What is Biotechnology?

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

What About the TermGenetic Engineering?

Genetic engineering involves:

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

Genetic engineering is the basic tool set of biotechnology

What is a transgenic?

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

Concept Based on the Term Transgene

Biotechnology Terms You Probably Heard

Transgene: the foreign gene added to a species

Ex. – modified EPSP synthase gene (encodes a protein thatfunctions even when plant treated with Roundup)

Transgenic: an organism containing a transgene introduced by technological (not breeding) methods

Ex. – Roundup Ready Crops

Biotechnology Develops

GMOs - Genetically modified organisms

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

• Also called transgenic organism

Important Terms

• Breeding

• Transformation

Source: USDA

Source: USDA

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

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

Let’s 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

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.

Wheat Rye

Triticale

X

Interspecific Cross

New species, but NOT biotechnology products

ATTCGA

ATTGGA

SusceptibleNormalGene

ResistantMutantGene

MutagenesisTreatment

Mutagenesis: New Trait, No Foreign Gene

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

Transformation Cassettes

Contains

1. Gene of interest

• The coding region and its controlling elements

2. Selectable marker

• Distinguishes transformed/untransformed plants

3. Insertion sequences• Aids Agrobacterium insertion

Transformation Steps

Prepare tissue for transformation

Introduce DNA

Culture plant tissue• Develop shoots• Root the shoots

Field test the plants

• Leaf, germinating seed, immature embryos

• Tissue must be capable of developing into normal plants

• Agrobacterium or gene gun

• Multiple sites, multiple years

• Transformation cassettes are developed in the lab

• They are then introduced into a plant

• Two major delivery methods

Delivering the Geneto the Plant

• Agrobacterium

• Gene GunTissue culturerequired to generatetransgenic plants

The Next Test Is The Field

Non-transgenics

Transgenics

Herbicide Resistance

Final Test of the TransgenicConsumer Acceptance

RoundUp Ready Corn

Before After

Crop Biotech Market Dominated By Four Countriesa

68%35.7 mha

22%11.8 mha

6%3.2 mha 3%

1.5 mha

Total = 99% of market

a2001 growing season data.

Agriculture Products On the Market

Source: USDA

Insect resistant cotton

Insect resistant corn

Normal Transgenic

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

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

Virus resistance

Source: Monsanto

Herbicide resistant crops current: soybean, corn, canola coming: sugarbeet, lettuce, strawberry, alfalfa, potato, wheat (2005) resistance gene from bacteria

papaya, squash, potato resistance gene from a virus

Economic Effect of Bt CottonIn China

$200/acre increase in income

$750 million increase nationally

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

What Are the Public Concerns?

EconomicsAre we changing the economics on the farm?

EnvironmentalAre we irreversibly modifying the environment?  

GlobalizationIs technology becoming centralized in too few hands?

 Social

Will we develop a class of genetic outcasts?

ReligiousAre we playing God?

Benefits of Plant Biotechnology1. Greater production efficiencies: It can help plant breeders

improve a crop’s yield

2. Less chemical damage

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

4. Improved food quality.

5. New crops

6. Improved protection against human and animal diseases

Concerns associated with GM crops

1. 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).

File to support registration of new crop variety- conventional breeding

Biological systems for transformation

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

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.

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.

Ti plasmid

Between the borders are several genes:

1. Isopentyl adenine transferase (IPT): synthesizes cytokinins.

2. Tryptophan monooxygenase

3. Indoleacetamide hydrogenase: both enzymes involved in

the biosynthesis of the auxin, indoleacetic acid.

4. 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.

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.

Course of events during Agrobacterium infection

1) 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.

2. 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).

3. 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.

4. 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.

5. 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.

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).

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. 

3. 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. 

4. 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.

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:

A. Antibiotic to kill remaining Agrobacterium.

B. Balance of plant hormones to allow leaf cells to divide and form callus tissue.

C. 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).

4. Transferring individual callus onto appropriate media with right hormone balance to allow regeneration of callus cells into intact plants.

5. Transformed plants will be hemizygous for inserted gene. Self pollination with convert some progeny into homozygous transformed lines.

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.

Problems with the use of Agrobacterium for transformation:

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.

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.

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.

Advantages:

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.

2.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.

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;

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.

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.

Advantages:

1. Bombardment is able to penetrate intact cells thus avoiding the need to remove the cell wall.

2. It can work with any plant species.

3. It was the first reliable technique to work with soybeans and moncots such as corn and rice.

Problems:

1. You need to be able to regenerate whole plant from the single bombarded cell.

2. If complex tissue is used for bombardment, you can get chimeric plants containing both transformed and non-transformed tissue.

3. Expensive.