GM Crop Research, Development, & Biosafety Considerations Fundamentals of Biosafety: Risk Assessment and Risk Management
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1 GM Crop Research, Development, & Biosafety Considerations Fundamentals of Biosafety: Risk Assessment and Risk Management Peter J. Raymond Technical Advisor Program for Biosafety System (PBS) Dar es Salaam, Tanzania September 2015
2 Background Education BS/Plant and Soil Sciences, UVM, Vermont. MS/Plant Pathology, Kansas State University Work Experience Kansas State University/Wheat Pathology Midwest Agricultural Research & Discovery Regulatory Trial Coordinator (maize, soy, cotton) Regulatory Affairs (Bt & VR potato) Program for Biosafety Systems (PBS) Biosafety Resource Network (BRN)
3 Science is continuously improving the quantity and quality of food production BC Fermentation & Leavening 1800 s Mendel s Pea, Darwin s Species, Pasteur s Microbes Antibiotics, Pasteurization, Preservation, Crop Breeding DNA, Human Nutrition, Fortification, Green Revolution Gene Sequencing, Biotech Crops, Human Insulin 2001 Human Genome, Plant Genome, Animal Genome 3 * Photo credit: AAAS, ARS, Nature
4 F M. Ac. Rapid Adoption of GM Crops since 1996 Rapid Adoption due to productivity benefits for small and large farmers m ac in m farmers in canola cotton corn soy BT MAIZE BT COTTON 0 // SOURCE: ISAAA Global Status of Commercialized Biotech Crops: 2008Brief No 38 HT MAIZE Seed delivery system- familiar, ease in adoption, and scale neutral.
5 GM CROPS: Unprecedented Adoption by Farmers 2016 marks two decades of the use of GM Crop technologies 2014: >181 million hectares planted globally Diversity of crops, including: 82% of global cotton 75% of global soybean 32% of global maize 26% of global canola 100% of papaya in Hawaii (20 million hectares) (70 million hectares) (70 million hectares) (8 million hectares) (100 hectares) Planted by farmers in 29 countries 10 countries in LATAM 8 countries in Europe 6 countries in Asia 3 countries in Africa 2 in North America Horizon: Ghana, Indonesia, Kenya, Nigeria, Uganda, Vietnam SOURCE: ISAAA Global Status of Commercialized Biotech Crops: 2014
6 GM Crop Technology is Progressing, Building Upon the Experience and Learning Acquired to enable Informed Decisions in Future The evolution of biotechnology benefits Bio-Materials Bio-fuels and bio-pharma Quality Traits Human nutrition and animal productivity 2 nd Generation Agronomic Traits Crop yield and drought tolerance 1 st Generation Agronomic Traits Crop production and pest management 6
7 Insect Protected (Bt) Crops European Corn Borer Control: Maize Substantial losses in the USA 1 st generation: 1-5% loss 2 nd generation: 2-15% loss ECB control difficult to contact with insecticides, so much so that less than 20% US acres treated. Estimated: $1B US loss Corn Rootworm Control: Maize
8 Drought Tolerance Leads Advance into 2004 Field Trials Corn without gene Corn with gene Mid Summer Vegetative Drought Tolerance 8 Source: Monsanto Biotechnology Pipeline, Dec 2004; Doug Rushing
9 GM Biofortified Crops β-carotene Enriched Rice: Golden Rice β-carotene Enriched Cassava Elevated Iron and Pro Vitamin A in Banana To address and find solutions for Vitamin and Mineral Deficiencies South and South East Asia Africa Cassava Banana Rice Sorghum
10 What is Biotechnology? Biotechnology is defined as a set of tools that uses living organisms (or parts of organisms) to make/ improve plants for specific uses.. Builds upon the traditions of: Plant Domestication: Selections since agriculture began (last 11,000-17,000 years before present): Wheat, Rice, Maize, Potato, Soya, Cassava. Crop Improvement- Controlled Crosses- Controlled Pollination. Solid gains since 1900 (115 years). Exploiting Variability and Diversity: (Exotic germplasm, wide/wild crosses (since 1960).
11 What is Biotechnology? Ag GM Crop Technology: to Plant Breeders: Interkingdom (eg. Bacteria to Higher plants) gene transfer to combine specific and unique traits from a myriad of sources into consideration for broad goal of sustainable agriculture and protection of environment. DNA/RNA as universal language of life- all life. Tap into other genomes for sources of useful traits.
