Progress 07/01/24 to 06/30/25

Outputs
Target Audience:Target Audiences Reached During This Reporting Period During the current reporting period, our efforts primarily engaged three key target audiences: Extension Agents and Forestry Educators in the Southeastern U.S. These individuals were reached through early-stage engagement to lay the groundwork for future workshops and train-the-trainer programming. This audience is central to our outreach strategy because they serve as the critical interface between scientific advancements and community adoption. They were identified as essential stakeholders for translating our CRISPR-edited poplar research into practical guidance and decision-making tools for landowners, consultants, and pulpwood producers. Through collaboration with Auburn University's College of Forestry and Wildlife Sciences (CFWE) and the Southern Forest Nursery Management Cooperative (SFNMC), we initiated the development of region-specific, science-based materials tailored to the information needs and regulatory frameworks familiar to extension specialists in the Southeastern forestry sector. Forest Landowners and Managers Interested in Short-Rotation Woody Crops (SRWC) Our target audience includes small and medium-sized private landowners in Alabama and surrounding southern states who own marginal or underutilized land. These individuals were identified and contacted during the early planning of our Alabama field trial. We focused outreach efforts on landowners interested in diversifying their forestry income streams with short-rotation hardwoods like poplar, especially those already producing for the pulp and paper industry. Landowners were engaged through field trial planning, site selection, and will be involved next year for initial invitations to upcoming workshops. This group matters to our work because they are the potential adopters of the elite CRISPR-edited poplar varieties and management practices that could deliver both economic returns and environmental benefits such as reduced carbon footprint and improved soil health. The first year we focus on the propagation of seedlings and establishment of field trial. Nursery Managers and Industry Stakeholders in the Forest Seedling Supply Chain We initiated conversations with nursery managers, seedling producers, and forestry input suppliers, especially those affiliated with SFNMC, which represents over 80% of U.S. seedling production across 12 southern states. These stakeholders are critical to scale-up efforts and long-term sustainability of our project goals. Their inclusion at this early stage ensures that propagation strategies, tissue culture protocols, and eventual seedling distribution align with real-world nursery capacities. We focused specifically on understanding their feedback on genotype scalability and any potential regulatory or logistical barriers they foresee for gene-edited poplar introduction. Their perspectives were incorporated into the design of propagation and greenhouse preparation tasks, ensuring smoother transition from lab to field. Why These Audiences Were Targeted These groups were chosen because they represent the full ecosystem of decision-makers and implementers in the Southern U.S. pulpwood production pipeline--from research translation to practical deployment. Each plays a vital role in ensuring that the CRISPR-edited poplar varieties, designed for improved pulping efficiency and lower lignin content, can be adopted responsibly, profitably, and at scale. Extension agents are trusted sources of guidance for landowners and can amplify the impact of our findings through their networks. Forest landowners are the end-users of our research outputs. Their interest in sustainable pulpwood crops that also restore degraded land aligns directly with our project goals. Nursery managers bridge the gap between research propagation and commercial seedling production. Their early involvement enables feedback loops that inform our propagation methodology and variety selection criteria. How These Audiences Were Reached While formal workshops and courses are scheduled for later phases, this reporting period focused on relationship-building and collaborative planning. Key efforts included: Coordination meetings with SFNMC representatives and faculty at AU's CFWE to begin tailoring training materials. Site visits and outreach to landowners during the field trial site selection process in Alabama. Initial distribution of CRISPR poplar propagation protocols and technical specifications to nursery stakeholders for feedback on scalability and viability. Planning for extension workshop curricula covering CRISPR gene editing, poplar plantation management, lignin biology, and pulp production optimization next reporting period. Although no formal workshops were conducted during this early period, these groundwork efforts are foundational to our future educational and extension impacts. The engaged stakeholders are already informing our research direction and will continue to shape downstream dissemination activities. Changes/Problems:During the current reporting period, the project encountered several logistical and regulatory challenges that led to delays and adjustments in timeline, particularly regarding field trial implementation and administrative coordination. These challenges were not deviations from the scientific goals but rather procedural and institutional steps required to ensure compliance with federal and institutional standards for gene-edited tree research. Below are the primary areas where significant effort was directed to overcome unforeseen complexities. 