What is Biotechnology?

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What is Biotechnology?
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What is Biotechnology?
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 organisms
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What About the Term Genetic Engineering?
Genetic engineering is the basic tool set of
biotechnology
Genetic engineering involves

• Isolating genes
• Modifying genes so they function better
• Preparing genes to be inserted into a new
  species
• Developing transgenes

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What is a transgenic?
Concept Based on the Term Transgene
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
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Biotechnology Terms You Probably Heard
Transgene the foreign gene added to a species
Ex. modified EPSP synthase gene (encodes a
protein that functions even when plant treated
with Roundup)
Transgenic an organism containing a transgene
introduced by technological (not breeding)
methods
Ex. Roundup Ready Crops
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Biotechnology Develops
GMOs - Genetically modified organisms

• GMO - an organism that expresses traits that
  result
• from the introduction of foreign DNA
• Also called transgenic organism

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Important Terms

• Breeding

• Beneficial gene added from the same species
• Gene delivered by mating within the species

Source USDA

• Transformation

• Beneficial gene added from another species
• Gene delivered by plant genetic engineering

Source USDA
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Lets Be Up Front

• Breeding ? Biotechnology
•  Breeding only exchanges genes found in the
  species.
• Breeding can transfer the transgene to other
  breeding materials
•  BUT it is not the same as biotechnology.
• Biotechnology adds traits not available in the
  species
•  Soybean does not have a gene to breakdown
  Roundup
• The gene comes from bacteria

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What are the structures in molecular genetics?

• Molecular genetics study of genes and how they
  are expressed.
• Chromosome part of cell nucleus that contains
  heredity information and promotes protein
  synthesis.
• Gene basic unit of heredity on a chromosome.
• DNA molecule in a chromosome that codes genetic
  information.

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Interspecific Cross
Wheat
Rye
X
Triticale
New species, but NOT biotechnology
products
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Mutagenesis New Trait, No Foreign Gene

• Mutagenesis changes the sequence of a gene
• New, useful traits can be obtained

Mutagenesis Treatment
Susceptible Normal Gene
ATTCGA
Resistant Mutant Gene
ATTGGA
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Transformation Cassettes
Contains
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Transformation Steps
Prepare tissue for transformation
Introduce DNA

• Agrobacterium or gene gun

Culture plant tissue
Field test the plants

• Multiple sites, multiple years

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Delivering the Gene to the Plant

• Transformation cassettes are developed in the lab

• They are then introduced into a plant

• Two major delivery methods

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The Next Test Is The Field
Herbicide Resistance
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Final Test of the Transgenic Consumer Acceptance
RoundUp Ready Corn
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Crop Biotech Market Dominated By Four Countriesa
6 3.2 mha
68 35.7 mha
3 1.5 mha
22 11.8 mha
Total 99 of market
a2001 growing season data.
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Agriculture Products On the Market
Insect resistant cotton

• Bt toxin kills the cotton boll worm
• toxin gene from a bacteria

Source USDA
Insect resistant corn

• Bt toxin kills the European corn borer
• toxin gene from a bacteria
• Rootworm GM approved (2/26/03)

Normal
Transgenic
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Herbicide resistant crops

• current soybean, corn, canola
• coming sugarbeet, lettuce, strawberry,
• alfalfa, potato, wheat (2005)
• resistance gene from bacteria

Source Monsanto
Virus resistance

• papaya, squash, potato
• resistance gene from a virus

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Economic Effect of Bt Cotton In China

• 200/acre increase in income

• 750 million increase nationally

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EU Labeling Regulations

• Foods with less than 0.9 of GM gene product
• Labeling not required
• Products derived from a GM crop
• Labeling required
• Applies even if the product does not contain the
  GM
• gene product
• Ex Corn syrup does not have the Bt protein,
  but must
• be labeled
• Animal feeds from GM crops
• Same guidelines apply

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What Are the Public Concerns?
Economics Are we changing the economics on the
farm? Environmental Are we irreversibly
modifying the environment?   Globalization Is
technology becoming centralized in too few hands?
  Social Will we develop a class of genetic
outcasts? Religious Are we playing God?
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Benefits of Plant Biotechnology

1. Greater production efficiencies It can help
   plant breeders improve a crops yield

1. Less chemical damage

1. Hardier plants it leads to produce plants that
   will resist diseases and unfavorable weather
   conditions.

1. Improved food quality.

1. New crops

1. Improved protection against human and animal
   diseases

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Concerns associated with GM crops

• Possible production of allergenic or toxic
  proteins not native to the crop.
• 2. Adverse effects on non-target organisms,
  especially pollinators and biological control
  organisms.
• 3. Loss of biodiversity.
• 4. Genetic pollution (unwanted transfer of genes
  to other species).
• 5. Development of pest resistance.
• 6. Global concentration of economic power and
  food production.
• 7. Lack of "right-to-know" (i.e., a desire for
  labeling transgenic foods).

