Greening-Resistant Genetically- Enhanced Trees: A case study. Michael S. Irey
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1 Greening-Resistant Genetically- Enhanced Trees: A case study Michael S. Irey
2 Huanglongbing aka Citrus Greening Found in Florida in 2005 Now found along with it s vector in: Florida (endemic) Georgia South Carolina Louisiana Texas California Considered to be the worst disease of citrus Bottom line Production costs up 40-50% Production losses Possible effects on quality Current management efforts are not sustainable..
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6 Varietal Resistance Most Tolerant Poncirus trifoliata, citranges Grapefruit, lemons, pummelos, sour orange Most Susceptible Sweet oranges, mandarins, tangelos
7 Varietal Resistance Bottom line: No real resistance in Citrus sp.
8 Sustainable Management Current pesticide usage can t continue Florida nursery capacity can t support increased tree mortality or increased roguing as trees begin to decline Varietal resistance Must be part of a long term strategy Resistance is generally thought to be foundation of a sustainable management program
9 Varietal Resistance How do we get there? Traditional breeding Need to identify adequate sources of resistance and then transfer the traits to commercial types Very long term approach (20+ years) Biotechnology Short to medium term approach The science may be the easy part.. Case study Development of a resistant plant using a gene from Spinach
10 Major steps Efficacy Which gene? How do you test? Greenhouse Field Regulations Federal State Freedom to operate Genetic elements Methods Regulatory USDA EPA FDA Cost of the projects and timeline
11 Efficacy Which gene? Want one that is not objectionable Not toxic to nontarget organisms Not allergenic No consumer acceptance issues How do we test for resistance? SGC Project Gene from Spinach Already in the food chain Contrasted with genes from human or pig Not allergenic Contrasted with genes from crustacean and bee venom Generally well received because it is spinach Toxicity?
12 Efficacy How do we test? Field trial vs. greenhouse trials If field trial, how long will it take If greenhouse trial, how will we inoculate and will it be representative of field resistance How do we get agronomic performance and yield data Juvenility Can t flower Trees/technology originates in Texas Florida quarantine procedures Had to develop a process to import to Florida
13 Efficacy Field trial USDA permit Yearly reporting requirements Yearly inspections Land intensive Expensive Carries $$$ penalties for violations Time consuming 3 years to get data 1 Mile radius = ~2,010 ac
14 Efficacy Field trial USDA permit Yearly reporting requirements Yearly inspections Land intensive Expensive Carries $$$ penalties for violations Time consuming Many years of data collection
15 Efficacy Field trial USDA permit Yearly reporting requirements Yearly inspections Land intensive Expensive Carries $$$ penalties for violations Time consuming Many years of data collection
16 Special considerations. A serious incident, or a history of lesser incidents, may prompt BRS to refer the situation to APHIS Investigative and Enforcement Services (IES) for further investigation. BRS also works closely with State Departments of Agriculture and other federal agencies, including the Food and Drug Administration, the Environmental Protection Agency and the Department of Justice, to ensure compliance with APHIS regulations. The Plant Protection Act provides for substantial penalties for serious infractions, including fines up to $500,000 and the possibility of criminal prosecution.
