Plant Genetic Engineering

Genetic Engineering

The process of manipulating and transferring instructions carried by genes from one cell to another

Why do scientists want to change gene instructions?

 

to produce needed chemicals



to carry out useful processes to give an organism desired characteristics

THE SCIENCE OF GENETIC ENGINEERING

Isolate desired gene for a new trait from any organism Isolate plasmid DNA Gene inserted into plasmid.

Introduce modified plasmid into

bacterium for replication.

Grow in culture to replicate.

DNA of interest is EVERYWHERE

Plant Genetic Engineering

 Product Concepts and Technical Feasibility  Building the Transformant  Plant Transformation  Selection  Plant Breeding  Seed Production and Marketing  Detection of GMO Crops in the Commodity Chain

Plant transformation

 getting DNA into a cell  getting it stably integrated  getting a plant back from the cell

Requirement

1. a suitable transformation method 2. a means of screening for transformants 3. an efficient regeneration system 4. genes/constructs Vectors reporter genes ‘genes of interest’ Promoter/terminator selectable marker genes

Plant Transformation

DNA Delivery to Target Cells Selection and Regeneration Event Selection

Transformation methods

DNA must be introduced into plant cells

Technique Methods

Indirect Direct 1. Agrobacterium mediated gene transfer 2. Viral vector 1. Lipid-mediated method, 2. Calcium-phosphate mediated, 3. Dextran-mediated, 4. Electroporation, 5. Biolistics, 6. Polybrene, 7. Laser transfection, 8. Gene transfection enhanced by elevated temperature.

Method depends on plant type, cost, application

Agrobacterium tumefaciens plasmid

• A large (~250kbp) plasmid called Tumor-inducing (Ti) plasmid) • Plasmid contains genes responsible for the crown gall disease • Portion of the Ti plasmid is transferred between bacterial cells and plant cells  T-DNA (Transfer DNA) • There are 2 x 23bp direct repeat border

Agrobacterium tumefaciens

Ti plasmid

vir genes

for transfer to the plant

T-DNA

23 kb

tra

bacterial conjugation pTi ~200 kb

opine catabolism

Genetic engineering of the Ti plasmid

Some considerations:  Ti plasmid is 200 kb - too big for easy cloning  need to remove tumor-inducing genes from T-DNA Ti plasmid

Disarm

Disarmed Ti plasmid

VIR

genes Remember:

– Only

VIR

transfer genes are required for T-DNA – Only LB and RB are required for T-DNA insertion into genome

T-DNA YFG

Cloning vector

Agrobacterium

The binary Ti plasmid system

Binary vector system

Binary vector system

Agrobacterium

-mediated transformation

Transformation by the help of agrobacterium

Agrobacterium

is a ‘natural genetic engineer’ i.e. it transfers some of its DNA to plants

Agrobacterium tumefaciens

Agrobacterium

Genomic DNA Genomic DNA (carries the gene of Plant cell interest ) Ti plasmid Restriction enzyme A Restriction enzyme A + Empty plasmid Gene of interest Ti plasmid with the gene of interest

Agrobacterium tumefaciens

Ti plasmid with

the new gene +

cell’s DNA Transformation

Agrobacterium

Plant cell

The new gene

Transgenic plant Cell division

Biolistics

 ‘gene gun’  DNA is coated onto gold (or tungsten) particles (inert)  Gold is propelled by helium into plant cells  if DNA goes into the nucleus it can be integrated into the plant chromosomes  Cells can be regenerated to whole plants

“Gene Gun” Technique

DNA coated golden particles Cell’s DNA Plant cell Gene gun A plant cell with

the new gene

Transgenic plant Cell division

Tearless Onion

Colorful Cauliflowers

Purple tomatoes

Blue Roses

Herbicide Resistance

A problem in agriculture is the reduced growth of crops imposed by the presence of unwanted weeds. Herbicides such as Roundup TM and Liberty Link TM are able to kill a wide range of weeds and have the advantage of breaking down easily. Development of herbicide resistant crops allows the elimination of surrounding weeds without harm to the crops .

ROUNDUP (Glyphosate) TOLERANCE ( HERBICIDE TOLERANCE IN CROPS) Glucose ROUNDUP (Glyphosate ) 3 phosphoglycerate Glycolysis EPSP SYNTHASE Phosphoenol pyruvate EPSP SYNTHASE Tryptophan Tyrosine Phenylalaline ROUNDUP (Glyphosate )

In transgenic plant, herbicide can not bind the mutant of EPSP synthase (Example: RR-Cotton, RR-Soybean)

Insect Resistance

Various insect resistant crops have been produced. Most of these make use of the Cry gene in the bacteria

Bacillus thuringiensis (Bt)

; this gene directs the production of a protein that causes paralysis and death to many insects .

Corn hybrid with a

Bt

gene Corn hybrid susceptible to European corn borer

δ -endotoxin gene (Cry gene) of Bacillus thuriengenesis GENE FOR Bt TOXIN WAS TRANSFERRED TO OBTAIN BT TRANSGENIC PLANTS INSECT FEEDS ON TRANSGENIC PLANT PLANT SYNTHESIZES INACTIVE PROTOXIN PROTEINASE DIGESTION IN INSECT GUT MAKES THE ACTIVE TOXIN

Toxin binds a receptor on the gut epithelial cells, forms a channel on the membrane. This causes electrolyte leakage and insect death

Virus Resistant Crops

Papaya infected with the papaya ringspot virus Virus resistance gene introduced The Freedom II squash has a modified coat protein that confer resistance to zucchini yellows mosaic virus and watermelon mosaic virus II.

Scientists are now trying to develop crops with as many as five virus resistance genes

Delayed Fruit Ripening

    Tomatoes are usually picked and sprayed with the plant hormone ethylene to induce ripening, although this does not improve taste Tomatoes have been engineered to produce less ethylene so they can develop more taste before ripening, and shipment to markets. It is produced by blocking the polygalacturonase (PG) gene, which is involved in spoilage. PG is an enzyme that breaks down pectin, which is found in plant cell walls. Plants were transformed with the anti-sense PG gene, which is mRNA that base pair with mRNA that the plant produces, essentially blocking the gene from translation.

Flav’r Sav’r tomato

“Rot-Resistant Tomato” Anti-sense gene  complementary to polygalacturonase (PG) PG = pectinase  accelerates plant decay/rotting

Golden Rice

Normal rice Transgenic technology produced a type of rice that accumulates beta-carotene in rice grains. Once inside the body, beta-carotene is converted to vitamin A. “Normal” rice “Golden” rice

Gernayl Gernayl diphosphate (GGPP) Phytoene synthase Phytoene Phytoene desaturase Lycopene Lycopene cyclase Beta carotene

Complete biochemical pathway in the rice for production of beta carotene, a precursor for vitamin A.

Edible Vaccines

Edible vaccines are vaccines produced in plants that can be administered directly through the ingestion of plant materials containing the vaccine. Eating the plant would then confer immunity against diseases.

The first human clinical trial took place in 1997. Vaccine against the toxin from the bacteria adverse effects.

E.coli

was produced in potato. Ingestion of this transgenic potato resulted in satisfactory vaccinations and no

Approved Transgenic plants

        Soybean Corn Cotton Oil Seed rape Sugarbeet Squash Tomato Tobacco        Carnations Potato Flax Papaya Chicory Rice Melon