1. Regulatory Approvals: USDA-APHIS Permitting and Institutional Compliance A critical step in our project involved obtaining the necessary regulatory approval from the United States Department of Agriculture--Animal and Plant Health Inspection Service (USDA-APHIS) to conduct confined field trials involving CRISPR-edited poplar trees. This process required the preparation of detailed permit applications outlining the genetic constructs, containment procedures, site management, and post-trial disposal plans. To meet APHIS biosafety standards, we worked closely with biosafety officers and regulatory consultants to ensure that the gene edits fell within exemption categories or were sufficiently justified for confined release. Simultaneously, we navigated Auburn University's internal review process, which included Institutional Biosafety Committee (IBC) oversight and coordination with campus facilities and compliance offices. These layered approval requirements extended the timeline for field deployment but were essential for ethical and legal adherence to federal guidelines on environmental releases of genetically engineered organisms. 2. Securing a Field Testing Site and Preparing for Long-Term Monitoring Identifying and preparing a suitable site for the long-term field evaluation of CRISPR-edited poplars posed another major logistical undertaking. We evaluated multiple candidate locations across central and southern Alabama, considering soil type, irrigation access, security, and long-term lease arrangements. After extensive consultation with university land management and external partners, we secured an Auburn-affiliated agricultural research site with the necessary acreage, fencing, and infrastructure. However, site readiness required additional steps, including soil testing, plot preparation, removal of legacy plantings, and installation of a new irrigation system tailored to short-rotation woody crops. These preparatory measures demanded unanticipated coordination with facilities and land-use planning personnel, resulting in schedule adjustments but ultimately improving the quality and research suitability of the field environment. 3. Training and Transition as a New PI As this project is led by a new Principal Investigator (PI), additional time and effort were dedicated to onboarding and training personnel, developing research protocols, and building institutional partnerships. The new PI led the training of graduate students and research staff in CRISPR propagation, field trial logistics, and biosafety protocols, often adapting instructional methods for participants unfamiliar with tree biotechnology or USDA compliance structures. Simultaneously, the PI worked to establish collaborations with extension personnel, forest industry stakeholders, and academic mentors to strengthen project execution and align activities with USDA NIFA's integrated research-extension-education goals. These training efforts added significant early-stage workload but were essential for building long-term project capacity and enhancing workforce development in forest biotechnology. 4. No Change in Scientific Objectives or Scope It is important to note that despite procedural delays, there were no changes to the scientific objectives, hypotheses, or scope of the project. The timeline for field trial deployment was adjusted to accommodate regulatory and logistical approvals, but all core research questions and evaluation metrics remain intact. The additional time has, in fact, enabled more robust preparation for data collection, pulping assay standardization, and stakeholder outreach. Special Reporting Requirements No additional reporting requirements beyond those specified in the original Terms and Conditions of the award have been triggered. However, we are maintaining detailed documentation of APHIS communications, field site compliance logs, and internal safety approvals for future reference, especially as field trials progress and more extensive plant material handling begins. What opportunities for training and professional development has the project provided?During this reporting period (the first year), the A1701 project provided diverse training and professional development opportunities to students, postdoctoral researchers, and early-career professionals engaged in fieldwork, laboratory analyses, and extension coordination. These opportunities supported skill-building in forest biotechnology, CRISPR applications, environmental field monitoring, and agroforestry system development. 