Download UNIT 5 NOTES 2012

UNIT 5 NOTES
1. Explain how DNA technology can be used to
improve the nutritional value of crops and to
develop plants that can produce pharmaceutical
products.
• Addresses the root
causes of micronutrient
BIOFORTIFICATION
deficiencies
• Produces genomic
foods
• EXAMPLES: Rice, maize,
wheat
• Process is transgenic
modifications
What is meant by transgenic
modification?
Inserting foreign
genes into an
existing
chromosome
The Golden Rice Story
• Vitamin A deficiency is a major health problem
– Causes blindness (particularly in children)
– Influences severity of diarrhea and measles
– Greater than 100 million children have Vit A deficiency
– In many countries, the infrastructure does not exist to
deliver vitamins in pill form
– One approachthat has beentaken is to improve the
Vitamin A content in widely consumed crops
-Carotene Pathway Problem in Plants
IPP
Geranylgeranyl diphosphate
Phytoene synthase
Phytoene
Problem:
Rice lacks
these enzymes
Phytoene desaturase
ξ-carotene desaturase
Lycopene
Lycopene-beta-cyclase
Normal
Vitamin A
“Deficient”
Rice
 -carotene
(vitamin A precursor)
The Golden Rice Solution
-Carotene Pathway Genes Added
IPP
Geranylgeranyl diphosphate
Daffodil gene
Phytoene synthase
Phytoene
Vitamin A
Pathway
is complete
and functional
Phytoene desaturase
Single bacterial gene;
performs both functions
ξ-carotene desaturase
Lycopene
Daffodil gene
Golden
Rice
Lycopene-beta-cyclase
 -carotene
(vitamin A precursor)
Other transgenic uses
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
•
Bt stands for Bacillus thuringiensis, a
bacteria that produces a toxin that kills
the insects. The gene that encodes the
toxin protein was inserted into plants
Herbicide resistant crops
Now: soybean, corn, canola
Coming: sugarbeet, lettuce, strawberry
alfalfa, potato, wheat
transgene = modified EPSP synthase or
phosphinothricin-N-acetyltransferase
Virus resistance - papya resistant to
papaya ringspot virus
transgene = virus coat protein
Glyophosate and glufosinate are the primary classes of herbicides that
the plants are resistant against. Virus resistance is obtained by inserting
the viral coat-protein gene into the plant. When this protein is
produced in the plant, the viral immune system is activated, and the
plant is resistant.
2. Identify various natural and artificial ways to propagate
plants to increase genetic variety or maintain the genetic
composition.
• Breeding – can be used to maintain or create new genetic
composition
Crossing two individuals from the same species;
produces a new, improved variety;
not a biotechnology procedure
Source: USDA
Source: USDA
• Transformation – used to create new genetic composotion
Adding a gene from another species; the
essential biotechnology procedure to produce
transgenics
• Cloning – used to maintain genetic composition, very
easy with plants , vegetative propagation or tissue culture.
• Plants are toipotent – a single cell can produce a
complete plant
•Grafting – causing two plants to grown together, genes
do not blend. Not biotechnology
Traditional Breeding example: Inter specific
Cross
Wheat
Rye
X
Triticale
New species, but
NOT biotechnology
products
3. Explain how genetic engineering supplements
traditional methods of plant breeding to generate new
traits in crop plants.
• The concepts may be
simple, but the reality is
time consuming and very
expensive
• High rate of failure,
especially with grain crops
such as wheat or rye which
are polyploid, this presents
challenges for transgenics!
Lets take a look at
the whole process
PLANT GENETIC ENGINEERING
•Product Concepts and Technical Feasibility
•Building the Transgenes
•Plant Transformation
•Event Selection
•Plant Breeding
•Seed Production and Marketing
•Detection of GMO Crops in the Commodity Chain
Product Concepts and Technical Feasibility
Market potential for GMO Crop
•alternatives for production inputs
•enhanced storage stability
•improved nutritional or processing qualities
Can the desired traits be engineered?
•How many genes must be introduced?
