Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
(N/A)
Non Technical Summary
developing commercial citrus varieties (rootstocks and scions) with genetic resistance to HLB using traditional techniques and gene editing, consolidating screening efforts to identify high-priority intervention targets for advanced testing and commercialization. CLas-induced callose and ROS hyperaccumulation is a significant breakthrough. This finding will facilitate the development of HLB solutions in these regions, potentially leading to future acceptance and integration of these transgenic varieties.
Animal Health Component
80%
Research Effort Categories
Basic
10%
Applied
80%
Developmental
10%
Goals / Objectives
We have created several transgenic lines expressing the Arabidopsis thaliana NPR1 (AtNPR1) gene, which have shown consistent and robust tolerance to Huanglongbing (HLB) through multiple generations of grafting-propagated progeny plants. Despite high levels of AtNPR1 protein in the leaves of these transgenic plants, minimal accumulation occurs in the fruit pulp and juice. Importantly, any residual AtNPR1 in the fruit can be rapidly broken down by digestive enzymes such as pepsin and trypsin in the stomach and intestines, ensuring that the transgenic citrus remains safe for consumption. Our primary objective here is to scale up production of these promising transgenic lines for large-scale field trials at two prominent locations in Florida. Concurrently, we will gather comprehensive data to support plant patent applications and secure regulatory approvals from USDA, EPA, and FDA. Furthermore, we will provide clean budwood resources to the industry through the FDACS/DPI Bureau of Citrus Budwood Registration Program, facilitating widespread adoption of these advanced transgenic citrus varieties. Addressing public concerns about genetically modified organisms (GMOs) is our secondary goal. To achieve this, we will conduct an extensive review of public attitudes and perceptions via literature analysis and environmental scans focused on genetic modification. This foundational research will guide a targeted survey designed to identify knowledge gaps and perceptions specifically related to transgenic citrus. Based on these insights, we will develop tailored communication tools and educational materials to effectively communicate the benefits and safety of AtNPR1 transgenic citrus to stakeholders, aiming to foster acceptance among consumers and citrus growers alike.
Project Methods
Objective 1: Conduct large-scale field trials(a) Generate plants for field trials. We have developed two HLB-tolerant AtNPR1 transgenic lines (26-36 and 35-30) from mature tissue transformation and three lines (13-3, 13- 29, and 57-28) from juvenile tissue transformation. We will collect budwood from lines 26-36 and 35-30, as these lines have flowered, and the mature budwood will enable immediate flower- ing after planting. The transgenic lines 13-3, 13-29, and 57-28 have not yet flowered and will only be propagated after they produce mature budwood, which is expected in Spring 2025. After the Spring 2024 flushes have hardened off, we will collect budwood/budsticks by thinning the canopy (rather than pruning) to ensure some fruits remain on the trees for harvesting and charac- terization. Transgenic buds will be grafted onto the superior rootstock 'US942'.(b) Plant the transgenic trees into the field. Field trials will be conducted at two locations in Florida to rigorously evaluate the performance of the transgenic and control trees. The experi- mental design will follow a randomized complete block format to ensure the reliability and re- producibility of the results. To prepare for the trials, we will select healthy and vigorous trees from our greenhouse preparation efforts. This selection process will involve assessing overall health, growth rate and absence of disease symptoms to ensure that only the most robust trees are used in the trials. Each plot at both trial locations will contain at least 20 trees per tested line, with up to six replicates/blocks per genotype (n = 120), to provide sufficient statistical power for our analysis. The randomized complete block design will help account for environmental varia- bility across the trial sites, thereby enabling more accurate comparisons between the transgenic lines and the control trees.(c) Management of the field trials. The trials will be managed following the established practices used in commercial citrus production groves to ensure the conditions reflect real-world agricultural settings. This includes adhering to standard irrigation, fertilization, as well as pest and disease control procedures.Objective 2: Collect data for patent application and regulatory concerns(a) Collect and analyze field trial data. HLB and other disease symptoms will be scored, and morphological phenotypes of the transgenic lines will be measured and recorded quarterly. These measurements will include various aspects of the trees (size, height, spread, vigor, density, form, growth habit, etc.), trunks (diameter, texture, bark color, etc.), branches (crotch angel, branch length, texture, color, spines, etc.), leaves (size, thickness, type, shape, apex, base, margin, surface, color, petiole, etc.), flowers and flower buds (type, bearing, diame- ter, depth, blooming period, flower bud size/shape, color, petal, sepal, pedicel, pistil, style, ovary, fragrance, etc.), and fruits (weight, size, shape, apex, base, harvesting time, fruit stem, core, skin, flesh, seeds, etc.). The yield of each tree will be determined at the end of each season.