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File to support registration of new crop variety-
conventional breeding
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Biological systems for transformation
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1. Agrobacterium tumefaciens

• Agrobacteria are soil bacteria.
• They naturally infect dicotyledonous plants.
• Because host range is limited, procedure has not
  been used for some major crops such as corn,
  wheat, rice, etc.

• Life cycle of Agrobacterium involves living in
  the soil until it encounters a plant and then
  infecting the plant.
• Infection causes a rapid proliferation of plant
  cells around the infection leading to formation
  of a crown gall tumor

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• For Agrobacterium hizogenes, masses of roots
  emerge from the gall forming hairy root disease.

• Once the gall is produces, it provides a haven
  for the bacteria to proliferate.
• To obtain food, the bacteria also subjugates the
  plant to produce an unusual class of compounds
  called opines.
• Opines are condensation products of the amino
  acid arginine and carbon compounds present in the
  Kreb cycle.
• Most common are octopine and nopaline. Opines
  cannot be metabolized by host plant but are used
  by the bacterium for food and amino acids.

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• Disease caused by the action of a plasmid in
  Agrobacterium called the Ti plasmid (Ti Tumor
  Inducing).
• Ti plasmid is a large circular plasmid, 180 kbp
  in size.
• Only one present in each bacterial cell.

• Ti plasmid contains several regions of
  importance
• 1)  Transfer or T-DNA Is a region of the plasmid
  that is transferred from the bacteria to the host
  plant cell during the infection process.
• Once in the host, it becomes stably integrated in
  one of the host's chromosomes.
• T-DNA is 25-kbp long bracketed by two 25-bp
  direct repeats called left and right borders.

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Ti plasmid
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• Between the borders are several genes
• Isopentyl adenine transferase (IPT) synthesizes
  cytokinins.
• Tryptophan monooxygenase
• Indoleacetamide hydrogenase both enzymes
  involved in the biosynthesis of the auxin,
  indoleacetic acid.
• Gene for synthesis of a specific type of opine,
  either the octopine or nopaline type.

• The first three genes are involved in making the
  plant hormones, cytokinin and auxin.
• Massive production of these hormones at the site
  of infection causes the surrounding plant cells
  to divide and create the gall tumor.

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• 2) virulence or vir region Region that contains
  many genes required for the infection process.
• Important ones are vir A, B, C, D1, D2, E, G, and
  pin F that are required for the transfer and
  integration of the T-DNA into host.
• Vir region does not have to be physically
  connected to the T-DNA region can work in trans
  on a separate plasmid. 
• Basis for the construction of binary Ti plasmids.
  
• 3)  Replication origin for Agrobacterium.
• 4) Genes responsible for opine metabolism Allows
  Agrobacterium to metabolize opines back into
  arginine and carbon compounds.

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Course of events during Agrobacterium infection

• Agrobacterium present in the soil detects dicot
  plants susceptible to infection by the secretion
  of polyphenols from the roots or from wound
  sites.
• Since such wounds are a site of easy infection by
  bacteria, so they use the polyphenol signal to
  identify good targets.
• Bacteria move up chemical gradient of polyphenols
  to find the plant.

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• Polyphenols binds to a receptor encoded by vir A
  gene.
• Binding activates vir A which then activates the
  vir G protein by phosphorylation.
• Both vir A and G are constitutively expressed.
• Vir G protein is a transcription factor which
  then initiates transcription of the rest of vir
  genes on Ti Plasmid as well as vir genes on the
  Agrobacterium chromosome (CHV genes).
• Specific vir gene products then cut T DNA at left
  and right borders (Vir D1, D2, C).
• Single stranded copies of the T DNA region are
  synthesized, creating the T-strand.

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1. T-strand is coated with single stranded DNA
   binding proteins (Vir E) and the ss DNA/Vir E
   complex is shuttled out of the bacterium and
   transferred to plant cell where it is integrated
   in the host chromosome. Process similar to
   bacterial conjugation.
2. Once integrated in the plant chromosome, T-DNA
   genes become active, producing the oncogenic
   proteins for the synthesis of auxins and
   cytokinins, thus forcing the cells to
   proliferate. The opine synthesis enzyme is also
   produced and the manufactured opines are used as
   food for bacteria.

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• Life cycle of Agrobacterium make it a perfect
  vehicle for the stable introduction of foreign
  DNA into plants.
• Method involves insertion of DNA to be introduced
  between left and right borders of T-DNA and then
  infect the plant.

• Early methods used natural Ti plasmids that
  contained the oncogenes for hormone biosynthesis
  and the opine bosynthesis genes.

• Created transformed plants but presence of
  oncogenes caused plants to remain as galls
  (Hardly useful as a crop).

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Newer methods for transformation use highly
modified version of the Ti-plasmid

1. Are disarmed ("non-obcogenic") by deletion of the
   oncogenes.
2. Ti-plasmid is divided into two plasmids, a larger
   one containing the vir region and a smaller one
   containing only the T-DNA region. 

1. Smaller T-DNA plasmid contains two replication
   origins, one for E. coli and one for
   Agrobacterium, and antibiotic resistance gene for
   selection in E. coli and Agrobacterium. 
2. All natural genes are removed from the between
   the T-DNA borders (including those for opines)
   and replaced with a multiple cloning site to
   faciliatate insertion of your gene, and a
   selectable marker. Some plasmids also contain
   reporters in the T-DNA region.