1. Hands-on Training in CRISPR Propagation and Tree Biotechnology Graduate students and research assistants received direct training in the propagation and management of CRISPR-edited poplar lines. Under the guidance of faculty mentors and senior technical staff, trainees learned tissue culture protocols, shoot proliferation techniques, rooting induction, and acclimatization procedures necessary for transferring genetically edited trees from lab to greenhouse and then to field conditions. This training deepened their technical proficiency in plant regeneration and in vitro propagation systems, which are foundational to forest biotechnology. 2. Mentorship in Field Trial Establishment and Data Collection Protocols Graduate students, including those pursuing thesis work, participated in the design and establishment of field plots for short-rotation poplar trials. Through one-on-one mentorship and small group instruction, trainees learned how to design field layouts, standardize field data collection for growth and resilience traits, and conduct measurements such as basal diameter, height, biomass estimates, photosynthetic activity, water-use efficiency, and pathogen scoring. These activities trained participants in field-based phenotyping, experimental forestry design, and long-term monitoring strategies relevant to climate-resilient tree production. 3. Cross-disciplinary Exposure to Forest Genetics and Environmental Modeling Students working on the project attended internal seminars and reading groups to enhance their understanding of the intersection between gene editing, wood quality, and climate-smart agroforestry. Participants were introduced to techniques in genomic prediction, lignin biochemistry, and downstream pulp yield modeling. These sessions fostered cross-training in plant genetics, environmental biophysics, and industrial processing relevance--broadening the interdisciplinary expertise of all involved. 4. Graduate-Level Course Integration The newly developed course Forestry and Environmental Biotechnology (FORY 4970/6970) provided structured academic instruction for undergraduate and graduate students. Taught by project PIs, the course incorporated examples, data, and protocols directly from this USDA-funded project. Students gained conceptual and applied knowledge of CRISPR applications in forestry, regulatory and ecological considerations, and the future of biotechnological innovation in sustainable land management. This course served both as a training platform and a recruitment pipeline into project activities. 5. Professional Development through Conferences and Symposia Graduate students and postdoctoral researchers were supported to attend regional and national professional meetings where they presented posters or participated in technical sessions. These included forestry, plant biology, and biotechnology conferences where they networked with professionals in both academic and industry sectors. The project team also participated in a USDA-supported workshop on climate-smart forestry, further enhancing the team's policy literacy and outreach planning skills. 6. Extension-Facing Preparation and Stakeholder Engagement Trainees were engaged in the early planning stages of extension materials and stakeholder engagement events. This included preparing preliminary technical briefs, meeting with extension agents, and planning field visit protocols. These experiences strengthened participants' ability to translate technical knowledge into accessible formats for landowners, nursery managers, and pulp mill representatives--skills essential for careers in translational science and science communication. 7. Laboratory Skills and Wood Property Analysis Training Several graduate students participated in training to prepare for upcoming pulp yield analysis and cell wall chemistry assays. Although full wood sampling will occur in later project phases, students were trained in wet lab methods including lignin content determination, basic chromatography, and micro-kraft pulping procedures using archived greenhouse-grown biomass. This preemptive training ensures readiness for the next phase of experimental analysis and supports professional development in wood science and industrial biotechnology. How have the results been disseminated to communities of interest?Scientific and Academic Community During this reporting period, results and methodologies from the A1701 project were shared extensively with the scientific and academic community through conference presentations, academic integration, and internal seminars. Project team members presented posters and talks at professional meetings, highlighting the propagation success and preparation of field establishment of CRISPR-edited poplar varieties. These engagements allowed the team to disseminate