The science-of-transgenics
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  • 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. 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. What About the Term Genetic Engineering? Genetic engineering involves:
    • Isolating genes
    • Modifying genes so they function better
    • Preparing genes to be inserted into a new species
    • Developing transgenes
    Genetic engineering is the basic tool set of biotechnology
  • 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. 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. Agriculture Transgenics On the Market Source: USDA
    • Insect resistant cotton – Bt toxin kills the
    • cotton boll worm
    • transgene = Bt protein
    • Insect resistant corn – Bt toxin kills the
    • European corn borer
    • transgene = Bt protein
    Normal Transgenic
  • 7.
    • Virus resistance - papya resistant to
    • papaya ringspot virus
    • transgene = virus coat protein
    Source: Monsanto
    • Herbicide resistant crops
    • Now: soybean, corn, canola
    • Coming: sugarbeet, lettuce, strawberry
    • alfalfa, potato, wheat (2005?)
    • transgene = modified EPSP synthase or
    • phosphinothricin-N-acetyltransferase
  • 8.
    • Biotech chymosin; the enzyme used
    • to curdle milk products
    • transgene = genetically engineered enzyme
    • bST; bovin somatotropin; used to increase
    • milk production
    • transgene = genetically engineered enzyme
    Source: Rent Mother Nature Source: Chr. Hansen
  • 9. Some Ag Biotech Products Are Discontinued
    • Poor Quality
    • FlavrSavr tomatoes (Calgene)
    • Negative Consumer Response
    • Tomato paste (Zeneca)
    • Negative Corporate Response
    • NewLeaf (Monsanto)
    • Universal Negative Publicity
    • StarLink corn (Aventis)
    Why???
  • 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. Turfgrass – herbicide resistance; slower growing (= reduced mowing) Bio Steel – spider silk expressed in goats; used to make soft-body bullet proof vests (Nexia)
  • 12. Biotechnology is Not Just on the Farm Disease Treatment Diagnostics   Environmental Cleanup   Human Applications
  • 13. Human Applications
    • Pharmaceutical products
    • New solutions to old problems
    •  
    • Disease diagnosis
    • Determine what disease you have or may get 
    • Gene therapy
    • Correcting disease by introducing a corrective gene
  • 14. Biotechnology and Health The genes for these proteins are:
    • Cloned
    • Inserted into bacteria
    • Product isolated using biofermentation
    Product Use Insulin Diabetes Interferon Cancer Interleukin Cancer Human growth hormone Dwarfism Neuroactive proteins Pain
  • 15. Environmental Applications Bioremediation - cleanup contaminated sites; uses microbes designed to degrade the pollution Indicator bacteria – contamination can be detected in the environment
  • 16. Tooth decay – engineered Streptococcus mutans, the bacteria that destroys enamel Future Health-related Biotech Products Vaccines – herpes, hepatitis C, AIDS, malaria
  • 17. Edible Vaccines Transgenic Plants Serving Human Health Needs
    • Works like any vaccine
    • A transgenic plant with a pathogen protein gene is developed
    • Potato, banana, and tomato are targets
    • Humans eat the plant
    • The body produces antibodies against pathogen protein
    • Humans are “immunized” against the pathogen
    • Examples:
      • Diarrhea
      • Hepatitis B
      • Measles
  • 18. A Popular Term We Need To Know GMOs - Genetically modified organisms
    • GMO - an organism that expresses traits that result
    • from the introduction of foreign DNA
    • Originally a term equivalent to transgenic organism
  • 19.
    • Some claim any improved biological product is a GMO
    • They feel this will
    • For example, some call plant varieties biotechnology products
    • This is a false claim
    The GMO Ruse
    • ease the publics fear
    • pave the way for product acceptance
  • 20. Let’s Be Up Front
    • Biotechnology adds traits not available in the species
      •   Soybean does not have a gene to breakdown Roundup
      • The gene comes from bacteria
    • Breeding  Biotechnology
      •   Breeding only exchanges genes found in the species
      • Breeding can transfer the transgene to other breeding materials
      •   BUT this does not make it a biotechnology procedure
  • 21. Important Plant Improvement Methods
    • Breeding
    • Crossing two individuals from the same species;
    • produces a new, improved variety;
    • not a biotechnology procedure
    • Transformation
    • Adding a gene from another species; the
    • essential biotechnology procedure to produce
    • transgenics
    Source: USDA Source: USDA
  • 22. Wheat Rye Triticale X Interspecific Cross New species, but NOT biotechnology products
  • 23. Mutagenesis
    • A useful procedure to produce a new trait
    • But the normal gene is modified
    • A transgene is not involved
    • The product of mutagenesis is not a GMO
  • 24. ATTCGA ATT G GA Susceptible Normal Gene Resistant Mutant Gene Mutagenesis Treatment Mutagenesis Changes the DNA Sequence
  • 25. BASF Clearfield Products
    • Herbicide resistance
      • imidazolinones
    • Mutant AHAS enzyme
      • developed by mutagenesis
    • Crops
      • Canola
      • Corn
      • Rice
      • Sunflower
      • Wheat
    • A Major Marketing Advantage
    • but lost when stacked with a transgene
  • 26. The Roundup Ready Story
    • Glyphosate is a broad-spectrum herbicide
    • Active ingredient in Roundup herbicide
    • Kills all plants it come in contact with
    • Inhibits a key enzyme ( EPSP synthase ) in an amino acid pathway
    • Plants die because they lack the key amino acids
    • A resistant EPSP synthase gene allows crops
    • to survive spraying
  • 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. 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. The Golden Rice Story
    • Vitamin A deficiency is a major health problem
    • Causes blindness
    • Influences severity of diarrhea, measles
    • >100 million children suffer from the problem
    • For many countries, the infrastructure doesn’t exist
    • to deliver vitamin pills
    • Improved vitamin A content in widely consumed crops
    • an attractive alternative
  • 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. 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. Introducing the Gene or Developing Transgenics Steps 1. Create transformation cassette 2. Introduce and select for transformants
  • 33. Transformation Cassettes Contains 1. Gene of interest
    • The coding region and its controlling elements
    2. Selectable marker
    • Distinguishes transformed/untransformed plants
    3. Insertion sequences
    • Aids Agrobacterium insertion
  • 34. Transformation Steps Prepare tissue for transformation Introduce DNA Culture plant tissue Field test the plants
    • Agrobacterium or gene gun
    • Multiple sites, multiple years
    • Develop shoots
    • Root the shoots
    • Leaf, germinating seed, immature embryos
    • Tissue must be capable of developing into normal plants
  • 35.
    • Transformation cassettes are developed in the lab
    • They are then introduced into a plant
    • Two major delivery methods
    Delivering the Gene to the Plant
    • Agrobacterium
    • Gene Gun
    Tissue culture required to generate transgenic plants
  • 36. The Lab Steps
  • 37. The Next Test Is The Field Herbicide Resistance Non-transgenics Transgenics
  • 38. Final Test of the Transgenic Consumer Acceptance RoundUp Ready Corn Before After