Performing Department
(N/A)
Non Technical Summary
Our long-term goal is to create suitable and efficient agroforestry systems aimed at fostering a more sustainable wood fiber bioeconomy. However, the major bottleneck in efficient production of wood fiber is the presence of lignin, a polymer that makes wood recalcitrant to be deconstructed into cellulose and sugars (Himmel et al., 2007; Ball et al., 2023). Within the pulp industry, delignification processes are resource-intensive, both in terms of reagents and energy consumption, resulting in high chemical recovery costs (Chen et al., 2016). Hence, an ideal agroforestry for wood fiber production needs the elite trees producing the wood without the recalcitrance of lignin. Through the precise manipulation of lignin levels and composition using gene editing tools, we have successful developed the elite poplar varieties exhibiting enhanced traits for fiber pulping and reduced carbon emissions in our greenhouse trial (Sulis et al., 2023). The cultivation of these climate-smart elite trees offers a promising solution to a significant operational challenge faced by the paper and pulp industry (Figure 1). It holds the potential to introduce remarkable operational efficiencies, create new bioeconomic prospects, and yield substantial environmental benefits for both forestry landowners and paper and pulp mills.The United States is a significant player in the global paper and pulp industry. As of 2021, the US was both the world's largest producer of pulp for paper and the second-largest consumer of paper and paperboard, utilizing approximately 64 million metric tons (FAO, 2022). Around 10 million tons of lignin need to be removed per year as a by-product in the US paper and pulp industry (Demuner et al., 2021). The lignin removal process incurs significant energy expenses and releases chemicals into the environment causing pollution (Monte et al., 2009). Given that paper and pulp mills in southern US accounting for 74% of the national pulping capacity (Piva et al., 2014), evaluating our edited poplar varieties in southern US field trial is a crucial step in transforming fundamental knowledge generated from lab and greenhouse to the condition closer to industrial exploitation. To offer more hands-on guidance to pulpwood landowners, we have planned a field trial for our low-lignin elite variety in Alabama. This state is the largest producer of pulpwood among all southern states (Winn et al., 2023), making it an ideal location for practical testing and guidance. Although loblollypine(Pinus taeda L.) monoculture plantations with long-rotation management are most predominated agroforest inAlabama as the softwood pulpwood feedstock for local mills (Schultz, 1999; Kandhola et al., 2022), hardwood roundwood production still increased by 8% between 2020 and 2021, accounting for 20% of total pulpwood production (~ 22 million cord) in Alabama (Winn et al., 2023). Our poplar varieties offer a sustainable source of hardwood pulpwood with optimized properties for mill (Sulis et al., 2023). They can be managed in short rotations in the Southern US, allowing for harvesting every three to five years (Stanturf et al., 2015; Zalesny et al., 2019; Ile et al., 2022). The short rotation of poplar agroforestry could provide consistent revenue as pulpwood and bioenergy resource for landowners while minimizing soil disruption and promoting soil health restoration (Fritsche et al., 2017; Cowie et al., 2017). Consequently, the field trial in Alabama utilizing our lignin-modified elite poplar varieties presents a valuable opportunity for Southern US mills and landowners to produce pulpwood feedstock in a more cost-effective, reliable, and sustainable manner. Agroforestry in southern US is often subject to extreme weather conditions, such as drought, hurricanes, and ice storms (Bragg et al., 2003; Marengo et al., 2023; Peterson et al., 2012; Schoeneberger et al., 2017; Sharma et al., 2021). Here, field-grown trees experience annual seasonal growth and dormancy cycles while interacting with various biotic and abiotic environmental factors, including wind, drought, cold, and pathogens (Bishaw et al., 2021). These environmental factors can significantly alter the wood property, composition, and growth of trees (Harfouche et al., 2014; Cesarino et al., 2019; De Meester et al., 2023). Therefore, we should not only assess the growth and performance of edited trees from the field trial, but also measure the wood property and pulp yield of the wood harvested from trial plantation.Our planned field trial of these modified poplar varieties, along with associated physiological