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Risk Assessment of GM Plants Assoc. Prof. Dr. Wichai Cherdshewasart Department of Biology, Faculty of Science, Chulalongkorn University Tel 02-2185033 Fax 02-2185034 Modes of plant gene modification 1. 2. 3. 4. 5. 6. Classical breeding (wild crossing) Mutation Somaclonal variation Protoplast fusion Embryo rescue Gene transfer 1. Classical breeding (wild crossing) Advantage: practical, low cost, stable, effective within species Disadvantage: time-consumed, ineffective within different species 2. Mutation Advantage: practical, low cost Disadvantage: randomized, needs long selection procedure, not totally stable, may initiate revertant 3. Somaclonal variation Advantage: in vitro manipulation Disadvantage: takes time, randomized, needs long selection procedure 4. Protoplast fusion Advantage: across species barrier Disadvantage: randomized, remote species may success but fail for further development 5. Embryo rescue Advantage: cross between different species is possible to initiate embryonic development. Disadvantage: transfer pre-mature embryo to new environment could initiate fully developed plants, but sterile 6. Gene transfer Advantage: precise genotype obtained, laboratory and industry practical Disadvantage: Not possible for all species, especially monocot Mode of gene transfer: 1. Vector-mediated gene transfer Agrobacterium-mediated Virus-mediated 2. Vectorless-mediated gene transfer (Direct gene transfer) Mechanical Physical Electrical Chemical Analysis of transgenic plants 1. Phenotypic analysis 2. Genotypic analysis 3. Greenhouse condition analysis 4. Field trial condition analysis Genotypic analysis PCR for rapid screening Southern blot for precise gene detection Northern blot for transcription analysis Western blot for translation analysis, together with Ab-binding or enzymatic analysis Mendelian analysis for insertion locus and linkage analysis In situ hybridization for precise insertion locus analysis DNA methylation analysis for silencing potential analysis A generally accepted risk assessment method*,**,*** 1. 2. 3. 4. 5. Identify potential adverse effects on human health and/or the environment Estimate the likelihood of these adverse effects being realized Evaluate the consequence should be identified effects be realized (the risk) Consider appropriate risk-management strategies Estimate the overall potential impact, including a consideration of potential impacts that may be beneficial to human health or the environment * UNEP International Technical Guideline for Safety in Biotechnology ** The Cartegena Protocol *** EC Directive 2001/18/EEC Approaches to risk assessment 1. Trait analysis characteristics of the modified organism; transgene, parental organisms, receiving environment less problem, if small scale more problem, if large scale 2. Familarity comparison of transgenic to similar organism(s) derived from classical genetic methods assume that small genetic changes (1-4 genes) exhibits no significant change in well-known organism, phenotype is still the same 3. Formulaic possible adverse effects; to human health or the environment R = H x E R; Risk, H; Hazard, E; Exposure facilitates consideration of risk-management options 4. Intuitive Reasoning use education, experience and reason to promote knowledge for making decision with complete information depends on what should be considered use of expert committees, independent reviewers/assessors without a conflict of interest Environment Safety Assessment For Transgenic Crops: Needs: 1. Environmental friendly products 2. Tight global regulatory requirements 3. Trade barrier Methods: 1. Product and country specification 2. Science-based assessment 3. Multi-tiered, complementary approaches Plant assessment: 1. Survival against wild type plants 2. Stability of gene expression, especially in the field vs. laboratory / greenhouse 3. Distinct genotype over wild type plant 4. Invasiveness of transgenic plants, the possibility to develop into weeds Trait assessment: 1. Toxicity to non-target organisms 2. In case of human consumption, no allergen / toxic substance 3. Ecological impacts (outcrossing) Guidelines for Plant Testing 1. Field obseravation Emergence Order of testing relies on degree of possible risk of the plant Growth Transgenic plant growth / wild type, not greater than 1 measurement Days of flowering Transgenic plant days of flowering / wild type, not greater than 1 Length of flowering period Transgenic plant length of flowering period / wild type, not greater than 1 Pollen dispersal distance This is the reason why buffer zone has to be set up) Shattering of seed from plant Reproductive success or yield (annual) Distance of seed shattering determines degree of risk Reproductive success determines transmission risk Reproductive success Perennial risk determines more risk or yield (perennial) Qualitative insect Non-target insects = 0 Qualitative pathogens Others Non-target pathogens = 0 2. Plant testing Dormacy/ germination -shorter dormancy / germination determine front running risk -longer dormancy / germination determine latent risk risk -increase longevity determines risk Field seedbank longevity (dormancy x viability) Competition (Replacement or addition series) -stronger competition determines risk Replacement capacity -higher replacement capacity determines risk Gene flow (through -wider pollen dispersal determines risk pollen movement) -outcrossing determines risk Introgression (hybrid weediness) -hybrid weediness determines long term risk Alleopathy -Competitive of survival risk Susceptibility to conventional management Genetic stability -Competitive of agricultural risk Epistasis -Epistasis determines unexpected genetics -Horizontal gene transfer Gene transfer between plant nucleus and organelle Gene transfer between plant nucleus and genome of consumer, predator, Gene transfer between nucleus and organelle Other -High genetic stability determines risk Regulatory principles: 1. Scientifically based, based on information of organism, used technology and effects to humans and environment 2. Product-based approach, use existing product-based legislation 3. Familiarity and substantial equivalence, experience with the use of that species. The determination is based on scientific literature and practical experience with the plant and similar plant varieties. 4. Case-by case, allow the development of knowledge that could inform criteria and requirement over time. Regulatory principles: 5. 6. 7. 8. Step-wise fashion, products should be assessed throughout the chain of development : From laboratory to greenhouse and finally large-scale field trial Transparency Precautionary principle/approach, derived from Rio Declaration, regulatory groups can make decisions about products based on scientific uncertainty. Harmonization, sharing of or acceptance of another group’s review 1. Good laboratory practice Tightly control of GM-vectors, plasmids and plant materials Apply no bacterial antibiotic resistant-derived gene Apply bioluminescence gene from animal as marker Apply antisense for pollen developmental gene Limit level of toxic gene, eg, cry family 2. Good agricultural practice Controlled plantation area with standard buffer zone and % sharing with wild type plants Emasculation Flower bud elimination Closed-bag control Net protection of fruits and seeds from insects, birds, bats, rodents Total fruit and seed collection Labeling and separation technique for transgenic plant and seed Whole plant elimination after harvest 3. Good manufacturing practice Labeling GM-products according to domestic and export regulations Testing for allergen and toxicity of the products containing GM-materials 4. Good marketing practice Fully-informed alien gene(s) and awareness of application Evaluated for allergen and toxic molecule Labeling Post marketing record 5. Good consumption practice For GM-food products: determine animals as primary consumer and human as secondary consumer Study labeling Food safety criteria References Head G. and Duan J. 2002. Environmental safety assessment for transgenic crops. Wolf K. 1994. Gene transfer between organelles and the nucleus in lower eukaryotes Copy P. Bazin C. Anxolabehere D. Langin T. 1994. Horizontal transfer and the evolution of transposable elements Landmann J. Graser E. Riedel-Preuss A. van der Hoeven C. 1994. Can Agrobacteria be eliminated from transgenic plants? Hoffmann T. Golz C. Schieder O. 1994. Preliminary findings of DNA transfer from transgenic plants to a wild-type strain of Aspergillus niger Hansen L. C. Obryeki J.-J. L. 2000 Field deposition of Bt transgenic corn pollen: lethal effects on the monarch butterfly.