preliminary findings--such as genotype-specific growth responses and transplant survival rates--and to receive feedback from forestry geneticists and plant biotechnologists. Additionally, the research has been incorporated into academic instruction through the new course Forestry and Environmental Biotechnology (FORY 4970/6970), where students engaged directly with real-world datasets and propagation protocols developed by the project. Internal seminars and lab group meetings further supported peer exchange and fostered cross-disciplinary learning among faculty, graduate students, and technical staff, contributing to a broader scientific dialogue on the role of gene editing in sustainable forestry. Forestry and Pulpwood Industry Stakeholders The project team also engaged forestry industry stakeholders--including nursery managers, cooperative members, and pulpwood processors--through targeted outreach designed to communicate the relevance of CRISPR poplar to commercial operations. Detailed propagation results, survival statistics from propogation,and early observations of environmental adaptability were shared during in-person meetings, virtual briefings, and site visits. For example, we collaborate closely with Alabama Center for Paper and Bioresource Engineering for checking how lignin perturbation affect plant intitial growth and possible fiber characteristic during their growth.These interactions provided a platform for two-way communication, allowing industry participants to offer input on genotype scalability, field deployment logistics, and anticipated regulatory concerns. Informational briefs and plain-language summaries were prepared to support these discussions, emphasizing the potential for enhanced pulping efficiency, faster rotations, and improved stress tolerance. Engagement with the Southern Forest Nursery Management Cooperative (SFNMC) in particular enabled broad dissemination across the seedling supply chain and helped ensure that project outputs are aligned with industry propagation capacity and field readiness expectations. What do you plan to do during the next reporting period to accomplish the goals?In the upcoming reporting period, the A1701 project will build upon the successful establishment of the CRISPR-edited poplar field trial by intensifying field-based monitoring and initiating physiological data collection. These efforts will directly address the project's core questions concerning plantation resilience, wood quality retention, and agroforestry value under Southern U.S. conditions. Objective 1: Establish the short-rotation plantation of CRISPR-edited poplar varieties Following the successful propagation, greenhouse acclimation, and field transplantation of CRISPR-edited and control poplar varieties, the next reporting period will focus on rigorous maintenance and early field performance tracking. We will implement a scheduled irrigation, fertilization, and weeding protocol across all plots to support uniform establishment and reduce abiotic stress variance between genotypes. Seasonal phenology observations--such as bud burst, leaf expansion, senescence, and overwintering signs--will be recorded to characterize how gene-edited lines differ from controls in adapting to the Southern climate. Additionally, we will develop a spatially resolved field map using GPS-tagged identifiers to track individual tree performance over time. This mapping system will enable cross-referencing of growth data, environmental gradients, and future biomass or harvest events. We also plan to install environmental sensors (e.g., soil moisture, temperature) to contextualize performance data with localized microclimatic variability. These detailed baseline observations are critical to identifying early growth bottlenecks or vigor advantages among the edited lines and to prepare for long-term site productivity assessment. Objective 2: Evaluate growth and resilience traits of CRISPR-edited elite poplar varieties in field trials The coming year will see the initiation of a full-scale phenotyping campaign for both productivity and resilience traits. Tree height, basal diameter, and crown width will be measured quarterly, while aboveground biomass estimates will be derived using site-specific allometric equations. Leaf area index (LAI) will be recorded using hemispherical photography or LAI meters to understand light interception and canopy development. For physiological traits, we will begin gas exchange measurements (photosynthetic rate, stomatal conductance, transpiration) using a portable LI-COR system, particularly under natural diurnal cycles and varying moisture conditions. We will assess water-use efficiency (WUE) through gravimetric and isotope-based methods and record stem relative water content and xylem-specific hydraulic conductivity using field and lab-based techniques. These indicators will help quantify how well CRISPR-edited genotypes maintain productivity under thermal or water stress conditions typical