Performing Department
(N/A)
Non Technical Summary
American chestnuts were once among the most prominent hardwood trees in eastern US forests. These long-lived trees were culturally, ecologically, and economically important: they produced a uniquely consistent mast crop, providing food to a variety of wildlife, as well as a marketable food product and plentiful rot-resistant lumber. An invasive fungus (Cryphonectria parasitica), introduced from eastern Asia to the US in the late 1800s, causes a disease called chestnut blight, which killed billions of American chestnut trees and effectively removed them from their entire native range by the 1950s. Several efforts have been previously undertaken attempting to restore American chestnuts to the landscape, starting with USDA-sponsored programs in the early 1900's to plant non-native Asian or hybrid chestnuts. By the 1990s, The American Chestnut Foundation (TACF) was leading efforts to enhance blight resistance through a backcross breeding program, but years of data and new genomic analyses have revealed that backcrossing alone is unlikely to produce restoration chestnut trees with optimal combinations of blight resistance and American chestnut traits. We successfully produced a transgenic tree that effectively tolerates chestnut blight while retaining its full complement of American chestnut traits. The enhanced blight tolerance comes from a gene from wheat called oxalate oxidase (OxO), which degrades a toxin (oxalic acid) produced by the blight fungus. This means that the transgenic tree is not actually preventing or resisting blight infections, but rather tolerating them with less damage. This tree, known as Darling 58, has been planted under USDA-APHIS permits and outcrossed to many wild American chestnuts and closely related species, and plans are underway to start restoration efforts pending regulatory approval, which is expected in 2023. With numerous safety assessments and environmental comparisons already completed (some under previous BRAG support), regulatory reviews mostly complete, and public distribution likely starting soon, the work now transitions from confined research plots to larger-scale plantings in preparation for range-wide reintroduction efforts. Some of the specific assessments we will conduct under this grant include monitoring growth, blight resistance, flowering development, and photosynthetic performance of Darling 58 chestnut trees as they start to mature in real-world conditions. These measurements will be focused on Darling 58 chestnuts and related controls in three locations across the chestnut's range (NY, PA, and VA), each of which has plots in two environments (open and wooded), for a total of six plots. We will also measure the dispersal distance of Darling 58 pollen in a one-of-a-kind plot in NY that was specifically designed for this purpose. These types of observations will be critically important to understand fitness and landscape-scale dispersal of reproductively mature genetically engineered trees, and they are currently not possible in almost any other context besides the established Darling 58 chestnut plots in this study. Results from this work will be informative to diverse audiences including restoration practitioners, forest land managers, individual chestnut enthusiasts, students of various ages, other university researchers, and federal biotechnology regulatory agencies as they prepare to evaluate other genetically engineered trees in the future. If successful American chestnut restoration can be initiated using Darling 58 trees, there would not only be societal benefits resulting directly from the chestnut reintroduction, but this would also facilitate tremendous potential for using similar biotechnologies to address other dire threats to native trees.
Animal Health Component
40%
Research Effort Categories
Basic
50%
Applied
40%
Developmental
10%
Goals / Objectives
The American Chestnut Research and Restoration Project at ESF has a long-term goal of restoring American chestnuts (Castanea dentata) throughout their native range with a population of blight-resistant trees. We have successfully produced a transgenic tree (Darling 58) that effectively tolerates chestnut blight while retaining its full complement of American chestnut traits. The work now transitions from genetic manipulation and characterization to fully assessing the performance of this genetically engineered (GE) organism in the wild, its interaction with related native trees via outcrossing, and initiating effective distribution of this tree throughout its historic range in the eastern USA. The restoration of this species and its associated habitat would represent a major achievement for plant biotechnology being applied toward environmental conservation, and would facilitate the use of modern and creative approaches toward restoring other threatened species. We propose three overarching goals to advance this work, followed by specific objectives for each:Goal 1. Monitor fitness metrics (blight tolerance, growth, and physiological performance) of GE American chestnut trees compared to related controls in