1. NDSU Extension Biotechnology: Principles and Products From Protein to Product Phil McClean Department of Plant Science North Dakota State University The techniques used by the biotechnology industry to modify genes and introduce them into transgenic organisms

2. NDSU Extension What is Biotechnology? How about some definitions General Definition The application of technology to improve a biological organism Detailed Definition The application of the technology to modify the biological function of an organism by adding genes from another organism

3. NDSU Extension But we know nature does not have all of the traits we need Here we see bean has many seedcoat colors and patterns in nature Nature has a rich source of variation These definitions imply biotechnology is needed because:

4. NDSU Extension Nature does not contain all the genetic variation man desires Fruits with vaccines Grains with improved nutrition

5. NDSU Extension What controls this natural variation? Allelic differences for a gene control variation for a specific trait Gene - a piece of DNA that controls the expression of a trait Allele - the alternate forms of a gene Important definitions for this statement:

6. NDSU Extension What is the difference between genes and alleles for Mendel’s Traits? Mendel’s Genes Plant height Seed shape Tall Short Allele Smooth Wrinkled Allele

7. NDSU Extension This Implies a Genetic Continuum A direct relationship exists between the gene, its alleles, and the phenotypes (different forms ) of the trait Alleles must be: similar enough to control the same trait but different enough to create phenotypic differences for that trait

8. NDSU Extension Allelic Differences for Mendel’s Genes Plant Height Gene Gene: gibberellin 3-  -hydroxylase Function: adds hydoxyl group to GA 20 to make GA 1 Role of GA 1 : regulates cell division and elongation Mutation in short allele: a single nucleotide converts an alanine to threonine in final protein Effect of mutation: mutant protein is 1/20 as active

9. NDSU Extension Gene: strach branching enzyme (SBE) isoform 1 Function: adds branch chains to starch Mutation in short allele: transposon insertion Effect of mutation: no SBE activity; less starch, more sucrose, more water; during maturation seed looses more water and wrinkles Allelic Differences for Mendel’s Seed Shape Gene

10. NDSU Extension Central Dogma of Molecular Genetics (The guiding principle that controls trait expression) DNA (gene) RNA ProteinTrait (or phenotype) Transcription Translation Plant height Seed shape

11. NDSU Extension Plant Biotechnology Requires Two Steps Identify a gene from another species which controls a trait of interest Or modify an existing gene (create a new allele) Gene Manipulation Introduces that gene into an organism Technique called transformation Forms transgenic organisms Gene Introduction

12. NDSU Extension Gene Manipulation Starts At the DNA Level The nucleus contains DNA Source: Access Excellence

13. NDSU Extension DNA Is Packaged Source: Access Excellence Double-stranded DNA Chromosomes is condensed into

14. NDSU Extension Chromosomes Contain Genes Chromosome Gene Source: Access Excellence

15. NDSU Extension Genes Are Cloned Based On: Similarity to known genes Homology cloning (mouse clone used to obtain human gene) Protein sequence Complementary genetics (predicting gene sequence from protein) Chromosomal location Map-based cloning (using genetic approach)

16. NDSU Extension Gene Manipulation It is now routine to isolate genes But the target gene must be carefully chosen Target gene is chosen based on desired phenotype Function: Glyphosate (RoundUp) resistance EPSP synthase enzyme Increased Vitamin A content Vitamin A biosynthetic pathway enzymes

17. NDSU Extension The RoundUp Ready Story Glyphosate is a broad-spectrum herbicide Active ingredient in RoundUp herbicide Kills all plants it comes in contact with Inhibits a key enzyme (EPSP synthase) in an amino acid pathway Plants die because they lack key amino acids A resistant EPSP synthase gene allows crops to survive spraying with RoundUp

18. NDSU Extension + Glyphosate X Biochemical Response of 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

19. 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

20. 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 is an attractive alternative

21. NDSU Extension  -Carotene Pathway 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

22. 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

23. NDSU Extension Metabolic Pathways are Complex and Interrelated Understanding pathways is critical to developing new products