12 DNA: Focus of Modern Biotechnology DNA can be viewed as a strand of pearls Each pearl represents a gene that encodes a protein
13 Plant Breeding TRADITIONAL BREEDING Desired Trait Desired Trait Donor Plant Undesirable Traits X Adapted Farmer Preferred Population Undesirable Traits Plant with Novel Trait Limited to accessing genes from sexually compatible species; lengthy and inefficient process Large segments of extra DNA are introduced which result in undesirable phenotypic or agronomic impacts Backcrossing necessary to acquire the adaptive traits of the recurrent parent (several generations) Modes of action of desired gene and undesirable gene are unknown 1 Recurrent parent contains genes for adaptation, yield, and quality that you want to recover and carry forward in a new variety.
14 Rice & Wild Relatives Barley and Wild Relatives Evolutionary interrelationships between Principal subfamilies and tribes within Gramineae: Relative size and diversity. Adapted from: G. Ledyard Stebbins, 1971)
15 Biotechnology TRANSGENIC ENGINEERING Desired Gene Commercial Cultivar Improved Commercial Cultivar Desirable genes can be accessed from many organisms The mode of action of desired gene is understood Absence of any undesirable DNA sequences Gene transfer is precise, rapid, & efficient Burdened by intensive regulation
16 Assembling a New Gene Construct The instructions : the gene gets made into a protein that confers the desired trait The stop sign Marker Promoter Gene Terminus To help identify the plants containing the new gene Serves as the switch and the engine, turning the gene on and off and driving expression of the gene
17 Agrobacterium-mediated Trait Transfer Agrobacterium is a natural deliver system for delivering foreign DNA into plants. Utilizing this common soil bacterium that causes crown gall, it infects and then and inserts a parcel of its own genetic material A. tumefaciens causes crown galls 1969 Tumor induction (Ti) responsible for galls 1974 Disarmed strains without Ti still infective 1983 Routine interkingdom transfer of DNA bacteria origin into higher plants and their expression
18 Agrobacterium-mediated Trait Transfer Agrobacterium is a natural deliver system for delivering foreign DNA into plants. Tools necessary: site specific restriction endonuclease And ligases that help repair sticky ends together to bond. T-DNA
19 Biolistic Transformation: the Gene Gun + DNA gold particles pressure and vacuum Select and regenerate plants with the new DNA. These are transgenic plants. Some of the DNA will integrate into the plant s DNA, or genome.
20 Regenerated Shoots Transgenic Plantlets
21 then out to the field for testing
22 CFT/MLT Regulated Field Trials in Africa Country Regulated Field Trials Burkina Faso Bt cowpea, 2008 Bt Cotton 1 Ghana Egypt Sudan Nigeria Cameroon Kenya Uganda Malawi Bt cotton, Bt cowpea DT wheat, 2008 Bt maize (supp.) 2012 Bt Cotton Bt cowpea, Vit enh. cassava, Vit enh. sorghum Bt/Ht cotton Bt maize, DT maize, Bt cotton VR CMV & CBSD cassava IR VR sweet potato Bt pigeon pea Enh. Vit. A, ZN, Fe sorghum Bt maize, DT maize Bacterial wilt resistance banana, VR CMV, VR CBSD cassava Bt/Ht cotton/ Bt Cowpea S. Africa DT maize, IR stack maize, IR/Ht cotton, Ht/mod oil soya, 1998 Bt maize 1 Commercial GM crops in bold font. Slide adapted from: Dr. Clive James, ISAAA/ Brief 46 Global Status of Commercialized Biotech/GM Crops 2013.
23 GM CROP STAGE PLAN Trait Discovery Product Development Commercialization Product Concept Gene Discovery Transformation GH & Field Evaluation Line Selection and Variety development Field (seed) Production Approval and Market Post Market Define product and choose genes Continuous Evaluation and Selection Biosafety/Regulatory Assessment Starts Here! Regulatory/Biosafety Dossier The three Thematic Questions relevant to safety: Is the GM Crop save to use? Safe to Grow? Safe to Consume? Compare to what? Non-gm parent, conventional agriculture and what is Currently known about the host species.
24 How safe is GM Crop Biotechnology? Examine the Record of Food, Feed, & Environmental Safety 1 billion hectares planted to GM crops equivalent to >90% of the entire land area of Europe Not a single scientifically validated occurrence of an adverse effect on humans, animal health, or to the environment Expert committees across the planet have concluded that food from GM crops is as safe and nutritious as food derived from crops created using century-old traditional breeding techniques. as safe as conventional European Commission (2110 A decade of EU-funded GMO research ( ) Luxembourg, Belgium: Publication Office of the European Union.