•Where must gene be expressed?
appropriate organs, tissues, developmental stage
localization within the cell
• Are genes and expression elements available to
modify trait?
•Will there be interactions with other genes?
Building the Transgenes
ON/OFF Switch
PROMOTER INTRON
Makes Protein
CODING SEQUENCE
Plant Transgene
Plant Selectable
Marker Gene
Plasmid DNA
Construct
bacterial genes
•antibiotic marker
•replication origin
stop sign
poly A signal
Plant Transformation
The introduction and expression of
genes into plants is a three step process:
DNA Delivery to Target Cells
Selection and Regeneration
Event Selection
Plant Transformation – DNA Delivery
microprojectile bombardment
“biolistics” or “gene gun”
Agrobacterium tumefaciens
natural property of Agrobacterium
tiny DNA-coated particles are shot to transfer DNA to host plant cells
into plant cells
is exploited to introduce genes of
interest
versatile method
difficult with cereal crops
complex DNA integration patterns:
tandem arrays of fragmented
simple DNA integration patterns
molecules
Plant Transformation – Target Cells
All Crop Transformation Protocols Deliver
DNA to Plant Cells in Tissue Culture
Tissue cultures allow regeneration of
fertile plants from single cells
Large number of target cells available for
DNA delivery in a compact form (callus)
Establishment, maintenance and plant
regeneration is labor intensive
Methods limited to a few genotypes,
usually not commercial varieties
Can introduce undesirable mutations
Plant Transformation – Selection
At best only 1 in 1000 cells integrate delivered
DNA
tissue culture cells
under selection
Transformed cells (events) are marked by cointroducing gene that provides resistance to
selective agents
Transformed cells are selected by killing nontransformed cells with selective agent.
Three main types of selective agents:
•antibiotics
•herbicides
•plant growth regulators
Selectable markers assist in following
inheritance of transgenes.
Herbicide Leaf Paint Assay
transgenic non-transgenic
resistant
susceptible
Event Selection
Goal: Identify transgenic lines that stably exhibit
desired phenotype
Typically only 1 in 100 events are commercialized
•Transgene expression varies with chromosome position
•Complex transgene insertions are generally unstable
•Transgene cannot have negative effects on other plant
phenotypes
•Transgenic line must satisfy regulatory requirements:
USDA, EPA, and FDA each review product
no novel toxic or allergenic proteins or metabolites
genetic stability
documented expression profile
THE MAKING OF A GMO CROP VARIETY
Backcrossing and selection (6 - 8 generations)
x
x
x
Transgenic
line
Commercial
variety
Biotechnology
Commercial Transgenic
Line
Seed Production
Target of 0.5% of U.S. Corn or Soybean Market
80 million acres x 0.005 = 400,000 acres
Corn (Cross-Pollinated Hybrids)
Planted at 30,000 plants/acre = 12 billion hybrid seed
Need 300 million seed of each inbred parent
Requires two field seasons to generate enough seed, one
season to produce hybrid seed
Soybean (Self-Pollinated Varieties)
require 3 seasons to generate enough seed
Maintaining Quality Control is a Challenge!!!
4. Analyze how changing the genome of an organism can
affect its ability to survive in different environments.
• Drought resistance in plants – University of California Davis has developed
plants that use 70% less water.
When water is scarce, plants are able to increase their chances of survival by minimizing water loss through
their leaves, increasing root growth while reducing leaf growth, and dropping their older leaves.
Scientists conjectured that the loss of leaves was the result of programmed cell death, a process by which the
plant triggers certain genes to initiate destruction of certain cells -- in this case, leaf cells.
The researchers set out to suppress the programmed death of leaf cells and equip the plants to survive severe
drought conditions.
Tobacco was chosen as an experimental plant because it is big, fast growing and a good model for many other
crop plants. The researchers inserted into the tobacco plants a gene that interrupted the biochemical chain of
events that normally leads to the loss of the plant's leaves during drought. It worked !
• Shortened growth season – Cambridge University, Eangland
Genetic researchers have sped up the growth of a plant by making its cells split faster, a technique that could
lead to heartier crops, shorter growing seasons and less use of herbicides.
