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Final report mst 5


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Final report mst 5

  1. 1. Principles and Practice of Environmental Safety Assessment of Transgenic Plants
  2. 3. <ul><li>Safety concerns prior to commercialization: </li></ul><ul><ul><li>stability of the genetic modification, gene transfer to related plants, gene transfer to unrelated organisms, weediness potential, secondary and non-target adverse effects </li></ul></ul><ul><ul><li>identify + evaluate  risk </li></ul></ul><ul><ul><li>release and cultivation of these plants  traditional plants. </li></ul></ul>
  3. 4.
  4. 5. <ul><li>In order to be able to assess the environmental safety of GM plant, one must be: </li></ul>1. Familiar with the biology of the plant 2. Familiar with the novelty trait 3. Familiar with agricultural/silvicultural practices employed it its cultivation
  5. 6. <ul><li>Can be used to identify species-specific characteristics that may be affected by the novelty trait </li></ul><ul><li>Provide details on significant interactions between the plant and other life forms </li></ul>
  6. 7. <ul><li>Environmental risk of GM plants </li></ul>Novelty trait of the genetic elements Used to transform it Novelty of the plant itself
  7. 8. <ul><li>Changes in agricultural practices associated with the introduction of new plant varieties, including, but not limited to, transgenic plants, can have significant environmental impacts. </li></ul>
  8. 9. <ul><li>Requires: </li></ul><ul><ul><ul><li>sufficient analytical data be available through experiment for effective comparison </li></ul></ul></ul><ul><li>Goal: </li></ul><ul><li>not to establish an absolute level of safety but a relative level of safety </li></ul>
  9. 10. Zea mays
  10. 11. <ul><li>No difference in seed/plant maturity observed </li></ul><ul><li>No significant difference in yield </li></ul><ul><li>No difference in agronomic quality, disease, insect susceptibility </li></ul>Other than European Corn Borer Control
  11. 12. <ul><li>MON 810 contains DNA sequences from the ff: </li></ul><ul><li>Bacillus Thuringiensis cry1Ab gene </li></ul><ul><li>-modification of the native gene </li></ul><ul><li>-truncated </li></ul><ul><li>Cauliflower mosaic virus (CaMV) enhanced 35s promoter </li></ul><ul><li>-with duplicated enhancer region </li></ul><ul><li>intron from maize hsp70 gene (heat shock protein) </li></ul><ul><li>-increased level of transcription </li></ul>
  12. 13. <ul><li>the 3’ untranslated region of the nopaline synthase gene </li></ul><ul><li>-from Ti plasmid of agribacterium tumefaciens </li></ul><ul><li>-provides polyadenylation signal for stable expression </li></ul>None of them are known to have any Pathogenic characteristics
  13. 14. <ul><li>The following are present only in plasmids for transformation process: </li></ul><ul><li>CP4EPSPS marker gene </li></ul><ul><li>Glyphosate oxidase marker gene </li></ul><ul><li>Neomycin Phospotransferase II encoding bacterial marker gene </li></ul><ul><li>Lac Z </li></ul><ul><li>ori-pUC </li></ul><ul><li>CTP 2 </li></ul>
  14. 15. <ul><li>Potential Pathogenicity of the Donor Organism </li></ul>-produce crystalline insecticidal proteins (delta endotoxins) -humans are not affected -no deleterious effects to non-target organisms HOW DOES Cry1Ab WORK?
  15. 16. (insect gut) Protein binds to specific receptors Inserts into membrane Forms cation-specific pores DEATH and PARALYSIS
  16. 17. Why is there a need to identify the potential pathogenicity of the donor organisms? (relation to ERA) - To identify if it has significant effects to humans, non-target animals
  17. 18. <ul><li>2. Microprojectile Transformation </li></ul><ul><li>-for direct delivery of DNA to host genome </li></ul><ul><li>- had intact copies of the transgene, as well as multiple, rearranged,and/or truncated transgene fragments </li></ul><ul><li>HOW it WORKS? </li></ul>Plasmid w/ gene of interest Tungsten/gold particles (microcarriers) Host cell (nucleus) DNA may separate with microcarrier Integrate with host genome 1. Agrobacterium mediated transformation
  18. 19. <ul><li>Description of the transformation method ( MON 810 ) </li></ul><ul><li>-microprojectile bombardment </li></ul><ul><li>-although DNA solution contained genes that code for glyphosate tolerance (CP4 EPSPS), no such gene in MON 810 genome </li></ul><ul><li>* glyphosate selection </li></ul>
  19. 20. <ul><li>While information on the integrity and copy number of the inserted DNA are generally required by regulatory authorities, there is no evidence to suggest that transgenic plants containing multiple copies of the inserted DNA are any less safe than comparable plants containing only a single copy. </li></ul>
  20. 21. <ul><li>-number of copies of the inserted DNA </li></ul><ul><li>*MON 810 contains 1 fragment of integrated DNA </li></ul><ul><li>-compositon and integrity of the inserted DNA </li></ul><ul><li>* MON810-produced by PAT with DNA solution composed of cry1Ab , CP4 EPSPS , gox and nptII genes </li></ul>MON 810:
  21. 22. <ul><li>Why is there a need to conduct a Molecular Characterization of the inserted DNA? </li></ul><ul><li>-to convey that plants with more than 1 transgene copy aren’t less safe than those with only 1. </li></ul><ul><li>-to address issues related to positional effects, pleiotropic effects, and gene silencing </li></ul>
  22. 23. <ul><li>Segregation Analysis of MON 810 </li></ul><ul><li>-segregation and stability data are consistent with a single active site of insertion of the cry1Ab gene into genomic DNA of maize line MON 810 </li></ul><ul><li>Stability of insertion: demonstrated through 7 generations of crossing Integron analysis </li></ul>Integron Analysis of MON 810 -Southern blot analysis: cry1ab insertion  stable in 3 generations of MON 810 breeding
  24. 26. <ul><li>Field Trials </li></ul><ul><li> US and European field trials assessed Cry1Ab, CP4 EPSPS, and GOX protein levels in maize tissues using validated ELISA s and Western Immunoblot Analysis specific for each protein. </li></ul><ul><li> for both trials, ELISAs for CP4 EPSPS and GOX proteins were not performed since the genes are not present in maize fine MON 810. </li></ul><ul><li> the Cry1Ab protein levels in MON810 are expressed in low level. The Cry1Ab protein in line MON 810 shows immunoreactive product of the expected size when compared with the purified protein standard. </li></ul>
  25. 28. <ul><li>Analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It provides more specific results than the ELISA protocol. </li></ul>There’s more..
  26. 32. For gene flow to occur via normal sexual transmission , certain conditions must exist:
  27. 34. <ul><li>Why is there a need to know the Gene transfer to Related plants? </li></ul><ul><li>- To assess environmental risks associated with outcrossing from transgenic plants  know reproductive biology of plant, distribution of sexually compatible relativesimpact of introduced trait. </li></ul>Up next: Gene Transfer to unrelated Plants organisms
  28. 35. <ul><li>Gene Transfer to Unrelated Organisms </li></ul><ul><li>Horizontal gene transfer (HGT) </li></ul><ul><li> non-sexual exchange of genetic material between organisms belonging to the same, or different species. </li></ul><ul><li> HGT between plants & bacteria has been seen as a risk associated with genetically engineered (GE) plants (possible transfer of gene encoding antibiotic resistance). </li></ul>
  29. 36. Up next: weediness potential <ul><li>Consequences & Environmental Impact of HGT </li></ul><ul><li>The transference of antibiotic resistance traits to bacteria, in either the soil or gut from transgenic plants has often been cited as a potential adverse outcome of HGT. </li></ul><ul><ul><ul><ul><ul><li>BUT…. </li></ul></ul></ul></ul></ul><ul><li>-MON810 has no novel phenotypic characteristics that would extend its use beyond the current geographic range of maize production. </li></ul><ul><ul><ul><ul><li>THUS… </li></ul></ul></ul></ul><ul><li>- risk of transferring genetic traits from MON810 maize to species in unmanaged environments was insignificant. </li></ul>
  30. 38. <ul><li>Weeds </li></ul><ul><li>= subset of plants </li></ul><ul><li>= considered pests </li></ul><ul><li> used to describe a plant that is a nuisance in managed ecosystems such as farms or forest plantations. </li></ul><ul><li> Typically weeds are plant species that spread easily in disturbed areas or among crops </li></ul>THEREFORE …………………
  31. 39. <ul><li>Weediness potential is really a measure of a plants ability to successfully colonize an ecosystem, especially when it may also lead to the displacement of other species. </li></ul><ul><li>In the case of MON810 maize…. </li></ul><ul><ul><ul><li>No competitive advantage was conferred to it other than resistance to ECB. </li></ul></ul></ul><ul><ul><ul><li>Does not render maize weedy or invasive of natural habitats since none of reproductive or growth characteristics were modified. </li></ul></ul></ul><ul><ul><ul><li>Unlikely to establish in non-cropped habitats </li></ul></ul></ul><ul><ul><ul><li>No reports of maize surviving as a weed </li></ul></ul></ul><ul><li>Thus.. </li></ul><ul><ul><ul><li>Zea mays is not invasive and is a weak competitor with very limited seed dispersal. </li></ul></ul></ul>Up next: secondary and non-target adverse effects
  32. 40. <ul><li>Secondary and Non-Target Adverse Effects </li></ul><ul><li>Environmental risk assessment must consider the unintended consequences of the environmental release of a transgenic plant, particularly as this may impact on existing agricultural and the agro-ecosystem. </li></ul><ul><li>In case of plant pesticides, EPA’s intent is to evaluate potential hazard to……. </li></ul><ul><li> ~terrestrial wildlife ~aquatic animals </li></ul><ul><li> ~plants and ~beneficial insect </li></ul>