of Southern summers. For biotic stress, we will document rust (e.g., Melampsora spp.) incidence, leaf damage ratings from insect herbivory, and note any observed pathogen load. This comparative dataset will enable the identification of superior genotypes not only in terms of growth potential, but also in disease tolerance and drought resilience--traits essential for long-term success in operational agroforestry systems. Objective 3: Assess the cell wall properties and pulp yield efficiency of wood sourced from field-grown elite poplar varieties As the field trial is still in its early stages, trees have not yet reached the biomass threshold required for destructive sampling or pulping analysis. Therefore, in the next reporting period, we will focus on preparing protocols and benchmarking standards for upcoming chemical assays and micro-kraft pulping tests. Laboratory procedures for lignin content analysis (Klason method), S/G monomer profiling via pyrolysis-GC/MS, and wood fiber morphology (e.g., fiber length, coarseness) will be validated using archived samples from greenhouse-grown trees. These standardizations will ensure technical readiness for downstream processing once the field-grown trees reach appropriate size for wood harvesting, anticipated in subsequent years. Meanwhile, we will monitor growth trajectories to determine when select genotypes can be targeted for early pulping evaluation. Objective 4: Disseminate training and outreach materials and technical support to stakeholders We will begin engaging directly with identified Alabama-based stakeholders, including landowners, extension agents, nursery operators, and industrial pulpwood processors, to build collaborative pathways for knowledge exchange and future adoption. We have initiated discussions with Talladega County and Macon County landowners interested in exploring diversified agroforestry models using short-rotation hardwoods, as well as with Westervelt Company and International Paper, both of which operate within the Southeastern forestry sector and have expressed interest in bio-based innovations and sustainable fiber sources. Extension programming will be expanded to include a landowner-focused workshop, co-hosted by Auburn University's College of Forestry and Alabama Cooperative Extension System, covering topics such as CRISPR forestry basics, field trial results, and opportunities for small-scale field test participation. We will also design a stakeholder newsletter and establish a project webpage to disseminate seasonal updates, practical insights, and engagement opportunities. These efforts are expected to strengthen translational pathways from field data to practical application and build capacity for climate-smart forestry deployment.

Impacts
What was accomplished under these goals? During this reporting period, we successfully launched the establishment of a short-rotation field trial to evaluate the performance of CRISPR-edited poplar varieties under real-world Southern U.S. environmental conditions. This work directly supports our three overarching research questions--assessing plantation stress tolerance, sustained wood quality, and agroforestry value--by initiating the experimental foundation needed to address each. For Objective 1, we completed the three-phase establishment pipeline. First, CRISPR-edited poplar clones were propagated via tissue culture under controlled sterile conditions, ensuring genetic fidelity and uniformity across genotypes. Following propagation, these elite lines were hardened and grown in greenhouse environments until they reached transplantable size. This controlled phase allowed us to evaluate early growth vigor, rooting efficiency, and morphological traits influenced by genetic edits. In the controlled phage, early survival and establishment metrics have been recorded, including transplant shock response, root anchorage, and leaf emergence timing, providing critical baseline data for understanding genotype-by-environment interactions. These efforts confirm the feasibility of transitioning gene-edited lines from lab to landscape and form the experimental basis for assessing their environmental adaptability. Detailedly, now we have already have 1500 three-months old gene-editing varieties that will be planted out in September 2025. Our USDAAPHIS permits (A-0000484376) are still pending. We expect to get the approval around Auguest 2025. For Objective 2, post-transplant monitoring of growth and resilience traits has commenced. Tree height, basal diameter, and biomass accumulation are being measured alongside physiological traits such as photosynthetic rate, respiration, and water use efficiency. While long-term evaluation is ongoing, initial observations suggest that several edited lines exhibit comparable or superior establishment vigor to wild-type controls. This indicates potential for enhanced productivity and climate resilience, especially under the hot, humid, and stress-prone field conditions typical of