The science-of-transgenics


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The science-of-transgenics

  1. 1. The Science of Transgenics Phil McClean Department of Plant Science North Dakota State University The Sociological, Economic, and Ethical Impact of Transgenic Organisms Workshop Fargo, ND February 21, 2003
  2. 2. Transgenics are a Biotechnology Product How about some definitions Biotechnology - General Definition The application of technology to improve a biological organism Biotechnology - Detailed Definition The application of the technology to improve the biological function of an organism by adding genes from another organisms
  3. 3. What About the Term Genetic Engineering? Genetic engineering involves: <ul><li>Isolating genes </li></ul><ul><li>Modifying genes so they function better </li></ul><ul><li>Preparing genes to be inserted into a new species </li></ul><ul><li>Developing transgenes </li></ul>Genetic engineering is the basic tool set of biotechnology
  4. 4. What is a transgenic? Transgene – the genetically engineered gene added to a species Ex. – modified EPSP synthase gene (encodes a protein that functions even when plant is treated with Roundup) Transgenic – an organism containing a transgene introduced by technological (not breeding) methods Ex. – Roundup Ready Crops Concept Based on the Term Transgene
  5. 5. We can develop organisms that express a “novel” trait not normally found in the species Why are transgenics important? Extended shelf-life tomato (Flavr-Savr) Herbicide resistant soybean (Roundup Ready)
  6. 6. Agriculture Transgenics On the Market Source: USDA <ul><li>Insect resistant cotton – Bt toxin kills the </li></ul><ul><li>cotton boll worm </li></ul><ul><li>transgene = Bt protein </li></ul><ul><li>Insect resistant corn – Bt toxin kills the </li></ul><ul><li>European corn borer </li></ul><ul><li>transgene = Bt protein </li></ul>Normal Transgenic
  7. 7. <ul><li>Virus resistance - papya resistant to </li></ul><ul><li>papaya ringspot virus </li></ul><ul><li>transgene = virus coat protein </li></ul>Source: Monsanto <ul><li>Herbicide resistant crops </li></ul><ul><li>Now: soybean, corn, canola </li></ul><ul><li>Coming: sugarbeet, lettuce, strawberry </li></ul><ul><li>alfalfa, potato, wheat (2005?) </li></ul><ul><li>transgene = modified EPSP synthase or </li></ul><ul><li>phosphinothricin-N-acetyltransferase </li></ul>
  8. 8. <ul><li>Biotech chymosin; the enzyme used </li></ul><ul><li>to curdle milk products </li></ul><ul><li>transgene = genetically engineered enzyme </li></ul><ul><li>bST; bovin somatotropin; used to increase </li></ul><ul><li>milk production </li></ul><ul><li>transgene = genetically engineered enzyme </li></ul>Source: Rent Mother Nature Source: Chr. Hansen
  9. 9. Some Ag Biotech Products Are Discontinued <ul><li>Poor Quality </li></ul><ul><li>FlavrSavr tomatoes (Calgene) </li></ul><ul><li>Negative Consumer Response </li></ul><ul><li>Tomato paste (Zeneca) </li></ul><ul><li>Negative Corporate Response </li></ul><ul><li>NewLeaf (Monsanto) </li></ul><ul><li>Universal Negative Publicity </li></ul><ul><li>StarLink corn (Aventis) </li></ul>Why???