24. NDSU Extension Modifying Pathway Components Can Produce New Products Modified Lipids = New Industrial Oils Turn On Vitamin Genes = Relieve Deficiency Increase amino acids = Improved Nutrition

25. NDSU Extension Trait/Gene Examples RoundUp ReadyBacterial EPSP Golden RiceComplete Pathway Plant Virus ResistanceViral Coat Protein Male SterilityBarnase Plant Bacterial Resistancep35 Salt toleranceAtNHX1 TraitGene

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

27. 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

28. NDSU Extension Gene of Interest Coding Region Encodes protein product ex.: EPSP  -carotene genes Promoter Region Controls when, where and how much the gene is expressed ex.: CaMV35S (constitutive; on always) Glutelin 1 (only in rice endosperm during seed development) Promoter Coding Region TP Transit Peptide Targets protein to correct organelle ex.: RbCS (RUBISCO small subunit; choloroplast target

29. NDSU Extension Selectable Marker Coding Region Gene that breaks down a toxic compound; non-transgenic plants die ex.: nptII [kanamycin (bacterial antibiotic) resistance] aphIV [hygromycin (bacterial antibiotic) resistance] Bar [glufosinate (herbicide) resistance] Promoter Region Normally constitutive ex.: CaMV35s (Cauliflower Mosaic Virus 35S RNA promoter Promoter Coding Region

30. NDSU Extension Effect of Selectable Marker Transgenic = Has NptII or Bar Gene Plant grows in presence of selective compound Plant dies in presence of selective compound Non-transgenic = Lacks NptII or Bar Gene X

31. NDSU Extension Insertion Sequences Used for Agrobacterium-transformation ex.: Right and Left borders of T-DNA Required for proper gene insertions TLTL TRTR

32. NDSU Extension Let’s Build A Complex Cassette pB19hpc (Golden Rice Cassette) TLTL TRTR aphIV35SGt1psy35SrbcScrtl Hygromycin Resistance Phytoene Synthase Phytoene Desaturase T-DNA Border T-DNA Border Selectable Marker Gene of Interest Gene of Interest Insertion Sequence Insertion Sequence

33. 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

34. NDSU Extension Plant Tissue Culture A Requirement for Transgenic Development A plant part Is cultured Callus grows Shoots develop Shoots are rooted; plant grows to maturity

35. NDSU Extension Agrobacterium A natural DNA delivery system A plant pathogen found in nature Hormone genes expressed and galls form at infection site Delivers DNA that encodes for plant hormones Infects many plant species Gall on stem Gall on leaf DNA incorporates into plant chromosome

36. NDSU Extension The Galls Can Be Huge

37. NDSU Extension Natural Infection Process Is Complex

38. NDSU Extension But Nature’s Agrobacterium Has Problems Infected tissues cannot be regenerated (via tissue culture) into new plants Transferred DNA (T-DNA) modified by Removing phytohormone genes Retaining essential transfer sequences Adding cloning site for gene of interest Phytohormone balance incorrect regeneration Solution? Why?

39. NDSU Extension The Gene Gun DNA vector is coated onto gold or tungsten particles Particles are accelerated at high speeds by the gun Particles enter plant tissue DNA enters the nucleus and incorporates into chromosome Integration process unknown

40. 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

41. NDSU Extension The Lab Steps

42. NDSU Extension Lab Testing The Transgenics Insect Resistance Transgene= Bt-toxin protein Cold Tolerance Transgene= CBF transcription factors

43. NDSU Extension Salt TolerantMercury Resistance More Modern Examples Transgene= Glyoxylase I Transgene= Mercuric ion reductase

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

45. NDSU Extension Final Test Consumer Acceptance RoundUp Ready Corn Before After

46. NDSU Extension The Public Controversy Should we develop transgenics? Should we release transgenics? Are transgenics safe? Are transgenics a threat to non-transgenic production systems? Are transgenics a threat to natural eco-systems?