17 Efficacy Greenhouse testing Can test many more lines Quicker (less than a year) Little permitting required Less inspections Needs a dedicated facility: very expensive
18 Efficacy Death house Material must be tested for all diseases of concern before arrival Must be inspected on arrival Once in the death house, can never be released No propagations other than for testing
19 Efficacy
20 Freedom to operate Multiple things to consider Process used to make transgenics Actual genetic elements Tweaks Process involved in conferring resistance Selectable element Gene of interest * * * Promoter (switch to turn it on) Gene (antibiotic selection, functional gene) Terminator (switch to turn it off) Leader (enhancing sequence)
21 Freedom to operate Agrobacterium transformation Syngenta Codon optimization Better expression Various
22 Regulatory PIP Plant incorporated protectant Plant-incorporated protectants are pesticidal substances produced by plants and the genetic material necessary for the plant to produce the substance Will be regulated by: EPA USDA FDA (voluntary)
23 Regulatory Roles of the agencies EPA regulates the sale, distribution, and use of pesticides in order to protect health and the environment (EUPs) USDA (APHIS) is responsible for protecting agriculture from pests and diseases and has regulatory oversight over products of modern biotechnology that could pose such a risk FDA is responsible for ensuring the safety and proper labeling of all plant-derived foods and feeds, including those developed through biotechnology
24 Regulatory - EPA Need to establish the safety of the PIP Tolerance or tolerance exemption Currently done by event Trying to work towards by construct Trying also to work toward inclusion of all citrus Why is this important? Scions (Hamlin, Valencia, Navel, Lemon, Red Grapefruit, White Grapefruit, Murcott, etc.) Rootstocks (Carrizo, Swingle, C35, Sour Orange, etc.) Data requirements
25 Regulatory - EPA Guideline Ref. Description Product Identity Manufacturing Process Discussion of Formulation of Impurities Analysis of Samples Certification of Limits Submittal of Samples Acute oral Toxicity Subchronic Oral Toxicity Avian Acute Oral Toxicity Avian Dietary Toxicity Wild Mammal Toxicity Fish Toxicity Freshwater Aquatic Organism Toxicity Estuarine and Marine Organism Toxicity Non-target Plant Studies Non-target Insect Studies Honeybee Acute Toxicity Non-Guideline Product Characterization: Plasmid Map & Transformation Non-Guideline Product Characterization: Insert Integrity Non-Guideline Product Characterization: Copy Number Non-Guideline Equivalency of SoD2 + SoD7 proteins (GE citrus v native) Non-Guideline Product Characterization: Quantitative ELISA Analysis in Plant Tissues Non-Guideline Non-Guideline Non-Guideline Non-Guideline Non-Guideline Non-Guideline Non-Guideline ELISA Method Validation Product Characterization: Inheritance Patterns Product Characterization: Event Specific PCR Product Characterization: Protein Efficacy and Activity In-vitro Digestion/ Thermolability Potential for Allergenicity Bioinformatics Equivalency of SoD2 + SoD7 proteins (GE citrus vs. recombinant) Required data for a tolerance exemption/eup Some we will petition to be exempt from Some in progress All must be addressed All are expensive (ex. $250K for 0.5g)
26 Regulatory - EPA In theory, the EPA does not have a problem with the by construct approach, however.. We would have to prove that no new ORFs were created (i.e. no new proteins were produced by mistake) If there were, then we would have to go through the process with the new proteins as well or START OVER
27 Regulatory - USDA-APHIS In addition to providing permission and regulating the field trials, APHIS is the agency that would issue the document that evaluates whether the proposed Federal action would have significant effects on the environment. EA, EIS, and FONSI EA Environmental Assessment EIS Environmental Impact Statement FONSI Finding of No Significant Impact
28 Regulatory USDA-APHIS An EA is a concise document that briefly provides evidence and analysis for determining the environmental impacts of a proposed Federal action and alternatives. An EIS is a detailed written statement on the environmental impact of a proposed action; it is prepared if significant impacts are likely and if an agency is proposing a major action that significantly affects the quality of the environment. A FONSI is a document that presents the reasons why the agency has concluded that there are no significant environmental impacts projected to occur upon implementation of a proposed action.
29 Regulatory USDA-APHIS 2004 Petition from Monsanto for Nonregulated status APHIS conducted an EA January, Months later sued by organic growers Dec 2009 APHIS published draft EIS Final EIS Dec 2010
30 Regulatory USDA-APHIS We are assuming that an EIS will be needed 89
31 Regulatory USDA-APHIS Three choices in response for a petition for non regulated status: Take no action Determine that the article is no longer regulated on the whole Determine that the article is no longer regulated in part Based on the above information, APHIS has considered that C5 HoneySweet plum is not a plant pest and herby approves APHIS pettition no p. Therefore, C5 HoneySweet plums will no longer be considered regulated articles under the regulations at 7 CFR part 340.