  33. 41. <ul><li>Non-Target Test Organisms </li></ul><ul><li>* Avian test species </li></ul><ul><li>*Aquatic animals … A. Freshwater fish species </li></ul><ul><li> B. Aquatic invertebrate species </li></ul><ul><li>*Non-target insect testing </li></ul><ul><li>Choice of appropriate indicator organisms is based on potential for…. </li></ul><ul><li>#field exposure to novel protein expressed in transgenic plants </li></ul><ul><li> #EPA has considerable background data </li></ul><ul><li> #standard method for care and handling are available </li></ul><ul><li> #species are widely distributed (available) </li></ul><ul><li> #have variety of food habits and habitat requirements </li></ul>
  34. 42. <ul><li>Plant pest potential </li></ul><ul><li> A plant may be considered a pest but not a weed </li></ul><ul><li>For example, a plant that produces an allelopathic substance may be considered a pest if the toxin produced has an undesirable environmental effect. Transgenic plants expressing novel toxins or potential allergens must be assessed accordingly. </li></ul>
  35. 43. <ul><li>Effects on Non-target organisms </li></ul><ul><li>Bacillus thuringiensis subsp. kurstaki ( B.t.k.) contains the Cry1Ab protein used to produce MON 810 </li></ul><ul><li>History of use  </li></ul><ul><li>bacterial BT protien is not toxic to humans, other vertebrates and beneficial insects.  </li></ul><ul><li>only active against specific lepidopteran insects </li></ul>Field Studies: United States
  36. 44. <ul><li>Field trials were conducted in the United States from 1993-95 to assess the impact of insect protected maize on beneficial arthropods. Maize inbreds and hybrids expressing the Cry1Ab protein were compared to their non-transformed counterparts for relative abundance of beneficial arthropods. </li></ul><ul><li> Studies demonstrated that CRY1Ab had neither a direct nor indirect effect on the beneficial arthropod populations. Field trials conducted at France also have the same result. </li></ul>#45. Slide 45
  37. 45. <ul><li>Laboratory Studies </li></ul><ul><li>-Honey bee larvae & adults -Collembola </li></ul><ul><li>-Parasitic Hymenopteran -Daphnia </li></ul><ul><li>-Ladybird beetles -Green lacewing </li></ul><ul><li>-Northern Bobwhite Quail -Earthworm </li></ul><ul><li>In all cases, there were no observavble adverse effects. </li></ul><ul><li>MON810 maize did not present an increased risk to or impact on interacting organisms, including humans with the exception of specific lepidopteran insect species. </li></ul>Up next: Insect Resistance Management
  38. 46. <ul><li>Insect Resistance Management </li></ul><ul><li>Bt toxin  control a range of insect pests including the European corn borer (ECB), cotton bollworm and the Colorado potato beetle. </li></ul>
  39. 47. <ul><li>Bt microbial insecticides have an enviable history of safe use thus, regulatory authorities in the US and Canada consider Bt toxins in microbial sprays and transgenic plants to be in the public good and therefore worthy of extra regulatory protection. </li></ul><ul><li>In order to prolong the effectiveness of plant-expressed BT toxins and the microbial spray formulations of these same toxins….. </li></ul><ul><li>+INSECT RESISTANCE MANAGEMENT+ programs </li></ul>
  40. 48. <ul><li>Insect Resistance Management Plans </li></ul><ul><li>high dose/refuge strategy  means of delaying the onset of Bt resistance. This strategy involves exposing a portion of the pest population to Bt plants with an extremely high concentration of toxin [25 times the amount needed to kill 99% of the susceptible insects while maintaining another part of the population in a refuge where the pests do not encounter any Bt toxin. </li></ul>
  41. 49. Fig. 1. How the high-dose/refuge strategy works to delay the increase in highly resistant (RR) insects in a pest population. Reproduced from Cohen et al. 2000. BT CROP NON-BT CROP 4 essential assumptions:
  42. 50. <ul><li>1. Resistance genes must be nearly recessive. </li></ul><ul><li>2. The genes conferring resistance are rare. </li></ul><ul><li>3. The refuge of non-transgenic plants will </li></ul><ul><li>maintain a sufficient number of susceptible individuals (SS homozygotes) to outnumber the resistant individuals (RR homozygotes) during mating so that their offspring will be RS heterozygotes, thus maintaining a population that is almost exclusively RS or SS. </li></ul><ul><li>4. The proximity of refuges to Bt crops is sufficient to ensure nearly random mating within the typical dispersal distance of the adults. </li></ul>
  43. 51. <ul><li>Why need IRM plan? </li></ul><ul><li>A specific IRM plan is necessary to ensure long term resistance management </li></ul><ul><li>IRM strategies should be tailored to address specific regional resistance management concerns, as appropriate. </li></ul><ul><li>Essential for sustainable pest management goals </li></ul>
  44. 52. <ul><li>Tapos na po. </li></ul><ul><li>Salamat.  </li></ul>