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Transformation with Ti plasmids involves

1. Preparation of Ti plasmid containing the gene to
   be transferred.
2. Incubate with plant tissue wounded in some way to
   facilitate entry of bacterium into the plant.
3. Plating leaf section on media containing

1. Antibiotic to kill remaining Agrobacterium.
2. Balance of plant hormones to allow leaf cells to
   divide and form callus tissue.
3. Suitable toxin (e.g., kanamycin,
   phosphinothricin) to kill all cells that begin
   dividing that are not transformed and thus do not
   contain the NPT II gene (Process called
   selection).

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1. Transferring individual callus onto appropriate
   media with right hormone balance to allow
   regeneration of callus cells into intact plants.
2. Transformed plants will be hemizygous for
   inserted gene. Self pollination with convert some
   progeny into homozygous transformed lines.

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Problems with the use of Agrobacterium for
transformation

1. Same DNA between T-DNA borders can be inserted
   into multiple chromosomal regions of the
   transformed plants. Easy to get as many as 10
   copies inserted during a single transformation.
   Makes generating of homozygous plant difficult.
2. Only able to transform dicotyledonous plants with
   sufficient efficiency. Attempts to expand the
   host range of bacterium has met with little
   success.

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Direct transfer of DNA in plant cells

1. Electroporation

• Electroporation involves the use of electrical
  discharges to make cell leaky.
• Leaks then provide avenues for DNA to enter cell.
  
• Technique cannot transport DNA across cell wall
  so it must be removed to generate protoplasts.
• Cell wall removed by fungal enzymes that
  specialize in digesting cellulose, pectins and
  other cell wall polymers.
• Once missing the cell wall, protoplast are very
  fragile and sensitive to osmotic shock.

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• Protoplasts are mixed with DNA to be introduced
  and placed in a cuvette lined with two
  electrodes.
• Both stable and transient expression increased as
  DNA concentration is increased. Electric shock
  (200-400 V) is given for 50-100 msec.
• Cuvettes are cooled to reduce heat.
• Then the protoplasts are allowed to recovered and
  regenerate their cell wall.
• When placed on hormone media containing a toxin
  suitable for selection only those cells that are
  transformed will multiply producing calli (stable
  transformants).
• Electroporation was the first technique to
  transform cereals like corn.

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Advantages

1. Works for any plant species and cell type.
2. Provides quick and accurate data on expression
   using transient assays.
3. Can test to see if a particular gene you have
   created will work once stably integrated without
   having to wait to regenerate a transgenic plant.
4. Delivery of the DNA is quick and relatively
   inexpensive so you can do lots of tests.

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Problems You need to produce protoplasts first.
Since for many species, you cannot regenerate
easily intact fertile plants from protoplasts,
this method may be not suitable for producing
stably transformed plants.
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1. Microprojectile bombardment

• Technique developed by Sanford at Cornell and the
  patent was sold to DuPont.
• It is a technique for the delivery of DNA in
  intact plant cells using DNA-coated particles
  accelerated to high velocities.
• Such particles have enough momentum to penetrate
  the cell wall and become lodged inside cells. 
• Following bombardment, cells repair the holes and
  can survive.
• To penetrate the cell wall, particles must have
  sufficient momentum (p). Because p mv, the
  faster and heavier the particle the better.

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• Small (10 uM diameter) particles made with dense
  metals such as tungsten or gold are used.
• Particles coated with naked DNA (usually plasmids
  containing the gene to be inserted) are made by
  mixing the bead with a solution containing the
  DNA and then the solution is dried.
• Usually the plasmid contains both a selectable
  marker and the DNA of interest. 
• Once particles are lodged in the cells, the
  DNA/RNA will dissolve.
• The RNA can be directly translated, and DNA can
  be transcribe and translated

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• If the particles carrying DNA become lodged in
  the nucleus, the released DNA can stably
  integrate into the host chromosomes (stable
  transformation).
• Occurs at a very low frequency, so a strong
  selection is necessary.
• Since the individual cells that become
  transformed must regenerate into a whole plant,
  tissue culture cells, callus, and embryonic cells
  are typically used.

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• If the particles carrying DNA become lodged in
  the nucleus, the released DNA can stably
  integrate into the host chromosomes (stable
  transformation).
• Occurs at a very low frequency, so a strong
  selection is necessary.
• Since the individual cells that become
  transformed must regenerate into a whole plant,
  tissue culture cells, callus, and embryonic cells
  are typically used.

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• Advantages
• Bombardment is able to penetrate intact cells
  thus avoiding the need to remove the cell wall.
• It can work with any plant species.
• It was the first reliable technique to work with
  soybeans and moncots such as corn and rice.

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• Problems
• You need to be able to regenerate whole plant
  from the single bombarded cell.
• If complex tissue is used for bombardment, you
  can get chimeric plants containing both
  transformed and non-transformed tissue.
• Expensive.

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