32 Regulatory - FDA Voluntary In the 1992 policy, FDA recommended that developers consult with FDA about bioengineered foods under development; since issuance of the 1992 policy, developers have routinely done so. So in effect, nothing goes commercial without going through the FDA process These procedures describe a process in which a developer who intends to commercialize a bioengineered food meets with the agency to identify and discuss relevant safety, nutritional, or other regulatory issues regarding the bioengineered food and then submits to FDA a summary of its scientific and regulatory assessment of the food; FDA evaluates the submission and responds to the developer by letter
33 Regulatory - FDA The name of the bioengineered food and the crop from which it is derived. A description of the various applications or uses of the bioengineered food, including animal feed uses. Information concerning the sources, identities, and functions of introduced genetic material. Information on the purpose or intended technical effect of the modification, and its expected effect on the composition or characteristic properties of the food or feed. Information concerning the identity and function of expression products encoded by the introduced genetic material, including an estimate of the concentration of any expression product in the bioengineered crop or food derived thereof.
34 Regulatory - FDA Information regarding any known or suspected allergenicity and toxicity of expression products and the basis for concluding that foods containing the expression products can be safely consumed. Information comparing the composition or characteristics of the bioengineered food to that of food derived from the parental variety or other commonly consumed varieties with special emphasis on important nutrients, and toxicants that occur naturally in the food. A discussion of the available information that addresses whether the potential for the bioengineered food to induce an allergic response has been altered by the genetic modification. Any other information relevant to the safety and nutritional assessment of the bioengineered food.
35 Regulatory - FDA Series of consultations Unofficial Official Initial Consultations Resolution of safety, regulatory and nutritional issues Multiple Final Consultations Submit and defend What you are looking for is a letter of Not materially different in composition, safety, wholesomeness, or any relevant parameter.
36 Regulatory - FDA Intended effect Regulatory considerations Genetic modifications Food/Feed use Compositional analysis
37 Regulatory - FDA Intended effect Regulatory considerations Genetic modifications Food/Feed use Compositional analysis USDA-ARS has concluded that plum line C5 is not materially different in composition, safety, wholesomeness, or any relevant parameter from plums now grown, marketed, and consumed. At this time, based on USDA-ARS's data and information, the agency considers USDA-ARS's consultation on plum line C5 to be complete.
38 Regulatory - FDA In theory, FDA does not have a problem with the by construct approach, however.. With each new plant, you would have to prove that the new plant is substantially equivalent to the one that received the letter of not materially different So, some of the testing is eliminated but there would still be a significant amount of data that would be needed
39 Consumer Acceptance After the EPA, FDA and USDA, we now know it is safe for the environment, people, and animals, but.. We have to get over some other things: Source of the gene is important: Crab Human Pig How it is presented is important Nice to have vs. necessary Other benefits
40 Consumer Acceptance Arctic Apple Uses gene sequences actually derived from apple Resistant to enzymatic browning (don t brown when bruised, sliced, etc.) 1932 Submissions Most were negative
41 Consumer Acceptance
42 Consumer Acceptance It has to be done right
43 Recovery of the Investment Citrus is a vegetatively propagated crop Nursery tree approach: charge a technology access fee at the point of sale ~3,000,000 trees available to growers per year Probably can t charge too much for trees or it may be cost prohibitive One time shot Based on production No system to capture value If good for grower, then good for tree supplier More potential for value recovery Hybrid TBD
44 So with all of this being said. We are moving forward Think we have an acceptable gene (consumer) Initial data shows that some lines are resistant We are producing synthetic peptide and working on equivalency Starting bee toxicity testing Starting to generate data for EUP and tolerance exemption Have done the freedom to operate due diligence and have acquired rights to necessary technology
45 It is a daunting task, but the end goal is in sight.
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