non-transgenic control trees were planted in a randomized block design in one open field plot and one shelterwood plot at each of two study sites, for a total of four separate plantings in central New York state. Growth and survival, ectomycorrhizal colonization, insect feeding, and vascular plant colonization around the trees were measured and analyzed statistically. The first year of the grant was used to produce the transgenic trees from tissue culture and get them (along with seedling controls) established in the field. General weed control (mechanical treatment) was used only during the first year to help establish the trees. Afterwards, only small (1ft sq) ground cloths were used to reduce competition around the base of the trees. Insert copy number and transgene expression were analyzed throughout the study. The second and third years were used to collect and analyze field data. Insect feeding studies were limited by the small size of the trees and the resulting scarcity of available leaves, yet interesting results were obtained. All the tissue culture-derived plants (transgenic and non-transgenic) started out smaller than the seedlings because of the lack of the large nut that provides additional nutrients and energy to the seedlings. Trees that did not survive through a given winter were replaced and tagged so that the data could be adjusted accordingly. PARTICIPANTS: In addition to the co-PIs, below are additional researchers: Andrew Newhouse (technician) Katherine D'Amico (graduate student) Samual Tourtellot (graduate student) James Johnson (graduate student) Keith Post (graduate student) Partner organizations: The American Chestnut Foundation The Forest Health Initiative ArborGen LLC TARGET AUDIENCES: Regulators, researchers and the general public are the target audiences. Presentations have been presented to: 1. Update on American chestnut research. 10/29/10, Annual meeting of the New York chapter of The American Chestnut Foundation, Syracuse, NY 2. Progress on transgenic chestnut research. 9/16 - 9/18/10, Annual NE1033 chestnut researchers meeting, Cataloochee Ranch in Maggie Valley, NC 3. American Chestnut Research and Restoration Project. 1/28/11. Invited speaker: Clemson University Seminar, Clemson, SC 4. Restoration of the American chestnut: Old problem, New solutions. 2/17/11. Fayetteville Free Library, Fayetteville, NY 5. Developing Blight Resistance in Transgenic American Chestnut for Agroforestry and Restoration. 3/1-3/2/11. Consortium for Plant Biotechnology Research (CPBR) symposium, Washington, DC 6. Restoration of the American chestnut: Old problem, New solutions. 3/15/11. Council on Environmental Health meeting. Layfayette, NY 7. Update on American chestnut research. 4/7 - 4/8/11. Forest Health Initiative, Science Advisory Committee's reverse site visit, Raleigh, NC 8. Update on American chestnut research. 4/15-4/16/11. The American Chestnut Foundation's annual Board and Cabinet meetings, Abingdon, VA 9. American Chestnut Research and Restoration Project. 4/28/11. Invited speaker. Missouri University of Science and Technology, MO 10. American Chestnut Research and Restoration Project. 4/29/11. Danforth Plant Science Center, St Louis, MO 11. American Chestnut Research and Restoration Project. 4/29/11. Monsanto, St Louis, MO 12. Restoration of the American chestnut: Old problem, New solutions. 4/30/11. Missouri Botanical Gardens, St Louis, MO 13. Update on American chestnut research. The American Chestnut Science Cabinet planning meeting. 5/4/11. Penn State, State College, PA 14. Restoration of the American chestnut. 5/18/11. Cary Institute of Ecosystem Studies, Millbrook, NY PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The primary outcome of this study was that no significant differences could be detected between any of the various American chestnut transgenic events and the non-transgenic controls. This lack of differences between the transgenic trees and the conventionally bred trees was not due to the inability to detect differences. For example, in the growth and survival study, significant differences were detected between tissue culture trees and seedling trees, with the seedlings having higher survival rates, growing faster the first year and staying ahead in subsequent years. There were also site differences, in which the lower-elevation open field site promoted the largest growth, and the higher-elevation open field site showed the lowest growth and survival rates. Likewise, both site type and plant size had an effect on ectomycorrhizal colonization, with shelterwood sites providing more root colonization than open field sites, and larger plants showing more root colonization than smaller ones. Despite limitations due to plant size, the insect studies detected feeding differences with gypsy moth larva but not fall webworms. The differences were attributed to genotype and not to transgene presence, because two transgenic events and one traditionally-bred backcross tree all showed marginally higher feeding by gypsy moth and were not significantly different from one another. The plant colonization studies found significant site differences, but no differences associated with transgene presence. So, differences were detected in all these studies, but none were due to the tree being transgenic. The impact of this study for regulators is to be aware that planting sites and characteristics of the plant, i.e., plant production methods, base genotypes, and plant size, can all influence environmental impact studies, and therefore proper controls and replications are necessary to distinguish these effects from any caused by the transgenes. This study examined the impacts of small transgenic trees, as they would appear in initial plantings. A useful follow-up study would track larger trees as they reach sexual maturity, and observe whether similar results are obtained. In conclusion, differences were detected for various reasons but none were due to transgene presence, copy number, expression level, or the number of different transgenes in a given event using the American chestnut transgenic events in this study.
