MONITORING DISPERSAL, FITNESS, AND DEPLOYMENT OF GE AMERICAN CHESTNUTS

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.