Understanding biotechnology
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Understanding biotechnology

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  • My thanks to Brian, and to Bill Young and Dan Curry of OSU for the opportunity to speak
  • The genes of interest are usually chimeric genes, that is they are made of many parts. The promoter determines when, where, and how much a gene will be expressed The terminator adds many adenines to the RNA, and make it stable within the cell The coding sequence codes for the desired protein The 3 parts can all come from different organisms
  • Plant genetic engineering involves 3 steps. First, there must be a way to obtain whole plants from single, cultured cells. Plants regenerate from culture via one of two developmental pathways. The first is called organogenesis, and the second organogenesis.
  • For organogenesis, pieces of tissue from a plant are taken, and placed into tissue culture. The presence of plant hormones in the tissue culture medium causes the cells to divide in an unorganized fashion. This cell growth is called a "callus"
  • When the mixture of plant hormones is changed, the callus can differentiate into a shoot. Because organ formation is part of the process, the whole process is termed "organogenesis" The resulting shoots can be allowed to root, transplanted into soil, and grow into plants. Organogenesis is good for crops like potato, tomato, tobacco, canola, broccoli, and poplar trees.
  • The second prerequesite is a way to get DNA into a plant cell prior to its regeneration There are 2 main ways by which this is accomplished: Agrobacterium tumefaciens and biolistics
  • One the genes of T-DNA have been replaced, the Agrobacterium will still transfer them to a plant cell
  • A diagram illustrating the point further Only one of the 4 cells is transgenic. Here the transgene is depicted by its orange color
  • If the tissue from the preceding slide was placed on plant tissue culture medium with the appropriate plant hormones, all cells would divide and grow, regardless of whether they were transgenic or not.
  • However, remember that a gene for antibiotic resistance was also added to the T-DNA. Hence, a transformed cell not only acquires the desired trait, it also becomes resistant to an antibiotic When the antibiotic is added to the plant cell culture medium, only the transgenic cells can grow. The remainder are killed.
  • My thanks to Brian, and to Bill Young and Dan Curry of OSU for the opportunity to speak
  • Breast meat yield and growth rate in broilers varies in much the same way as it does in turkeys. These birds are part of a broiler test done recently at the AAF test facility in Albertville, Alabama.

Understanding biotechnology Understanding biotechnology Presentation Transcript

  • Understanding Biotechnology
    • Steve Strauss, Professor, OSU
    • Forest Science, Genetics, Molecular and Cellular Biology
    • Director, Outreach in Biotechnology
    • http://wwwdata.forestry.oregonstate.edu/orb/
    • [email_address]
  • Outreach website http://wwwdata.forestry.oregonstate.edu/orb/
  • Educational activities Food for Thought Lecture Series / 2005-2008
    • Streaming video - OPAN/OPB usage
  • The plan
    • What is biotechnology
    • GMOs
      • State of usage in the world
      • How it works
      • The general concerns surrounding them
    • Non-GMO biotechnologies (Dave Harry)
      • Genomics and DNA markers
    • Break-outs for grass seed specifics
      • Commercialization issues, GMO testing, grass industry biotechnologies
  • What is biotechnology? Amer. Heritage Dictionary (2000)
    • 1. The use of microorganisms or biological substances such as enzymes , to perform industrial processes.
    • 2a. The application of the principles of engineering and technology to the life sciences; bioengineering.