different environments. G1 - Objective 1. Measure annual growth (height and diameter) and flowering phenology G1 - Objective 2. Assess natural blight infections and general tree health annually G1 - Objective 3. Assess photosynthetic and respiratory physiologyGoal 2. Evaluate natural dispersal of viable GE pollen to sexually compatible wild relatives. G2 - Objective 1. Quantify effective pollination distance from flowering American chestnut trees G2 - Objective 2. Evaluate offspring from in inter- and intraspecific crosses of Castanea species, including both native and introduced American chestnut relatives, for transgene inheritance and seedling developmentGoal 3. Distribute GE American chestnuts to various stakeholders in an equitable and collaborative manner. (Pending regulatory approval by USDA-APHIS and EPA-BPPD) G3 - Objective 1. Distribute GE seedlings to individuals (i.e., citizen scientists) and educational partners G3 - Objective 2. Track these distributions to collect data on tree survival, growth, and environmental interactions in environments outside research plots G3 - Objective 3. Work with partners to evaluate and optimize education and outreach activities associated with demonstration plantings of GE chestnuts. In order to evaluate the performance, efficacy, and safety of transgenic blight-tolerant chestnuts compared to related controls (Goals 1 and 2), we previously established long-term research plots at three locations (BRAG Project #NYZ1148200) under APHIS permits. These consist of six genotypes in a complete block design with six replicate blocks in both orchard-style (open field) plantings as well as plantings in forest shelterwoods at each site (NY, PA, VA). We have also established a separate plot (NY) specifically to quantify effective pollination distance (Goal 2). We currently employ a full-time distribution manager, whose position combines education and outreach with planning and implementation of distribution plans for trees and other plant material (Goal 3). This includes work with partner organizations and other related groups to prepare for distribution of Darling 58 chestnuts. Work with various organizations including arboreta, botanical gardens, nature centers, and universities will establish small plantings with the goal of public outreach and education. Other staff at both TACF and ESF are planning for initial small-scale distribution of Darling 58 trees to the public. This includes a planned citizen scientist pollination effort, which involves both technical training (how to handle pollen and perform pollinations) and logistical management (shipping pollen and tracking pedigree data for crosses performed). Once initial distributions and pollination efforts begin with transgenic trees, we will work even more closely with members of the public, project collaborators, and TACF to see that basic information about new plantings is recorded and that observations are collected over time. While the APHIS permits covering these sites allow for meaningful research to take place, regulatory reviews of Darling 58 and the OxO transgene are underway by all three agencies under the U.S. Coordinated Framework for Regulation of Biotechnology, which would allow for unconfined release at these and other locations. First, the USDA-APHIS is evaluating our Petition for Nonregulated Status of Darling 58, and recently posted draft decision documents indicating a lack of plant pest risks or detrimental environmental impacts. A final decision is expected by August of 2023. The EPA is currently reviewing documentation for a FIFRA Section 3 Registration and an exemption from tolerance limits under the Food, Drug, and Cosmetics Act, and a request for broader exemption from FIFRA registration of the OxO PIP has recently been submitted to EPA as well. Third, we have submitted a dossier on Darling 58 chestnuts to the FDA's Center for Food Safety and Applied Nutrition under the Plant Biotechnology Consultation Program, which rigorously evaluates food and feed safety. All three reviews may conclude as early as the summer of 2023, which will allow initial public distribution of transgenic chestnuts and additional research plots to be installed outside of APHIS-permitted locations. We also continue to produce, grow, and test other transgenic chestnut events besides Darling 58, which will still be covered by APHIS permits unless they also are granted nonregulated status. While these regulatory reviews are ongoing, we continue to work in fenced locations specifically allowed via APHIS confined-release permits and are able to pursue goals 1-2 regardless of regulatory approval. This work will advance our specific goal of restoring ecologically important American chestnuts to their natural habitat in eastern US forests. It will also help regulators and restoration practitioners understand how newly developed material including transgenic trees interacts with wild relatives and disperses on the landscape. Finally, pending regulatory approval, this work will provide a unique opportunity to evaluate and track public participation in the distribution of culturally relevant trees.
