1. NDSU Extension 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. NDSU Extension 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. NDSU Extension 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. NDSU Extension 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. NDSU Extension 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. NDSU Extension 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 NormalTransgenic

7. NDSU Extension 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. NDSU Extension 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. NDSU Extension 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. NDSU Extension 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. NDSU Extension Turfgrass – herbicide resistance; slower growing (= reduced mowing) Bio Steel – spider silk expressed in goats; used to make soft-body bullet proof vests (Nexia)

12. NDSU Extension Biotechnology is Not Just on the Farm Disease Treatment Diagnostics Environmental Cleanup Human Applications

13. NDSU Extension 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. NDSU Extension Biotechnology and Health ProductUse InsulinDiabetes InterferonCancer InterleukinCancer Human growth hormoneDwarfism Neuroactive proteinsPain The genes for these proteins are: Cloned Inserted into bacteria Product isolated using biofermentation

15. NDSU Extension Environmental Applications Bioremediation - cleanup contaminated sites; uses microbes designed to degrade the pollution Indicator bacteria – contamination can be detected in the environment

16. NDSU Extension Tooth decay – engineered Streptococcus mutans, the bacteria that destroys enamel Future Health-related Biotech Products Vaccines – herpes, hepatitis C, AIDS, malaria

17. NDSU Extension 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. NDSU Extension 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. NDSU Extension 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. NDSU Extension 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. NDSU Extension 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

22. NDSU Extension WheatRye Triticale X Interspecific Cross New species, but NOT biotechnology products

23. NDSU Extension 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. NDSU Extension ATTCGA ATTGGA Susceptible Normal Gene Resistant Mutant Gene Mutagenesis Treatment Mutagenesis Changes the DNA Sequence

25. NDSU Extension 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. NDSU Extension 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. NDSU Extension + Glyphosate X Roundup Sensitive Plants X X Shikimic acid + Phosphoenol pyruvate 3-Enolpyruvyl shikimic acid-5-phosphate (EPSP) Plant EPSP synthase Aromatic amino acids Without amino acids, plant dies X

28. NDSU Extension 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. NDSU Extension 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. NDSU Extension  -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. NDSU Extension The Golden Rice Solution IPP Geranylgeranyl diphosphate Phytoene Lycopene  -carotene (vitamin A precursor) Phytoene synthase Phytoene desaturase Lycopene-beta-cyclase ξ-carotene desaturase Daffodil gene Single bacterial gene; performs both functions Daffodil gene  -Carotene Pathway Genes Added Vitamin A Pathway is complete and functional Golden Rice

32. NDSU Extension Introducing the Gene or Developing Transgenics Steps 1. Create transformation cassette 2. Introduce and select for transformants

33. NDSU Extension 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. NDSU Extension Transformation Steps Prepare tissue for transformation Introduce DNA Culture plant tissue Develop shoots Root the shoots Field test the plants Leaf, germinating seed, immature embryos Tissue must be capable of developing into normal plants Agrobacterium or gene gun Multiple sites, multiple years

35. NDSU Extension 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. NDSU Extension The Lab Steps

37. NDSU Extension The Next Test Is The Field Non-transgenics Transgenics Herbicide Resistance

38. NDSU Extension Final Test of the Transgenic Consumer Acceptance RoundUp Ready Corn Before After