  10. 10. Next Generation of Ag Biotech Products Source: Minnesota Microscopy Society Golden Rice – increased Vitamin A content (but not without controversy) transgene = three pathway enzymes Sunflower – white mold resistance transgene = oxalate oxidase from wheat
  11. 11. Turfgrass – herbicide resistance; slower growing (= reduced mowing) Bio Steel – spider silk expressed in goats; used to make soft-body bullet proof vests (Nexia)
  12. 12. Biotechnology is Not Just on the Farm Disease Treatment Diagnostics   Environmental Cleanup   Human Applications
  13. 13. Human Applications <ul><li>Pharmaceutical products </li></ul><ul><li>New solutions to old problems </li></ul><ul><li>  </li></ul><ul><li>Disease diagnosis </li></ul><ul><li>Determine what disease you have or may get  </li></ul><ul><li>Gene therapy </li></ul><ul><li>Correcting disease by introducing a corrective gene </li></ul>
  14. 14. Biotechnology and Health The genes for these proteins are: <ul><li>Cloned </li></ul><ul><li>Inserted into bacteria </li></ul><ul><li>Product isolated using biofermentation </li></ul>Product Use Insulin Diabetes Interferon Cancer Interleukin Cancer Human growth hormone Dwarfism Neuroactive proteins Pain
  15. 15. Environmental Applications Bioremediation - cleanup contaminated sites; uses microbes designed to degrade the pollution Indicator bacteria – contamination can be detected in the environment
  16. 16. Tooth decay – engineered Streptococcus mutans, the bacteria that destroys enamel Future Health-related Biotech Products Vaccines – herpes, hepatitis C, AIDS, malaria
  17. 17. Edible Vaccines Transgenic Plants Serving Human Health Needs <ul><li>Works like any vaccine </li></ul><ul><li>A transgenic plant with a pathogen protein gene is developed </li></ul><ul><li>Potato, banana, and tomato are targets </li></ul><ul><li>Humans eat the plant </li></ul><ul><li>The body produces antibodies against pathogen protein </li></ul><ul><li>Humans are “immunized” against the pathogen </li></ul><ul><li>Examples: </li></ul><ul><ul><li>Diarrhea </li></ul></ul><ul><ul><li>Hepatitis B </li></ul></ul><ul><ul><li>Measles </li></ul></ul>
  18. 18. A Popular Term We Need To Know GMOs - Genetically modified organisms <ul><li>GMO - an organism that expresses traits that result </li></ul><ul><li>from the introduction of foreign DNA </li></ul><ul><li>Originally a term equivalent to transgenic organism </li></ul>
  19. 19. <ul><li>Some claim any improved biological product is a GMO </li></ul><ul><li>They feel this will </li></ul><ul><li>For example, some call plant varieties biotechnology products </li></ul><ul><li>This is a false claim </li></ul>The GMO Ruse <ul><li>ease the publics fear </li></ul><ul><li>pave the way for product acceptance </li></ul>
  20. 20. Let’s Be Up Front <ul><li>Biotechnology adds traits not available in the species </li></ul><ul><ul><li>  Soybean does not have a gene to breakdown Roundup </li></ul></ul><ul><ul><li>The gene comes from bacteria </li></ul></ul><ul><li>Breeding  Biotechnology </li></ul><ul><ul><li>  Breeding only exchanges genes found in the species </li></ul></ul><ul><ul><li>Breeding can transfer the transgene to other breeding materials </li></ul></ul><ul><ul><li>  BUT this does not make it a biotechnology procedure </li></ul></ul>
  21. 21. Important Plant Improvement Methods <ul><li>Breeding </li></ul><ul><li>Crossing two individuals from the same species; </li></ul><ul><li>produces a new, improved variety; </li></ul><ul><li>not a biotechnology procedure </li></ul><ul><li>Transformation </li></ul><ul><li>Adding a gene from another species; the </li></ul><ul><li>essential biotechnology procedure to produce </li></ul><ul><li>transgenics </li></ul>Source: USDA Source: USDA
  22. 22. Wheat Rye Triticale X Interspecific Cross New species, but NOT biotechnology products
  23. 23. Mutagenesis <ul><li>A useful procedure to produce a new trait </li></ul><ul><li>But the normal gene is modified </li></ul><ul><li>A transgene is not involved </li></ul><ul><li>The product of mutagenesis is not a GMO </li></ul>