the Southeastern U.S. rust disease symptoms and insect damage are also being scored visually, providing the first real-world pathogen and pest response data for edited lines. We identified several lignin knock-out varieties, such as CAD1 single knock-out, multiplex knockouts of C3H, CCoAOMT, COMT1, and CAD1, have significant higher root biomass, early root development in propogation, and enhanced root development, compared to the wild-type. Additionally, we also measured photosynthesis parameter of lignin pathway gene knock-out mutant. We identified two gene-editing varieties (C3H3 CAD1 AldOIMT1 C4H1 Knockout-2 and C4H1 CCoAOMT1 Knockout-1) having Jmax and Kmax when temperature increased (from 25 degree to 40 degree) compared to the wild-type, which suggested these varieties could have higher photosynthetsis activities when suffering to the heat stress. For Objective 3, although wood biomass from these field-planted trees is not yet mature enough for pulping analysis, we have finalized protocols and set benchmarks for future sampling. The early-stage data from the field will guide selection of candidate genotypes for destructive harvest in subsequent seasons. This ensures that only the most promising performers--based on field-derived growth and resilience data--are moved forward for cell wall characterization and micro-pulping assays. In preparation, we have calibrated lab-based lignin assays and established quality control metrics for downstream processing. For Objective 4, the field site has also served as an anchor point for early outreach to stakeholders. We have hosted initial site visits and collaborative meetings with extension agents and nursery managers to introduce them to the goals of the trial and preview future workshop content. These engagements are helping build a stakeholder network in anticipation of future workshops and online resources focused on climate-smart poplar agroforestry. In summary, the successful propagation, greenhouse cultivation, and field transplantation of CRISPR-edited poplar lines represents a major milestone for this integrated project. These foundational steps enable rigorous testing of performance traits that matter most to stakeholders--survivability, productivity, pulp quality, and environmental benefits--thereby advancing the practical deployment of gene-edited forestry resources in the Southeastern United States.

Publications

• Type: Book Chapters Status: Published Year Published: 2024 Citation: Developing tree improvement strategies for challenging environmental stresses under global climate change: a review from traditional tree breeding to genomics of adaptive traits for the quaking aspen." The Poplar Genome (2024): 153-182.
• Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Leites, L., McCafferty, M., LaDuke, N., Ayers, A., Storm, A., Penn, A., Clark, S., Ding, C., Warwell, M., Nelson, D., 2025. From seed to seedling: adaptation to climate in early life stages of Quercus alba populations within an assisted migration framework. WFGA-SFTIC-NFGA 2025 Joint Conference, The Pennsylvania State University, University Park, PA, June 2025.
• Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Xu, L-L. , Chen, H., Ding, C., Shearman, T., Qin, X. 2025. Evaluating Environmental Drivers of Southern Yellow Pine Niche Divergence in the Southeastern U.S. Using Species Distribution Models. WFGA-SFTIC-NFGA 2025 Joint Conference, The Pennsylvania State University, University Park, PA, June 2025.
• Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Ding, C., Chen., H., Dahal, R.P., Brouard, J.S. 2025. Assisted migration is plausible for a boreal tree species under climate change: A quantitative and population genetics study of trembling aspen. WFGA-SFTIC-NFGA 2025 Joint Conference, The Pennsylvania State University, University Park, PA, June 2025.
• Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Din, H.U., Dahal, R.P., KC, S., Furton, R., Latimer, W., Sanz-Saez, �., Wang, J., Chen., H., Ding, C. 2025. Photosynthetic efficiency and gas exchange dynamics in genetically modified poplar variants. WFGA-SFTIC-NFGA 2025 Joint Conference, The Pennsylvania State University, University Park, PA, June 2025.
• Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Dahal, R.P., KC, S., Din,H.U., Furton, R., Chen, H., DeWald, L.E., Nelson, C.D., Ding, C., 2025. Early multi-site genetic evaluation of growth and phenological traits in juvenile white oak (Quercus alba): Insights into heritability, genetic correlations, and adaptive potential. WFGA-SFTIC-NFGA 2025 Joint Conference, The Pennsylvania State University, University Park, PA, June 2025.
• Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Yang, P, Ding, C.*, L, Y., Chen, H., Narine, L., Cristan, R., Ji, Y-D. 2025. ForestAI: An AI-Powered Platform for Enhanced Forest Resource Assessment and Health Monitoring. Conference on Artificial Intelligence Applied to Agriculture, Mississippi State University Mar 31 to April 2nd. * corresponding author.