(b) Quantification of AtNPR1 in transgenic fruit and juice by MS analysis. We will develop an absolute quantification method to precisely determine protein concentrations in biological sam- ples (Gerber et al., 2003; Villanueva et al., 2014). This method involves adding defined amounts of isotope-labeled standards, known as AQUA peptides, which share properties such as chroma- tographic behavior and fragmentation pattern with their biological counterparts but can be distinguished by their mass difference.Objective 3: Produce clean budwood resources for industry usea) Bureau of Citrus Budwood Registration Program. Budwoods collected from the transgenic trees in the field will be brought to the Winter Haven Budwood Laboratory in Winter Haven, FL. To ensure the highest likelihood of success, all budwoods will be properly hardened off and prepared for grafting. For the initial parent propagations, at least 16 viable bud eyes will be included. Additionally, six budsticks, each approximately 10 inches long, will be provided for tissue culture purposes.(b) Clean the budwoods in the Mou lab. We will also clean the budwood in the laboratory by treating transgenic plants in a growth chamber with alternating temperatures of 25°C for 4 hours and 42°C for 4 hours. This treatment regimen is designed to promote the formation of new shoots that are free of pathogens. We have successfully used this method to eliminate CLas and CTV infection from citrus plants, demonstrating its effectiveness.Objective 4: Acquire regulatory approvals(a) Prepare and submit the documents regulatory agencies need. Preparation of the documents required by APHIS, EPA, and FDA will necessitate both scientific expertise and ad- ministrative experience. The dossier supporting our request for a Regulatory Status Review (RSR) has been submitted to APHIS and is currently under review.(b) Respond to regulatory agencies promptly and acquire new data as needed: Timely regulatory approval will hinge on our ability to respond to agency requests. If new data are required, we will promptly take the necessary steps to acquire them. Should written responses be needed to address concerns raised by the regulatory agencies, we will provide thorough and swift replies.Objective 5: Identify the potential market and any perceived barriers(a) Literature review and environmental scan. A literature review of recent peer-reviewed studies focusing on public perception of terms related to "transgenic" or "genetic modifi-cation" - including but not limited to "genetically engineered foods," "GMOs," and "CRISPR" - will be conducted.(b) Survey (U.S. and Canada). The literature review and environmental scan results will guide researchers in developing of a survey to identify current knowledge gaps and perceptions of genetic modification related to agriculture and food.(C) Communication toolkit development. Based on the research results, the PIE Center will cre- ate a communication toolkit plan and materials for communicating about transgenic citrus to tar- geted audiences.Objective 6: Engage stakeholders in project development and execution(a) Engage stakeholders. We are evaluating transgenic lines at Picos Farm, Fort Pierce, FL, and acquiring permits for field trials of the AtNPR1 transgenic lines. Grower collaborators will aid in plant propagation and field tests. We will establish a stakeholder advisory board at the start of the project in the first quarter to guide the project, and identify and prioritize research and extension objectives. Annual meetings will be held to update the Advisory Board, present find- ings, and gather feedback.(b) Develop teaching materials. We will develop teaching materials, including PowerPoint slides, extension articles, and a website, catering to diverse audiences such as citrus growers, in- dustry representatives, consumers, and state/federal regulators. These resources will offercomprehensive insights into transgenic technologies, their role in combating HLB, and their safe utilization. Our primary message will emphasize that transgenic approaches involve inserting a small plant DNA fragment into the citrus genome, akin to methods used in traditional breeding.(c) Create flyers, handouts, and fact sheets. These materials will utilize accessible language in both English and Spanish, covering two key topics.(d) Organize workshops, seminars, and meetings. We will capitalize on the well-established cit- rus grower and industry meetings in FL and CA to engage with and educate a diverse audience, including citrus growers, industry representatives, students, and the general public, regarding the significance of the transgenic lines developed in our research for combating HLB in the citrus industry.