  • A more crop oriented definition of biotechnology
    • Use of technologies that affect physiology, genetics, management, or propagation
    • Most common uses
      • Microorganisms for fermentation of plant products
      • Plant tissue culture for propagation
      • DNA sequencing and indexing for identification (DNA fingerprinting)
      • Gene isolation, modification, and insertion (genetic engineering, “modern biotechnology”)
        • GE, GEO or GM, GMO
  • Why emphasize GE forms of biotechnology? GE crops have been taken up rapidly by farmers when available, have had large benefits, and have great economic and humanitarian potential Exploding science of genomics fuels rapid discovery, innovation
  • Rapid rise of GE crops in developed and developing world http://www.isaaa.org
  • Many social issues with major impacts on use / acceptance
    • Few GMO crop types in production
      • Maize, soy, cotton, canola
      • Insect, herbicide tolerance traits
      • Small amounts of viral resistance (squash, papaya)
    • Benefits of reduced tillage, reduced pesticide use, improved yields, reduced costs
    • But other traits and crops mostly on hold
      • Substantial social resistance and obstacles to their use
  • Defining GMOs
    • GEO / GMO = creation of a “recombinant DNA modified organism”
      • It’s the method, can use native or foreign genes
    • DNA isolated, changed/joined in a test tube, and re-inserted asexually
      • Vs. making crosses or random mutations in conventional breeding
    • Powerful breeding tool but can generally handle one to a few genes at a time
      • Simple traits can be designed , but without constraints from native gene pools
      • That’s why its called genetic engineering , though we are modifying, not building, a new organism
    • Assembling a gene
    • Controls level of expression, and
    • Where and when expressed
    • Provides stability to messenger RNA, and
    • Guides processing into protein
    Can mix and match parts & can change sequences to improve properties Protein Coding sequence Promoter Terminator
  • Promoter (controls expression) Gene (encodes protein) Examples of promoter : gene combinations produced via recombinant DNA methods Phenolic pathway enzyme (bacteria) 35 S-CAMV (plant virus) RNA degrading enzyme (bacteria) Pollen sac (tobacco) Herbicide tolerant Male-sterile FMV (plant virus) Insect toxin protein (bacteria) Insect resistance Oilseed (canola) Insulin (human) Improved nutrition
  • Recombinant DNA modification of native plant genes
    • How are GE plants produced?
    Step 1 Getting whole plants back from cultured cells = cloning
    • Differentiation of new plant organs from single cells
    Leaf-discs First step is de-differentiation into “callus” after treatment with the plant hormone auxin
    • Shoots, roots, or embryos produced from callus cells using plant hormones
    • Step 2
    Getting DNA into plant cells Main methods - Agrobacterium tumefaciens - Biolistics [gene gun]
  • Agrobacterium is a natural plant genetic engineer
    • Agrobacterium gene insertion
    Gene of interest Agrobacterium tumefaciens Engineered plant cell T-DNA Ti Plasmid
    • Only a few cells get modified so need to identify and enrich for the engineered cells
    Not all cells are engineered, or engineered the same. Thus need to recover plants from that one cell so the new plant is not chimeric (i.e., not genetically variable within the organism)
    • Hormones in plant tissue culture
    • stimulate division from plant cells
    • Antibiotics in plant tissue culture
    • limit growth to engineered cells
    • Other kinds of genes can also be used to favor transgenic cells (e.g., sugar uptake, herbicide resistance)
  • Transformation of bentgrass (Wang and Ge 2006)
  • Glyphosate-tolerant Fescue Conventionally-bred Patented Varieties
  • GE traits under development in forage and turfgrasses Wang and Ge, In Vitro Cell Develop. Biol. 42, 1-18 (2006)
    • Nutritional quality
      • Lignin reduction, increase of sulfur-rich proteins
    • Abiotic stress tolerance
      • Drought, frost, salt
    • Disease/pest management
      • Fungal, viral, herbicide tolerance
    • Growth and nutrient use
      • Flowering time, phosporus uptake
    • Hypoallergenic pollen
    • Bioethanol processability
  •  
  • Problems and obstacles to wider use of GE crops
    • Regulations complex, uncertain, changing, and very costly
      • Three agencies can be involved
      • Environmental and food/feed acceptability criteria complex, stringent compared to all other forms of breeding
    • Unresolved legal issues of gene spread, safety assessment, liability, marketing, and trade restrictions
  •  
  • Legal actions
    • USDA sued over process for granting field trial permit for GE bentgrass and GE biopharma crops
    • USDA sued over deregulated Roundup- resistant alfalfa
      • First time an authorized crop forced to be removed from market
    • USDA required to do EIS for alfalfa, one was already underway for bentgrass
    • Scotts fined $ 500K over Roundup Ready bentgrass field trial
  • Strong and well funded political and legal resistance
  • Intellectual property issues
    • New, costly, overlapping “utility patents” issued for genes and crops since 1980
    • Patent “anticommons”
      • Major costs, uncertainties for use of best technologies and usually need several licenses for an improved crop
    • Major litigations ongoing for years to decades