measurements, will be conducted at Auburn University (AU)'s granted plantation in Alabama. This choice is due to Alabama's prominent position as the leading pulpwood producer in the US. Wood chemistry and pulping tests for field-harvested wood will be carried out at North Carolina State University (NCSU). It's worth noting that these poplar varieties were originally created and characterized by Co-PI Wang at NCSU using a multiplex editing approach, and relevant platforms, such as nuclear magnetic resonance spectroscopy (NMR) and micro pulping, which were established to assess the efficiency of wood harvested for fiber pulping (Sulis et al., 2023). For practicality, we obtained 6-month-old edited poplar varieties from a greenhouse. Examining these trees revealed a remarkable reduction in lignin content, reaching up to 50% in certain varieties, as well as a 228% increase in the C-L ratio in others, while maintaining crucial wood growth and properties such as elasticity and density (Sulis et al., 2023). Furthermore, conducting pulp yield and carbon footprint assessments could enable mills to produce up to 40% more sustainable fibers while reducing greenhouse gas emissions associated with pulp production by up to 20% (Sulis et al., 2023). The large-scale cultivation of these elite poplar varieties represents a significant step toward a more sustainable fiber production system, aligning with the United Nations Sustainable Development Goals (Kümmerer et al., 2020). Additionally, the results from our field trial can be swiftly adopted by agroforestry landowners and mills to establish a novel fiber production system in the Southern US.
Animal Health Component
0%
Research Effort Categories
Basic
45%
Applied
45%
Developmental
10%
Goals / Objectives
?We aim to test the field performance of edited poplar varieties and the pulping efficiency of their wood harvesting outdoors in Southern US. Whether it is worth promoting our poplar varieties to stakeholders depends on the answers to these three questions. 1. Can our elite poplar varieties handle the stresses in the plantation in Southern US, outside the controlled greenhouse environment? 2. Can the wood from our elite poplar varieties consistently maintain its improved pulp yield performance through the recurring cycles of growth and dormancy and environmental stresses in the plantation? 3. Does agroforestry system using our elite poplar varieties gain more productivity, profitability, and sustainability merits in the southeast US? All three questions will be experimentally tested via field trials, laboratory analyses, and in silico modeling. The specific objectives of this A1701 Integrated standard project proposal are:1. Establish the short-rotation plantation of CRISPR-edited poplar varieties. Our plantation will be established in three steps: (1) Propagation of Edited Poplar Seedlings: Initially, we will propagate the edited poplar seedlings using tissue culture. (2) Cultivation in Greenhouse: Following propagation, the seedlings will be carefully cultivated in a greenhouse to attain a specific size. (3) Transplantation into the field: Finally, the well-grown seedlings will be transplanted into the field for further growth and development. Through these processes, we assessed the suitability of our CRISPR-edited poplar plants, propagated from tissue culture, for adaptability and their capacity to thrive in the Southern US agroforestry lands. These evaluations offer insights into the effect of our genetic edits on seedling development and tree survival outdoors.2. Evaluate growth and resilience related traits of the CRISPR-edited elite poplar varieties in the field trials. To better evaluate the growth and resilience performance of the edited poplar varieties in the Southern US field trial, we will monitor several physiological traits. 1. For the tree productivity, tree heights, basal diameters, aboveground biomass, photosynthetic and respiration phenotypes are monitored. 2. For tree resilience, xylem specific conductivity, water use efficiency, and relative water content of stem are investigated. 