25 How safe is GM Crop Biotechnology? The potential risks posed by genetically engineered crops are not fundamentally different from those posed by conventional crops. How are risks of conventional products managed today? What would be different due to the GM trait? How does it fit into the conventional scheme of crop improvement? Regulation should be science-based and oversight conducted on a case-by-case basis
26 Regulation and Oversight Essentials Functional and Science-based regulatory system in place with protection goals defined and assessment endpoints understood Careful studies on relevant issues regarding intended use, agronomic fit, food safety and environmental risk assessment High quality scientific data that characterizes the technology, a relevant risk assessment, balance risk against product benefits Open relevant dialogue in a publicly transparent manner to address: Farmers and households who grow the crop Regulators who review safety data and dossiers NGOs who have competing agendas Consumers who purchase the harvest Ministers who approve the product
27 GM Crop Product Development Timeline 6 months Plant transformation and PCR screen (discard 50%) 1 year Greenhouse evaluation (discard 50%) 1 year Confined field trials (discard 50%) 2 years Multi-location trials (retain 2 events) 1 year Biosafety evaluation & agency review ~2.5 years Deployment to farmers Total: ~8years
28 Event Selection: A Reductive Process Plant transformation and event selection: (1-2 years) 100s to 1000s events generated in lab discard ~50% due to phenotype or multiple DNA inserts Remainder sent to greenhouse for evaluation discard ~50% due to poor trait expression or undesirable phenotype Small-scale Confined Field Trials (1-2 years) Conducted over multiple growing seasons Increased scrutiny of performance Retain 2-5 superior events for multi-location trials Multi-location trials (1-2 years) Narrow down to 2 events for commercial promotion Initiate rigorous regulatory assessment Approval and Forward Breeding into Farmer Preferred and Adapted Varieties (Commercialization) (many years)
29 Event Selection through R&D and Regulatory Process Research Proof-of-Concept Regulatory Studies Commercial Product Concept Gene Discovery Transformation GH & Field Evaluation Event Selection Variety Development Seed Production Approvals Market Post Market Molecular Characterization Flanking Sequence ID Preservation Event Specific Studies Implement event-specific QC analytics and best practices Protein Safety Studies Product Characterization And Food / Feed Safety Product Development Trials and Environmental Studies
30 Development Phase Biosafety Effort????? Gene selection/cloning (6 mos) *safety of gene source Safe to Use *bioinformatic assessment Safe to Use *gastric stability Safe to Eat Transformation (12 mos) *unintended phenotypes Safe to Grow *integrity of DNA insert Safe to Use *novel protein as intended? Safe to Eat Greenhouse (12 mos) *off types/phenotypes culled Safe to Grow Confined Field trials (36 mos) *Compliance Safe to Use *Agronomic assessment Safe to Grow *impact of environment Safe to Use *Mendelian inheritance Safe to Use *PCR analysis of insert Safe to Use *off types/phenotypes culled Safe to Use Biosafety Assessment (30mos) *acute toxicity Safe to Eat *DNA insert sequencing Safe to Eat *novel protein analysis Safe to Eat *nutrient composition Safe to Eat *environmental risk Safe to Grow
31
32 Foundation of Safety Assessment Process Elements derived from expert consultations conducted by Food & Agriculture Organization of the United Nations World Health Organization of the United Nations United Stated Food and Drug Administration International Life Sciences Institute Organization of Economic Co-operation & Development Blue Book Codex Alimentarius Commission: GUIDELINE FOR THE CONDUCT OF FOOD SAFETY ASSESSMENTOF FOODS DERIVED FROM RECOMBINANT-DNA PLANTS (CAC/GL )
33 OECD & the Blue Book In 1986, issued report: Safety Considerations for industrial, agricultural, and environmental applications of organisms by recombinant DNA techniques Provided a general framework for conducting food safety and environmental risk assessments It is the framework used by technology developers to guide their safety assessment
34 Blue Book: Elements of a Safety Assessment The report advised developers to take into account Characteristics of donor and recipient organism Nature and function of inserted DNA Rate and level of expression from that DNA Human health considerations Ecological considerations Interaction of the engineered organism with other biological systems These remain the essential elements of how risk assessments are conducted, globally
35 Codex Contributions Codex Alimentarius Commission Develops food standards, guidelines, and codes of practice Established task force in 1999 to consider health and nutritional implications of foods derived from GM crops Conducted a series of expert consultations, from a purely scientific perspective Issued a set of guidelines in 2003 that further define a framework for conducting a risk assessment