      • Basic Agrobacterium gene transfer method
      • Bt insect resistance gene innovations
    • Regulatory risks make large companies very reluctant to license to small companies, academics
    • Public sector, small companies find it very hard to cope with the costs, obstacles
    • Strong polarization on benefits vs. risks
      • A highly vocal, concerned minority (~20%)
    • A majority whose level of acceptance varies widely among applications depending on benefits and ethical views
      • Strong resistance to animal applications, and to impacts that appear to harm biological diversity
    • Very low knowledge of the science, technology
    Varied public approval
  • Rutgers survey data - USA (2005) http://www.foodpolicyinstitute.org/resultpub.php http://www.foodpolicyinstitute.org/docs/reports/NationalStudy2003.pdf
    • Seven in ten (70%) don't believe it is possible to transfer animal genes into plants
    • Six in ten (60%) don't realize that ordinary tomatoes contain genes
    • More than half (58%) believe that tomatoes modified with genes from a catfish would probably taste fishy
    • Fewer than half (45%) understand that eating a genetically modified fruit would not cause their own genes to become modified
  • Education needs: Gullibility
    • "People seem to have a great number of misconceptions about the technology. As a result, they seem to be willing to believe just about anything they hear about GM foods.“
    • Very few universities take an active role in outreach, education
      • University of California system an exception
  • Summary
    • GE is a method, not a product
    • GE crops a major presence and with major science and technology push forward
    • GE method highly regulated, causing great costs and uncertainties both for field research and commercial development
    • Social/legal obstacles slowing or blocking investment outside of the major crops and large corporations
  • Understanding Biotechnology Part 2: Genomics and DNA Markers
    • David Harry
    • Department of Forest Science
    • Assoc. Director, Outreach in Biotechnology
    • http://wwwdata.forestry.oregonstate.edu/orb/
    • [email_address]
  • DNA-based Biotechnologies
    • Genetic engineering (GE, GMO)
      • direct intervention and manipulation
      • gene manipulation and insertion through an asexual process
    • Genomics & DNA markers
      • are generally descriptive, examining the structure and function of genes and genomes
      • manipulating genes and genomes is indirect, through selection and breeding
  • Some definitions
    • Genes
      • a piece of DNA (usually 100’s to 1000’s of bases long)
      • collected together along chromosomes
      • serves as a structural blueprint or a regulatory switch
    • Genome
      • an entire complement of genetic material in the nucleus of an individual (excluding mitochondria and chloroplasts)
      • genes, regulatory elements, non-coding regions, etc
      • tools for describing genomes include maps and sequence
    • DNA marker
      • some type of discernable DNA variant (variation, or polymorphism) that can be tracked
      • tracking the +/- of markers offers powerful tools for managing breeding populations and, increasingly, for predicting offspring growth performance
  • For today:
    • Basics of DNA markers
    • DNA markers & fingerprints
      • are fixed for the life of an individual
      • can be used to identify individuals
    • Marker inheritance (parent to offspring)
      • nuclear markers
      • parentage verification
      • genome mapping
    • Associating markers and traits
      • maps and associations
      • marker breeding (MAS/MAB)
  • Genomes, genes, and DNA Genes are located on packaging platforms called chromosomes
  • DNA markers reveal subtle differences in DNA sequence A A A T T C G G A A A T T C C G T C C A C G T G T C C A C G T T G<>C G<>T Marker “1” Marker “2”
  • A DNA fingerprint is fixed throughout an individual’s life Age
  • MCW-305 MCW-184 MCW-087 DNA Fingerprints to Verify Identities 22 Paired Samples Collected at Different Times
  • Pedigree errors: “non-parental” marker types S D Progeny
  • Genetic Map: Perennial Ryegrass Gill et al. 2006
  • How might genetic markers accelerate breeding? X X Then, evaluate genetic makeup early to select young birds First, associate performance and genetic makeup X X
  • a b c d e f g h i j k l m Hypothetical genes (QTLs) affecting economic traits Linkage Map 1 2 3 4 Trait 2 Trait 1
  • Mapping loci affecting quantitative traits (QTL) in chickens Distance along chromosome Gga 3 (cM) Genes in the circled region appear to affect breast-meat yield
  • High-throughput Genotyping Illumina- BeadStation500G-BeadLab ~150,000 data points per week at UCDavis Genome Center
  •  
  • Marker Assisted Breeding in Conifers
    • Quantitative Trait Locus (QTL) Mapping
    • Association Mapping
    Pinus taeda (loblolly pine) Pseudotsuga menziesii (Douglas-fir) Pinus elliottii (slash pine)
  • Genomics & DNA Markers: Summary
    • DNA markers can be used as fingerprints to distinguish individuals, and
      • cultivars, varieties, etc
      • increasingly used to protect intellectual property (utility patents, PVP)
    • Marker inheritance allows parentage to be verified, facilitating pedigree control
    • DNA markers can be associated with phenotypic traits
    • Once marker-trait associations have been established, marker data can augment phenotypic observations to accelerate breeding