  24. 24. ATTCGA ATT G GA Susceptible Normal Gene Resistant Mutant Gene Mutagenesis Treatment Mutagenesis Changes the DNA Sequence
  25. 25. BASF Clearfield Products <ul><li>Herbicide resistance </li></ul><ul><ul><li>imidazolinones </li></ul></ul><ul><li>Mutant AHAS enzyme </li></ul><ul><ul><li>developed by mutagenesis </li></ul></ul><ul><li>Crops </li></ul><ul><ul><li>Canola </li></ul></ul><ul><ul><li>Corn </li></ul></ul><ul><ul><li>Rice </li></ul></ul><ul><ul><li>Sunflower </li></ul></ul><ul><ul><li>Wheat </li></ul></ul><ul><li>A Major Marketing Advantage </li></ul><ul><li>but lost when stacked with a transgene </li></ul>
  26. 26. The Roundup Ready Story <ul><li>Glyphosate is a broad-spectrum herbicide </li></ul><ul><li>Active ingredient in Roundup herbicide </li></ul><ul><li>Kills all plants it come in contact with </li></ul><ul><li>Inhibits a key enzyme ( EPSP synthase ) in an amino acid pathway </li></ul><ul><li>Plants die because they lack the key amino acids </li></ul><ul><li>A resistant EPSP synthase gene allows crops </li></ul><ul><li>to survive spraying </li></ul>
  27. 27. + Glyphosate X Roundup Sensitive Plants X X Without amino acids, plant dies X Shikimic acid + Phosphoenol pyruvate 3-Enolpyruvyl shikimic acid-5-phosphate (EPSP) Plant EPSP synthase Aromatic amino acids
  28. 28. Bacterial EPSP synthase Shikimic acid + Phosphoenol pyruvate 3-enolpyruvyl shikimic acid-5-phosphate (EPSP) Aromatic amino acids Roundup Resistant Plants + Glyphosate With amino acids, plant lives RoundUp has no effect; enzyme is resistant to herbicide
  29. 29. The Golden Rice Story <ul><li>Vitamin A deficiency is a major health problem </li></ul><ul><li>Causes blindness </li></ul><ul><li>Influences severity of diarrhea, measles </li></ul><ul><li>>100 million children suffer from the problem </li></ul><ul><li>For many countries, the infrastructure doesn’t exist </li></ul><ul><li>to deliver vitamin pills </li></ul><ul><li>Improved vitamin A content in widely consumed crops </li></ul><ul><li>an attractive alternative </li></ul>
  30. 30.  -Carotene Pathway Problem in Plants IPP Geranylgeranyl diphosphate Phytoene Lycopene  -carotene (vitamin A precursor) Phytoene synthase Phytoene desaturase Lycopene-beta-cyclase ξ-carotene desaturase Problem: Rice lacks these enzymes Normal Vitamin A “ Deficient” Rice
  31. 31. The Golden Rice Solution Daffodil gene Single bacterial gene; performs both functions Daffodil gene  -Carotene Pathway Genes Added IPP Geranylgeranyl diphosphate Phytoene Lycopene  -carotene (vitamin A precursor) Phytoene synthase Phytoene desaturase Lycopene-beta-cyclase ξ-carotene desaturase Vitamin A Pathway is complete and functional Golden Rice
  32. 32. Introducing the Gene or Developing Transgenics Steps 1. Create transformation cassette 2. Introduce and select for transformants
  33. 33. Transformation Cassettes Contains 1. Gene of interest <ul><li>The coding region and its controlling elements </li></ul>2. Selectable marker <ul><li>Distinguishes transformed/untransformed plants </li></ul>3. Insertion sequences <ul><li>Aids Agrobacterium insertion </li></ul>
  34. 34. Transformation Steps Prepare tissue for transformation Introduce DNA Culture plant tissue Field test the plants <ul><li>Agrobacterium or gene gun </li></ul><ul><li>Multiple sites, multiple years </li></ul><ul><li>Develop shoots </li></ul><ul><li>Root the shoots </li></ul><ul><li>Leaf, germinating seed, immature embryos </li></ul><ul><li>Tissue must be capable of developing into normal plants </li></ul>
  35. 35. <ul><li>Transformation cassettes are developed in the lab </li></ul><ul><li>They are then introduced into a plant </li></ul><ul><li>Two major delivery methods </li></ul>Delivering the Gene to the Plant <ul><li>Agrobacterium </li></ul><ul><li>Gene Gun </li></ul>Tissue culture required to generate transgenic plants
  36. 36. The Lab Steps
  37. 37. The Next Test Is The Field Herbicide Resistance Non-transgenics Transgenics
  38. 38. Final Test of the Transgenic Consumer Acceptance RoundUp Ready Corn Before After