3. For tree tolerance to pathogen, we will score rust infection and insect damage according to the observation. By comparatively measuring these traits between our edited varieties and wild-type poplars in the field trial we will evaluate not only the productivity, profitability, resilience, and sustainability of our poplar varieties but will also provide guidelines for our stakeholders who will utilize our varieties for pulpwood production. Poplar varieties displaying strong fitness and robust growth during the field trial will be chosen for further tests of their wood's pulping yield and properties.3. Assess the cell wall properties and pulp yield efficiency of wood sourced from field-grown elite poplar varieties. The wood harvested from the chosen poplar varieties, as well as the control group, will undergo analysis to assess their wood cell wall properties, including lignin content and composition. These properties serve as potential indicators for the suitability of these woods for various industries. Subsequently, micro kraft pulping will be conducted to assess how well the modified poplar trees accommodate wood samples. Taken together, the implementation of these practices and the subsequent analysis of the results hold the potential to provide comprehensive and actionable insights for forest landowners and mills regarding the optimization of our modified poplar resources. This information includes comprehensive insights into optimizing harvesting cycles, identifying industries poised to reap the benefits of these unique wood varieties, and recommending adjustments to current procedures. This empowers stakeholders to make well-informed choices that not only maximize economic gains but also enhance environmental sustainability through these innovative poplar resources.4. Disseminate training and outreach materials and technical support for Extension agents, landowners, pulp mill managers, dealers, nursery managers, focused on forest growth and pulpwood production for a climate-smart agroforestry. This program includes face-to-face workshops, landowner workshops, and an online forum facilitated by AU's College of Forestry and Wildlife Sciences. Stakeholders can connect with researchers, consultants, and others in the agroforestry and pulp production community, applying knowledge gained during field-testing. Small landowners may conduct their own trials or participate in larger ones with pulp industries. A forum will facilitate knowledge exchange, fostering sustainable impact in the region, with continuous technical support provided by the co-PIs.
Project Methods
We described methods based on the objectives.Objective 1: Establish the short-rotation plantation of CRISPR-edited poplar varieties We hypothesize that:Edited poplar varieties will thrive under field conditions as in greenhouse and tissue-culture settings.Varieties will demonstrate superior wood properties and enhanced pulp yield without growth penalties.?Selection and Propagation (1a): Elite poplar seedlings, chosen based on their S/G ratio, lignin content, and greenhouse growth performance, will undergo propagation in Dr. Hao Chen's lab. After PCR validation and deep-amplicon sequencing, approximately 1,120 clonal propagules (70 per variety) and 200 wildtype trees will be produced for field trials.Cultivation in Greenhouse (1b): Post-propagation, seedlings will be cultivated to reach a height of 0.5 to 0.7 meters. Assessments of photosynthesis, photorespiration, and respiration will be conducted using LI-COR LI-6800XT, ensuring plant health prior to field transplantation.Field Transplantation and Trial (1c): 1,320 trees (1,120 edited and 200 wildtype) will be planted in Auburn, AL. The field trial will employ a detailed row-column design, with physiological measurements conducted biennially.Objective 2: Evaluate growth and resilience of CRISPR-edited poplar varieties in field trialsInitial Assessment: Two months post-transplantation, survival rates will be recorded to gauge adaptability.Productivity Evaluation (2a): Monthly measurements of tree height, basal diameter, and biomass, alongside photosynthesis, photorespiration, and respiration assessments will be conducted.Resilience to Abiotic Stress (2b): Wood density, MOE, and water content assessments will be made from year-old and two-year-old trees. Xylem functional traits and anatomical properties will be closely monitored and analyzed.Pathogen Tolerance (2c): Rust infection and insect damage will be scored during peak susceptibility, with data aiding in the overall assessment of biotic resilience.Objective 3: Assess cell wall properties and pulp yield efficiencyWood Chemistry Analysis (3a): Harvested wood from selected varieties and wildtypes will undergo rigorous testing for cellulose and lignin content to determine pulping efficiency and bioproduct potential.Lignin Composition and Linkages (3b): 2D HSQC NMR analysis will quantify lignin composition and interunit linkages to evaluate enhancements in wood processing for bioenergy and paper industries.Pulp Yield Testing (3c): Micro-Kraft pulping experiments will assess the pulp yield, with extensive analysis of fiber quality and crystallinity.