The researchers first took a gene promoting cell division from inside the arabidopsis plant, a flowering weed
often used for genetic experiments. They transplanted that gene into a tobacco plant. There, in an especially
potent form, the gene produced large amounts of a protein that, in combination with other chemicals naturally
in the tobacco, made the plant's cells divide more quickly at the tips of roots and shoots. Within a month after
planting, the altered tobacco grew as much as twice as tall as other tobacco plants.
5. Predict how GM crops will interact with other plants
and insects in the environment
• Hot topic! – research being done, but long term
consequences unknown
• Gene transfer can occur between plants, no way to
stop pollen and seed spread – loss of diversity? GMO
crops push out native plants? Super weeds?
• Transgenic salmon have life spans about 70% shorter
than wild type, very hard to keep isolated.
• Human error and unforeseeable human factor!
6. Discuss the use of genetically engineered growth hormones in
animal production.
• bST; bovin somatotropin; used to
increase milk production
transgene = genetically
engineered enzyme
• Transgenic fish
Salmon,tilapia, catfish
Extra copies of growth
hormone that allow fish to
continue growing in cold
temperatures.
Concerns about effect of hormones on individuals who consume
the products, what effects do extra hormones have on children?
Animal “Pharming”
• 1997, Tracy the sheep,
the first transgenic animal
to produce a recombinant
protein drug in her milk
• alpha-1-antitrypsin (AAT)
treatment for emphysema
& cystic fibrosis
• Created by PPL
Therapeutics & The
Roslin Institute
http://nolswf.bbc.net.uk/science/genes/gene_safari/pharm/a_pharming.shtml
Nexia Biotechnologies transfered the silk gene from Orb spiders
into goats
The resulting male goats were used to sire silk-producing
Webster and Peter
female goats
Each goat produces several grams of silk protein in her milk
The silk is extracted, dried to a white powder, and spun into
fibers. Called BioSteel
The fibers are stronger and more flexible than steel
Transgenic male kids
carrying silk gene
GTC Biotherapeutics has
received approval to sell
human anti-thrombin
(ATryn) purified from goat’s
milk in Europe
Technology is not restricted
to cows, goats, & sheep
There is interest in using
rabbits since housing costs
are significantly less &
generation time is faster
Chickens which produce
recombinant drugs in their
eggs have been produced
by The Roslin Institute
7. Analyze the major challenges that face scientists in the
application of biotechnology in the animal production industry.
Think – pair – share
• Genetic engineering creates novel genetic combinations
• We saw the cost and time it takes to develop plants, which
propagate and grow fast (under a year), now compare that to
the time it would take to raise the same 6-8 generations of
animals such as cows!!
– Potential exists for undesirable effects of allergenicity or toxicity from
animal products.
– All GMOs are tested extensively for food safety prior to sale
• foods for human consumption and animal feed
• agricultural products (meat, dairy, fresh produce)
8. Propose how GM food technology is a solution to the
problems of population growth and environmental damage.
• GMO Crops Have Many Significant Environmental
Benefits
–
–
–
–
–
Reduced chemical pesticide and herbicide use
More sustainable pest management
Better erosion control through no-till practices
Enviro pig – pigs engineered for reduced phosphate in feces
Increased efficiency of production / unit fossil fuel energy expended
• GMO crops can be engineered to have
– optimal nutritional content – golden rice
– Deliver vaccinations and pharmaceuticals at a fraction of the cost
– Features that make it grow in environments which are not conducive to
crop growth (drought resistance, poor soil conditions, etc)
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
EnviroPig
TM
Transgenic pigs express phytase in their salivary glands
Phytic acid in the pig meal is degraded releasing phosphorus
The phosphorus is absorbed by the pig
Normally the phytic acid/phosphorus complex passes through the pig
and is excreted as waste
Pig waste is a major pollutant & can cause eutrophication of lakes &
streams
http://www.nature.com/news/2005/050221/images/nbt0305-283-I1
9. Discuss the use of organisms in a variety of industries
such as waste management, bioremediation and
energy production"