1. UNIT 5 NOTES

2. 1. Explain how DNA technology can be used to improve the nutritional value of crops and to develop plants that can produce pharmaceutical products. BIOFORTIFICATION
Addresses the root causes of micronutrient deficiencies
Produces genomic foods
EXAMPLES: Rice, maize, wheat
Process is transgenic modifications

3. What is meant by transgenic modification?
Inserting foreign genes into an existing chromosome

4. 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
The second major plant biotechnology product is more recent and was developed to address the vitamin A deficiency problems prevalent throughout the world. This vitamin deficiency is very critical because it can cause blindness and affects the severity of many diseases including diarrhea and measles. This is a severe problem that affects more than 100 million children worldwide. A simple solution would be to distribute vitamins to the affected children. Unfortunately, many countries where the deficiency is chronic do not have the necessary infrastructure to deliver the vitamin tablets to the most needed.
The solution that is currently being promoted is to improve the vitamin content in widely-consumed, and readily available to the consumer. Transgenic rice plants were developed that contain elevated levels of the precursor to vitamin A. This GMO is called “Golden Rice” because of its color: it is yellow rather than white. It is yellow because β-carotene, a yellow precursor to vitamin A is abundant in the seed.

5. Lycopene-beta-cyclase
-Carotene Pathway Problem 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
Unlike the single-step RoundUp Ready pathway, the β–carotene synthesis pathway involves multiple enzymes. This important vitamin A precursor cannot be synthsized in rice because it lacks four of the key enzymes. Therefore, the precursor is not made, and the plant contains white kernels.
Normal
Vitamin A
“Deficient”
Rice

6. The Golden Rice Solution
-Carotene Pathway Genes Added
IPP
Geranylgeranyl diphosphate
Phytoene
Lycopene
 -carotene
(vitamin A precursor)
Phytoene synthase
Phytoene desaturase
Lycopene-beta-cyclase
ξ-carotene desaturase
Vitamin A
Pathway
is complete
and functional
Daffodil gene
Single bacterial gene;
performs both functions
In a major feat of genetic engineering, scientists inserted a complete functioning -carotene biosynthetic pathway into the rice plant. They did this by inserting genes from daffodil the produce functioniong versions of the first and last enzymes of the pathway. In addition, a single bacterial gene that provides the same function as the second and third enzymes of the pathway, was also introduced. With a functioning pathway, the transgenic rice is able to produce the vitamin A precursor β-carotene. It is this product that gives "Golden Rice" its characteristic yellow color.
Daffodil gene
Golden
Rice

7. 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
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

8. 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.

9. 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
Transformation – used to create new genetic composotion
Adding a gene from another species; the
essential biotechnology procedure to produce
transgenics
Source: USDA
These are important definitions related to crop improvement. Not the distinction; one is a biotechnology procedure, the other is not.
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
Source: USDA

10. Traditional Breeding example: Inter specific Cross
Wheat
Rye X
Triticale
New species, but
NOT biotechnology
products
These photos illustrate the use of plant breeding to produce a new crop of agronomic utility.

11. Lets take a look at the whole process
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

12. 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

13. 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?

14. Building the Transgenes
ON/OFF Switch
Makes Protein
stop sign
CODING SEQUENCE
INTRON
poly A signal
PROMOTER
Plant Selectable
Marker Gene
Plasmid DNA
Construct
Plant Transgene
bacterial genes
antibiotic marker
replication origin

15. DNA Delivery to Target Cells
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

16. Plant Transformation – DNA Delivery
microprojectile bombardment
“biolistics” or “gene gun”
Agrobacterium tumefaciens
natural property of Agrobacterium to transfer DNA to host plant cells is exploited to introduce genes of interest
difficult with cereal crops
simple DNA integration patterns
tiny DNA-coated particles are shot into plant cells
versatile method
complex DNA integration patterns: tandem arrays of fragmented molecules

17. 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

18. Plant Transformation – Selection
At best only 1 in 1000 cells integrate delivered DNA
Transformed cells (events) are marked by co-introducing gene that provides resistance to selective agents
Transformed cells are selected by killing non-transformed cells with selective agent.
Three main types of selective agents:
antibiotics
herbicides
plant growth regulators
Selectable markers assist in following inheritance of transgenes.
tissue culture cells
under selection
Herbicide Leaf Paint Assay
transgenic non-transgenic
resistant susceptible

19. 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

20. THE MAKING OF A GMO CROP VARIETY
Backcrossing and selection (6 - 8 generations) x x x
Transgenic
line
Commercial
variety
Commercial Transgenic
Line
Biotechnology

21. Seed Production Target of 0.5% of U.S. Corn or Soybean Market
80 million acres x = 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!!!

22. 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.

23. 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!

24. Salmon,tilapia, catfish Extra copies of growth
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?

25. 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

26. The resulting male goats were used to sire silk-producing female goats
Nexia Biotechnologies transfered the silk gene from Orb spiders into goats
The resulting male goats were used to sire silk-producing 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
Webster and Peter
Transgenic male kids carrying silk gene

27. 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
Billy and Lilly

28. Genetic engineering creates novel genetic combinations
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)

29. GMO Crops Have Many Significant Environmental Benefits
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)

30. Transgenic Plants Serving Human Health Needs
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
Edible vaccines may be the most important and accepted biotech product. The principles are the same as those used for normal vaccines: a protein enters the body in some manner, and the human immune system produces antibodies against that protein. When the human is then exposed to the pathogen, the immune system is turned on and destroys the pathogen. or

31. 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

32. 9. Discuss the use of organisms in a variety of industries such as waste management, bioremediation and energy production"