UNIT 5 NOTES

1. Explain how DNA technology can be used to 1. Explain how DNA technology can be used to improve the nutritional value of crops and to improve the nutritional value of crops and to develop plants that can produce pharmaceutical develop plants that can produce pharmaceutical products.products.

BIOFORTIFICATIONBIOFORTIFICATION

• Addresses the root causes of micronutrient deficiencies

• Produces genomic foods

• EXAMPLES: Rice, maize, wheat

• Process is transgenic modifications

What is meant by transgenic modification?

Inserting foreign Inserting foreign

genes into an genes into an existing existing chromosome chromosome

The Golden Rice StoryThe 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

Lycopene

-carotene(vitamin A precursor)

Phytoene synthase

Phytoene desaturase

Lycopene-beta-cyclase

ξ-carotene desaturase

Problem:Rice lacks

these enzymes

NormalVitamin A“Deficient”

Rice

The Golden Rice Solution

IPP

Geranylgeranyl diphosphate

Phytoene

Lycopene

-carotene(vitamin A precursor)

Phytoene synthase

Phytoene desaturase

Lycopene-beta-cyclase

ξ-carotene desaturase

Daffodil gene

Single bacterial gene;performs both functions

Daffodil gene

-Carotene Pathway Genes Added

Vitamin APathway

is completeand functional

GoldenRice

Insect resistant cotton – Bt toxin kills the cotton boll worm

transgene = Bt proteinInsect 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

Other transgenic uses

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.

Herbicide resistant crops

Now: soybean, corn, canolaComing: sugarbeet, lettuce, strawberry alfalfa, potato, wheattransgene = modified EPSP synthase orphosphinothricin-N-acetyltransferase

Virus resistance - papya resistant to papaya ringspot virus transgene = virus coat protein

2. Identify various natural and artificial ways to propagate 2. Identify various natural and artificial ways to propagate plants to increase genetic variety or maintain the genetic plants to increase genetic variety or maintain the genetic composition.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

Source: USDA

• 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

Wheat Rye

Triticale

X

Traditional Breeding example: Inter Traditional Breeding example: Inter specific Crossspecific Cross

New species, butNew species, but NOT biotechnologyNOT biotechnology productsproducts

3. Explain how genetic engineering supplements 3. Explain how genetic engineering supplements traditional methods of plant breeding to generate new traditional methods of plant breeding to generate new traits in crop plants.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 Lets take a look at

the whole process 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?

CODING SEQUENCEINTRON poly A signalPROMOTER

Building the Transgenes

Plant Transgene

bacterial genes•antibiotic marker•replication origin

Plant Selectable Marker Gene

Plasmid DNA Construct

ON/OFF Switch Makes Protein stop sign

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”

tiny DNA-coated particles are shot into plant cells

versatile method

complex DNA integration patterns: tandem arrays of fragmented molecules

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

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

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 Assaytransgenic 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 productno novel toxic or allergenic proteins or metabolitesgenetic stabilitydocumented expression profile

THE MAKING OF A GMO CROP VARIETYBackcrossing and selection (6 - 8 generations)

Transgenic line

Commercial variety

x

x

x

Commercial Transgenic Line

Biotechnology

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 4. Analyze how changing the genome of an organism can affect its ability to survive in different environments.affect its ability to survive in different environments.

• Drought resistance in plants – University of California Davis has developed Drought resistance in plants – University of California Davis has developed plants that use 70% less water.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, EanglandShortened growth season – Cambridge University, EanglandGenetic 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 5. Predict how GM crops will interact with other plants and insects in the environmentand insects in the environment

• Hot topic! – research being done, but long term Hot topic! – research being done, but long term consequences unknownconsequences unknown

• Gene transfer can occur between plants, no way to Gene transfer can occur between plants, no way to stop pollen and seed spread – loss of diversity? GMO stop pollen and seed spread – loss of diversity? GMO crops push out native plants? Super weeds?crops push out native plants? Super weeds?

• Transgenic salmon have life spans about 70% shorter Transgenic salmon have life spans about 70% shorter than wild type, very hard to keep isolated. than wild type, very hard to keep isolated.

• Human error and unforeseeable human factor! 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 fishSalmon,tilapia, catfishExtra 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?

http://nolswf.bbc.net.uk/science/genes/gene_safari/pharm/a_pharming.shtml

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

Webster and Peter

Nexia Biotechnologies transfered the silk gene from Orb spiders into goatsThe resulting male goats were used to sire silk-producing female goatsEach goat produces several grams of silk protein in her milkThe silk is extracted, dried to a white powder, and spun into fibers. Called BioSteelThe 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 7. Analyze the major challenges that face scientists in the application of biotechnology in the animal production industry. application of biotechnology in the animal production industry.

Think – pair – share 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 VaccinesEdible VaccinesTransgenic Plants Serving Human Health NeedsTransgenic Plants Serving Human Health Needs

• Works like any vaccine Works like any vaccine • A transgenic plant with a pathogen protein gene is developedA transgenic plant with a pathogen protein gene is developed• Potato, banana, and tomato are targetsPotato, banana, and tomato are targets• Humans eat the plant Humans eat the plant • The body produces antibodies against pathogen proteinThe body produces antibodies against pathogen protein• Humans are “immunized” against the pathogenHumans are “immunized” against the pathogen• Examples:Examples:

DiarrheaDiarrheaHepatitis BHepatitis BMeaslesMeasles

EnviroPig EnviroPig TMTM

Transgenic pigs express phytase in their salivary glandsTransgenic pigs express phytase in their salivary glands

Phytic acid in the pig meal is degraded releasing phosphorusPhytic acid in the pig meal is degraded releasing phosphorus

The phosphorus is absorbed by the pigThe phosphorus is absorbed by the pig

Normally the phytic acid/phosphorus complex passes through Normally the phytic acid/phosphorus complex passes through the pig and is excreted as wastethe pig and is excreted as waste

Pig waste is a major pollutant & can cause eutrophication of Pig waste is a major pollutant & can cause eutrophication of lakes & streamslakes & streams

http://www.nature.com/news/2005/050221/images/nbt0305-283-I1.jpg

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