36 A fully integrated approach to the hazard assessment and characterization of all elements involved in producing a new GM variety Adapted from: König et al., Food and Chemical Toxicology 42: Parent Crop Donor, Transgene(s) and Delivery Process Characterization of Gene Product(s) Safety Assessment of New GM Crop/Food Identity, Phenotypic & Agronomic Performance Description of Donor Structure, Identity and Characterization Identity, Phenotypic & Agronomic Performance Geographical Distribution Description of Vector DNA Mode of Action/ Specificity Compositional Analysis History of Safe Use Transgene Delivery Process Toxicity Nutritional Analysis Compositional Analysis Characterization of Introduced DNA Allergenicity Safety Analysis (Animal Studies) Characterization of Insertion Site
37 Food & Feed Safety Considerations What categories of biosafety-related data are typically submitted to a regulatory agency? Mammalian Toxicity Allergenic Potential Nutritional Composition
38 Food Safety Assessment: Mammalian Toxicity A process conducted according to international Codex stds Source of the introduced gene Known to be toxic or allergenic? Mode of action of the novel protein Similar to any known toxins? Amino acid sequence of novel protein Homology to known toxins or allergens? A toxicity evaluation (Acute Oral Toxicity): Characterization of the novel protein and DNA insert
39 Food Safety Assessment Characterization of the DNA insert a. Is the DNA sequence as intended Characterization of the novel protein(s) a. Is the protein expressed in the plant as intended? i. Size on SDS-PAGE ii. Reactive to antibodies on Westerns iii. Glycosylation pattern iv. Amino Acid sequence
40 Inserted DNA Is the novel gene DNA sequence Intact? Rearranged? Unintended novel proteins? How it is done? Using molecular techniques Gene sequencing Hybridization studies Southern Blot Analysis, PCR analysis
41 DNA Insert Characterization Insert characterization Number of inserts Sequence of entire insert Sequence junctions Presence of backbone Presence of small inserts LB Transfer DNA found in GM Plant RB
42 Food Safety Assessment: Allergenicity The evaluation process as per Codex Guidelines 1. Bioinformatics: a. Assess amino acid similarity of the novel protein to known allergens 2. Gastric and intestinal stability of the novel protein a. Laboratory method (in vitro) established by international teams b. Labile proteins are less likely to be absorbed upon ingestion and pose unlikely allergenic risk
43 Investigations regarding Digestive Fate: Simulated gastric and intestinal fluids an in vitro digestibility study Novel protein quickly digested in simulated gastric fluids Labile proteins are less likely to be absorbed upon ingestion and pose unlikely allergenic risk
44 Allergenicity Sequence searches Allergenonline Allermatch Assessment of Amino Acid Sequence homology With known allergens
45 Compositional analysis Compare with other varieties Compare with literature values
46 OECD Guidance: Compositional Analysis CONSENSUS DOCUMENT ON COMPOSITIONAL CONSIDERATIONS FOR NEW VARIETIES OF CASSAVA (Manihot esculenta Crantz): KEY FOOD AND FEED NUTRIENTS, ANTI-NUTRIENTS,TOXICANTS AND ALLERGENS Suggested constituents to be analyzed in fresh roots and leaves Constituent Fresh leaves Fresh roots Proximate X X Starch X X Fatty acids X X Amino acids X X Mineralsa X X Vitaminsb X X Cyanogenic glycosides X X
47 Compositional Analysis Comparative Analysis CONSENSUS DOCUMENT ON COMPOSITIONAL CONSIDERATIONS FOR NEW VARIETIES OF CASSAVA (Manihot esculenta Crantz): KEY FOOD AND FEED NUTRIENTS, ANTI-NUTRIENTS,TOXICANTS AND ALLERGENS Suggested constituents to be analyzed in fresh roots and leaves Constituent Gm NASE3 NASE3 Range in Cassava Proximate X X Published Values Starch X X Fatty acids X X Amino acids X X Mineralsa X X Vitamins X X Cyanogenic glycosides X X Published Values
48 Environmental Risks and GM Crops The risks from GM crops are not fundamentally different from risks posed by conventionally bred varieties The risk assessment of a GM crops should focus on the novel trait and its potential to pose an environmental hazard Pathways to harm: Problem formulation (ie. What cascade of circumstance lead to greater harm?) Environmental risk assessments are conducted on GM crops intended for commercial cultivation These assessments are traditionally not conducted on harvested products (e.g., grain) intended to be used for food, feed, or processing.
49 Risk = hazard x exposure Risk is the possibility to suffer harm For there to be risk, both of the following must be true: there must be a hazard that can result in harm there must be sufficient exposure to that hazard to actually cause harm Risk Assessment in context of relevant policy protection goals.