Publications
- Post, K. H. and D. Parry. 2011. Non-Target Effects of Transgenic Blight-Resistant American Chestnut (Fagales: Fagaceae) on Insect Herbivores. Transgenic Plants & Insects. 40:955-963
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: Objective 1: A total of 120 transgenic and 160 non-transformed (tissue culture derived and seedlings) were planted (APHIS BRS Permit #08-011-105r-a2) in 2009 using a completely-randomized design on 3 x 3-meter spacing in 2 open-field and 2 shelterwood plots of 70 trees each. Deer fencing protected all planting areas. The transgenic events were Wirsig, Darling2, Darling3, Hinchee1, Hinchee2, and AN-2G3. The non-transgenic controls were tissue culture derived WB and seedlings of Zoar and Lasdon (Castanea dentata), Luvall's Monster, KLBC, GR68-B1 (hybrid chestnuts), Cropper and Qing (C. mollisima). Total field mortality the first year was 8.3% with 73% of this mortality in the tissue culture derived trees. This higher mortality was likely due to the smaller initial size when planted. The dead trees were replaced in the spring 2010. Objective 2: Roots were collected and assessed for mycorrhizal colonization from 209 trees at the 4 field sites. This equates to roughly 4 samples from each genotype. From these samples 1242 root tips were selected and stored in CTAB for RFLP analysis and DNA sequencing to determine species. Preliminary trends were evident. There does not appear to be any difference in mycorrhizal colonization rate between the chestnut genotypes. However, trees growing in shelterwood appear to have a greater diversity of morphological types (species) than those growing in open fields. DNA work and statistical analysis of the data are in progress. Objective 3: The diminutive size coupled with a late spring frost, significantly constrained our ability to conduct insect feeding experiments. Thus, statistical power for separating genotype differences was reduced. Nonetheless, we conducted a short-term growth bioassay with gypsy moth (Lymantria dispar L.), a species that we had previously found to be sensitive to differences among genotypes of Castanea. We found a marginally significant (p < 0.09) difference among the three sites tested. We did not detect a statistical difference in insect growth among the genotypes Cropper, Lasdon, KLBC, Darling4, Darling5, and WB. Ranked on percent growth rate differences, gypsy moth did best on the two transgenic genotypes and the tissue culture control and poorest on Cropper, similar to results from a previous study. In 2011, we will repeat our growth rate study, incorporating all sites and genotypes, and adding additional insect species. Objective 4: To examine the possible effects of transgenic chestnut on native plant growth, 3x3 ft water permeable mats were laid down around each tree to kill off any plants that may have already colonized the locations. The following spring, the mats for all of the 2009 planted trees were removed, leaving a 1x1 ft mat in place around the stem to prevent direct competition with the growing trees. After the mats were pulled, no plants were seen. During the end of July, the areas were checked for plant colonization. Out of 280 plots, less than 1% had any plants growing in the location of where the mat was pulled. In order to determine what the effects, if any, of the transgenic trees are on native vegetation, another season of observation will be required. PARTICIPANTS: William A. Powell (PI) Charles A. Maynard (Co-PI) Thomas Horton (Co-PI) Dylan Parry (Co-PI) Donald J. Leopold (Co-PI) Andrew Newhouse (technician) Katie D'Amico (graduate student) James Johnson (graduate student) Andrew Underwood (graduate student) Sam Tourtelott (graduate student) David Youngentob (undergraduate) Carrie Miller (undergraduate) TARGET AUDIENCES: The target audiences are Federal regulatory agencies to assist them in making science-based decisions about the effects of introducing genetically engineered organisms into the environment and also to other researchers doing field studies with transgenic plants targeted for eventual deregulation. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This second year was to establish the field plants and to take initial data for the mychorrizal, insect, and plant interaction studies. All the trees needed are in place for the rest of the objectives and the studies have begun. This year's studies were hampered somewhat by the small size of the trees but next summer this is not expected to be a problem. This project also reached out to the general public by holding the initial planting day with over 30 volunteers participating and an article in the local news. We also give routine tours of the field plantings to campus visitors. The annual meeting of the New York Chapter of The American Chestnut Foundation is being held at SUNY-ESF and it is expected that approximately 50 participants will tour the field plots.
Publications
- None to date. This project was designed to establish the field plots the first year with data collection in subsequent years. The data collection has just begun. But preliminary data has been presented at the 2010 annual meeting of the American Phytopathological Society meeting, the USDA regional NE1033 Chestnut meeting. They will also be presented at the annual meeting of the New York Chapter of The American Chestnut Foundation.
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: The first year we accomplished objective one. Two shelterwood sites were prepared by performing a selective cut to partially open the forest canopy. These two sites and two open field sites were fenced to protect the trees from deer. Randomized planting maps were prepared and the plots flagged. Prior to the field season, three transgenic events containing an oxalate oxidase gene from wheat, one empty vector transgenic event with only the GFP reporter gene and the BAR selectable marker gene, and non-transformed tissue culture derived plants of the same clone were propagated in tissue culture, rooted, acclimatized, and tested for transgene expression. Also during this time, seedling controls from two sources each of wild-type American and Chinese chestnut, two sources of AmericanXChineseXAmerican backcross chestnuts, and a mixed hybrid chestnut were grown. The initial planting was a public event with lay participants from The American Chestnut Foundation NY chapter members and volunteers from SUNY-ESF. The four field sites were maintained by mowing, weeding, and irrigation as needed through the summer. In the fall, trees that did not survive were replaced with newly produced trees. A total of 504 trees (transgenic plus controls) were planted initially. Two new transgenic events were produced during this time, multiplied, regenerated, rooted, and acclimatized. These plants are ready for the next spring planting and will increase the number of transgenic event test trees over the original 120 described in the proposal. PARTICIPANTS: William A. Powell - PI Charles A. Maynard - Co-PI Donald J. Leopold - Co-PI Thomas Horton - Co-PI Dylan Parry - Co-PI Christopher Nowak - collaborator Andrew Newhouse - technician Katie D'Amico - graduate student Tim Kuss - undergraduate field crew David Meyer - undergraduate field crew Meagan Collins - undergraduate research aide Lee Scriber - undergraduate research aide Carrie Miller - undergraduate research aide The American Chestnut Foundation, NY chapter- partner organization TARGET AUDIENCES: A field planting day was organized to allow the general public to handle and plant our transgenic and control trees. This was to demonstrate that there were no obvious differences in these trees. Approximately 30 lay people from TACF and SUNY-ESF participated in the planting. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This first year was to establish the field plants needed for the mychorrizal, insect, and plant interaction studies the following year. All the trees needed are in place for the rest of the objectives. This project also reached out to the general public by holding the planting day with over 30 volunteers participating and an article in the local news.
Publications
- No publications reported this period
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