Genetically Engineered Crops I

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Genetically Engineered Crops I

• Core 218
• Spring 2007

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Why Genetically Engineer Plants?

• Reduce the use of chemicals
• Protect the environment
• Increase the value of crops
• Reduce the risk of human medicines
• Increase the supply of human medicines
• Address difficult to solve problems

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Why Genetically Engineer Plants?

• Field Performance
• Herbicide tolerance Roundup ready plants.
• Insect tolerance Newleaf potato
• Virus tolerance Freedom II Summer squash
• Quality Enhancement
• Flavr Savr Tomatoes
• Canola with enhanced laurate content

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AgBiotechs first baby, the terminator tomato

• Calgene, 1994.
• How they did it.
• Copied polygalactruonase gene.
• Inserted the gene copy backwards.
• To help identify successful insertions, an
  antibiotic marker gene for kanamycin was
  attached.
• Plants that have successful insertions are
  kanamycin resistant.
• Why it failed.
• Sold for 1.99 a pound when regular tomatoes sold
  for 0.49 a pound.
• Genetically modified a cultivar that was not very
  tasty and had a tougher skin.

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What Is Genetic Engineering?

• A technique used to alter or move genetic
  material (genes) of living cells.
• Narrower definitions are used by agencies that
  regulate genetically modified organisms (GMO's).
• USDA's Animal and Plant Health Inspection
  Service, genetic engineering is defined as the
  genetic modification of organisms by recombinant
  DNA techniques (7CFR340 340.1), while
  definitions used in Europe are somewhat broader.

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What Are Transgenic Plants?

• Transgenic plants result from the insertion of
  genetic material from another organism so that
  the plant will exhibit a desired trait.
  Recombinant DNA techniques (DNA formed by
  combining segments of DNA from different
  organisms) are usually used.

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How Prevalent are Bioengineered Plants?

• It is estimated that nearly 70 of all foods
  consumed in the US has some element from a
  genetically modified crop.
• Herbicide tolerance followed by insect tolerance
  are the two most prevalent traits.
• GMO Soybean, corn, cotton, and canola have the
  highest acreage in the US.
• The US grows the most (by acreage) GMO crops.

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1985
1st transgenic plants produced
1988
Particle bombardment developed
1992
GM crops considered substantially equivalent to
hybrid varieties
1994
Flavr-Savr tomato is released
1996
Herbicide- and insect-resistant crops approved
for cultivation
4.3 million acres of GM crops planted
1998
UK TV reports that GM food is dangerous
Monarch butterfly paper causes uproar
1999
Gerber excludes GM corn from its baby food
Greenpeace starts anti-GM campaign
75 million acres of GM crops planted
2000
Golden rice with ß-carotene developed
McDonalds rejects GM potatoes
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Total Number of Field Tests (permits
notifications) in the US by Year
YEAR (total)
Source http//www.nbiap.vt.edu/ As of 8/28/02
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Approved Releases by Phenotype Category (1987-
present)
Source http//www.nbiap.vt.edu/ As of 8/28/02
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GM Crop Acreage
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GM CROPS AROUND THE WORLD
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77 HR 15 IR 8 HR IR
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From www.isb.vt.com
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How Are Transgenic Plants Made?

• Transgenic plants are made by introducing new or
  foreign DNA into the genome of the plant by using
  recombinant techniques.
• Fours steps are involved in developing transgenic
  plants.
• Construction of the recombinant molecule.
• Transformation techniques.
• Selection and testing.
• Backcross and gene stability.

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Plant Transformation Vectors

• Based on the Ti plasmid from Agrobacterium
  tumefaciens, a bacterial pathogen that causes
  crown gall disease of many woody plants.
• Uses a virus promoter (CaMV) that functions in
  plants.

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Agrobacterium is

• A soil-borne Gram negative bacterium.
• A plant pathogen that causes crown gall diseases
  on woody plants

Side Note Alan Kerr, in the 1970s found an
Agrobacterium strain called Agrobacterium
radiobacter strain 84 that was antagonistic to
the pathogenic strain Agrobacterium tumefaciens.
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The Ti plasmid (pTi) Controls Pathogenicity in
Agrobacterium

• T-DNA, transferred to plant genome
• Tumor inducing genes (auxins, cytokinins)
• Opine synthesis genes
• tra bacterial conjugation
• Opine catabolism
• Vir genes

T-DNA
tra
pTI 200 kb
Opine catabolism
vir genes
ori
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• Acetosyringone - wound phenolic, attracts A.
  tumefaciens, activates vir genes, activates
  permeases (opine uptake), activates endonuclease
  (excise T-DNA), vir genes nick and stabilize
  T-DNA.
• T-DNA integration into plant DNA, opines,
  agrocinopines, cytokinins, IAA produced

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Vector Construction
Eliminate opine catabolism genes Eliminate
tra Eliminate auxin and cytokinin genes
T-DNA
tra
pTI 200 kb
Opine catabolism
vir genes
ori
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Vector Construction
Eliminate opine catabolism genes Eliminate
tra Eliminate auxin and cytokinin genes Add plant
promoter Add target DNA Transform A. tumefaciens
T-DNA
vir genes
ori
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How do we transform plants?

• Biological.
• Agrobacterium mediated transformation.
• Mechanical.
• Particle bombardment.
• Electroporation.
• Microinjection.
• Chemical.
• Polyethylene glycol.

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Agrobacterium Mediated Plant Transformation

• Based on the natural ability of a Gram negative
  bacterium (Agrobacterium tumefaciens) to insert
  the T-DNA from the pTi plasmid into a plant
  cells genome.

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Agrobacterium Mediated Plant Transformation

• Plant hormone genes (cytokinins, auxins) and
  opine synthesis and catabolism genes are removed
  from the pTi.
• Recombinant genes (gene of interest, selectable
  marker, plant promoter) are inserted into the
  pTi.
• Agrobacterium is transformed with the recombinant
  pTi.
• Recombinant Agrobacterium is mixed with plant
  cells.
• Transformed plant cells are selected on tissue
  culture media containing the selectable marker
  (antibiotic resistance).
• Plant cells are regenerated into plants.
• Note only the nucleus receives the recombinant
  DNA.

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Agrobacterium Mediated Plant Transformation

• Vacuum infiltration (floral dip)
• Wounded explants
• Cultured cells

A. Bent. 2000. Plant Physiol. 1241540-1547.
Plants grown to just flowering (A), dipped into
Agrobacterium (B), grown until mature (C), seeds
harvested and grown on selective medium
containing kanamycin.
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Pros and Cons of Agrobacterium Mediated
Transformation

• Pros
• The bacterium does all the work.
• Transformation in chromosomes only.
• Cons
• Doesnt work well for grasses.
• Selection method also carries a gene for
  kanamycin resistance.