50 Risk Assessment General Process Step #1: describe what s new or different when compared to the history and experience of growing conventional crop. Step #2: Does what s new have the potential to cause harm? (a hazard) Step #3: If yes, estimate the likelihood of exposure to that hazard Step #4: Will there be adverse consequences resulting from that level of exposure to the hazard? Step #5: If yes, develop risk mitigation and/or monitoring programs
51 Case Study: Phosphomannose Isomerase Mode of action: converts mannose-6-p to fructose-6-p Function in GM crop Used as a selectable marker during the plant transformation process Source of gene: an non-pathogenic bacterium, E. coli K12 PMI gene present across kingdoms, including yeast, soybean, & humans Protein characteristics Bioinformatics: no homology to known toxins or allergens High substrate specificity: mode of action well understood Previous history of dietary exposure Conclusions Phosphomannose Isomerase is not a hazard, exposure now moot. NOT A RISK TO HUMANS, ANIMALS, OR TO THE ENVIRONMENT
52 Bt corn: A risk to Monarch butterflies? Step #1: What s different? Plants express a Bt protein, toxic to a narrow spectrum of caterpillars insects Step #2: Is the Bt protein a potential hazard? YES In addition to target pest, other Lepidoptera including the Monarch butterfly larvae are sensitive to Bt protein if digested in sufficient amounts. Pollen from Bt maize is toxic to Monarch larvae when exposed in a controlled laboratory environment. Step #3: Exposure? NO Monarch larvae feed exclusively on milkweed, a weed rarely present in maize fields Pollen levels on milkweed located at Bt maize margins and edges are too low to be harmful. Step #4: A risk to Monarch populations? NONE
53 Gene Flow Considerations Fact: gene flow occurs between traditionally cultivated varieties of crops and their wild relatives Key question: what novel risk, if any, is associated with gene flow from transgenic crops into commercial varieties or wild relatives? Will gene flow confer a benefit resulting in increased weediness potential? Will gene flow lead to extinction of wild plant populations?
54 Glyphosate-tolerant Beet : An environmental risk? Step #1: What s different? Introduced gene confers resistance to glyphosate (e.g., Roundup) Step #2: Would trait entering weed populations via gene flow be a hazard? Glyphosate-resistant beet become pervasive and unmanageable, impacting agriculture? Step #3: estimate the likelihood of exposure to that hazard Pollen transfer is likely into sexually compatible weedy species within 200 meter zone where HT-beet is grown Step #4: A risk to agriculture? NO (1) glyphosate not historically used to control weed species outside of agriculture fields (2) glyphosate-tolerant weeds remain susceptible to alternate herbicides, cultivation, tillage, and crop rotation practices.
55 Environmental Risks and GM Crops Invasive Potential/ Persistence in unmanaged environments. Any adverse effects on relevant non-target and beneficial species Relevant Impact on Biodiversity- relative to conventional crop cultivation. Risk Assessment needs to be expanded to anticipated benefits associated with technology. For GM crops already commercialized elsewhere, consider all relevant data, weight of evidence, and regulatory authorizations.
56 Safety Assessment Findings to Date Proteins expressed in currently commercialized transgenic crops are highly labile Readily degrade under soil conditions, similar to other proteins No evidence of accumulation after years of cultivation Neither lab nor field studies have shown any lethal or sub-lethal effects on non-target soil organisms or microorganisms Bees, earthworms, collembola, mites, woodlice, nematodes No toxicity towards mammals, avians, fish, aquatic invertebrates
57 Safety Assessment Findings to Date Since 1996, more than 1 billion acres of GM food crops have been planted and consumed in dozens of countries on nearly every continent There has not been a single documented occurrence of adverse effect to human or animal health, or to the environment, resulting from the cultivation and consumption of these crops Expert committees across the planet have concluded this technology is as safe as centuryold traditional breeding techniques used for crop improvement
58 Additional GM Insect Protected Maize Technologies 58 Bt Product Concepts In planta solution for corn borers, fall armyworm and corn earworm Benefits Dual mode of action enhances IRM and durability Increased insect spectrum Expanded Range in Efficacy ECB=European Corn Borer CEW= Corn Ear Worm FAW= Fall Army Worm BCW= Black Cut Worm ECB CEW FAW BCW ECB IRM FAW IRM Non-Bt cry1ab (Mon810) MON 810 cry1f cry1ab Vip3A cry1ab cry3bb1 (lab) YGII
59 Useful Internet Resources Center for Environmental Risk Assessment GM Crop Database Biosafety Clearing House Policy Documents Decision Documents ISAAA (Clive James) Annual Global Status Briefs International Life Sciences Instituted Compositional Analysis USDA/ Biotechnology Regulatory Services GM crop petitions, EA, Final Decisions
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