Project Methods
Goal 1: Monitor fitness metrics (blight tolerance, growth, and physiological performance) of GE American chestnut trees compared to related controls in different environments. Observations and data collection will be conducted in the existing BRAG plots, and compared among tree types, plot environments (open vs. shelterwood), and locations (NY, PA, VA). Additional observations on related GE & non-GE trees nearby may be included to increase statistical power. Chestnuts will be monitored yearly for blight and other factors of seedling mortality (e.g., frost damage or herbivory). Blight will be quantified by number of cankers per seedling, girdling status, apparent canker severity (e.g., swollen vs sunken), and stem survival above a canker. Additionally, unique canker phenotypes found on GE trees will be evaluated and tracked over time to assess correlations to stem survival relative to traditional cankers. We will measure tree growth yearly using metrics of basal diameter at 5-cm-height, height, and stem length. Relative growth will be analyzed with a statistical comparison of the relative change in plant size that is strongly correlated with total mass (diameter2 * height; accounts for initial seedling size variation). Phenology of male (catkins) and female (developing burs) flowers will be evaluated by assessing presence of both flower types, age at first flower production, and development success of pollinated female flowers (number of viable nuts per flower). Photosynthetic performance of all taxa will be evaluated at each site with measurements of light-saturated photosynthetic rates (Asat) and light-response curves at ambient CO2 concentration. Biochemical parameters will be extracted from these curves and compared across taxa and sites. We will also assess the respiration rates of leaves and fine roots (Rleaf and Rroot) of GE and non-GE American chestnuts at least once per year. Assessing respiration is particularly important to this project, given the previous work documenting a stimulation of Rleaf in transgenic trees. We will measure Rroot for the first time by placing root systems in a custom chamber designed by PI Drake. Leaf and root samples measured for respiration rates will be excised, and processed for physical and chemical analysis (e.g, area, mass, and N concentration).Goal 2. Evaluate natural dispersal of viable GE pollen to sexually compatible wild relatives. This project will address several aspects of pollen dispersal from transgenic trees, which will benefit practitioners and regulators by increasing our understanding of impacts of gene flow from GE plants to sexually compatible wild relatives. Effective pollination distance will be evaluated at a unique plot in NY containing a central hub of 25 Darling 58 trees, and several cleared "spokes" (oriented primarily downwind of the hub) each with several clusters of three male-sterile hybrid chestnuts (sentinel trees). The central hub trees are the only source of viable chestnut pollen in a radius of more than 1.5km. Female flowers (developing burs) observed on the sentinel trees will be protected from herbivory and all viable nuts will be collected when ripe. Effective pollination distance will be determined according to the number of viable nuts collected at each spoke position. Pollination distance may also be assessed in other plots, using genotyping to identify both parents of a given nut or seedling. If necessary, viable pollination distance can also be inferred from single flowering trees by sampling fresh pollen at different distances and microscopically assessing its viability. We will also investigate crosses between GE American chestnuts and other Castanea species, including both native and introduced relatives. Ozark chinquapins (C. ozarkensis) are an important native relative, with which we previously performed controlled crosses with Darling 58 pollen. We will continue this work and include other Castanea species (C. pumila, C. sativa, C. mollissima & hybrids) as well. Pollinations with non-GE pollen will allow comparisons of pollination efficiency between GE and non-GE crosses. Pollination efficiency rates and transgene inheritance rates will be calculated in each of these cross types. Additionally, to understand if the OxO transgene could impact fitness of Ozark chinquapin hybrids, we will monitor the health and development of 78 transgenic hybrid seedlings (produced in 2021) by comparing relative growth rates between GE and non-GE full siblings. Relative blight resistance will be assessed with a leaf inoculation assay, including wild-type C. dentata and C. mollissima controls. This will provide valuable information about blight resistance and development of C. ozarkensis hybrids with and without OxO. In the following years we will plant more GE and non-GE C. ozarkensis hybrids, as well as crosses with other Castanea species and hybrids. Many parts of this Goal depend on tree development, are strongly seasonal, and need regulatory approval. If regulatory approval or tree maturity is delayed, questions involving inheritance and hybridization with relatives can be instead addressed under permits with controlled pollination.Goal 3. Distribute GE American chestnuts to various stakeholders in an equitable and collaborative manner. (Pending regulatory approval by USDA-APHIS and EPA-BPPD). We will manage distribution of Darling 58 chestnuts as efficiently and equitably as possible, while tracking outcomes and incorporating feedback to guide evidence-based decision making. We will work closely with TACF to engage with enthusiasts and key planting partners, using their "dentataBase" online tree tracking system which has recently been upgraded specifically for easier use by individuals installing small plantings. Using this system, we will collect and track observations (survival, growth, environmental interactions) reported by citizen scientists across the American chestnut's native range, on a scale that is not possible using university-managed research plots. Initial distributions will be divided among the research, demonstration, production, and public distribution priorities described previously, but outcomes from most of these priorities will be applicable to the data collection proposed here. Partnerships with various organizations including arboreta, botanical gardens, nature centers, and universities will establish small plantings with the goal of public outreach and education. Related distribution efforts include a planned citizen scientist pollination effort, involving technical training (how to handle pollen and pollinate) and logistical management (shipping pollen and tracking pedigree). Once initial distributions and pollination efforts begin with GE trees, we will work even more closely with members of the public, project collaborators, and TACF to see that basic information about new plantings is recorded and observations are collected over time. The timing of Objective 3 depends largely on regulatory approval from USDA-APHIS and EPA, so its sequence relative to the other Objectives is not entirely predictable.