Plant Genetic Engineering

Genetic EngineeringGenetic Engineering

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

Why do scientists want to change gene Why do scientists want to change gene instructions?instructions?

to produce needed chemicals to carry out useful processes

to give an organism desired characteristics

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.

THE SCIENCE OF GENETIC THE SCIENCE OF GENETIC ENGINEERINGENGINEERING

DNA of interest DNA of interest is EVERYWHEREis EVERYWHERE

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 Genetic Engineering

getting DNA into a cell

getting it stably integrated

getting a plant back from the cell

Plant transformation

Requirement1. a suitable transformation method

2.2. a means of screening for transformantsa means of screening for transformants

3. an efficient regeneration system

4. genes/constructs Vectors Promoter/terminator reporter genes selectable marker genes ‘genes of interest’

DNA Delivery to Target Cells

Selection and Regeneration

Event Selection

Plant Transformation

Transformation methods

DNA must be introduced into plant cells

Method depends on plant type, cost, application

Technique Methods

Indirect 1. Agrobacterium mediated gene transfer2. Viral vector

Direct 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.

AgrobacteriumAgrobacterium-mediated -mediated transformationtransformation

Transformation by the help of agrobacteriumTransformation by the help of agrobacterium

Agrobacterium is a ‘natural genetic engineer’

i.e. it transfers some of its DNA to plants

Agrobacterium tumefaciens

Ti plasmid

AgrobacteriumGenomic DNA

Plant cellGenomic DNA

(carries the gene of the gene of interestinterest)

+

Ti plasmid with the gene of interest

Gene of interest

Empty plasmid

Restriction enzyme AA

Restriction enzyme AA

Agrobacterium

Ti plasmid with the new gene

Plant cell

cell’s DNA

Transgenic plant Cell division

The new gene

+ Transformation

Agrobacterium tumefaciens

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” TechniqueGene Gun” TechniqueDNA coated

golden particles

Gene gun

Cell division

A plant cell withthe new gene

Transgenic plant

Plant cell

Cell’s DNA

Herbicide Resistance

A problem in agriculture is the reduced growth of crops imposed by the presence of unwanted weeds.

Herbicides such as RoundupTM and Liberty LinkTM 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.

EPSP SYNTHASE

Phosphoenol pyruvate

Tryptophan

Tyrosine

Phenylalaline

ROUNDUP(Glyphosate )

Glucose

3 phosphoglycerate

GlycolysisEPSP SYNTHASE

ROUNDUP(Glyphosate )

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

ROUNDUP (Glyphosate) TOLERANCE( HERBICIDE TOLERANCE IN CROPS)

Insect Resistance

Corn hybrid with a Bt gene Corn hybrid susceptible to European corn borer

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.

δ -endotoxin gene (Cry gene) of Bacillus thuriengenesis

GENE FOR Bt TOXIN WAS TRANSFERREDTO OBTAIN BT TRANSGENIC PLANTS

PLANT SYNTHESIZES INACTIVE PROTOXIN

PROTEINASEDIGESTION ININSECT GUT MAKES THEACTIVE TOXIN

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

leakage and insect death

INSECT FEEDS ONTRANSGENIC PLANT

Papaya infected with the papaya ringspot virus

Virus resistance gene introduced

Virus Resistant Crops

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

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 It is produced by blocking the polygalacturonase (PG) gene, which is involved in spoilage. PG is an (PG) gene, which is involved in spoilage. PG is an enzyme that breaks down pectin, which is found enzyme that breaks down pectin, which is found in plant cell walls. in plant cell walls.

Plants were transformed with the anti-sense PG Plants were transformed with the anti-sense PG gene, which is mRNA that base pair with mRNA gene, which is mRNA that base pair with mRNA that the plant produces, essentially blocking the that the plant produces, essentially blocking the gene from translation. gene from translation.

Delayed Fruit RipeningDelayed Fruit Ripening

“Rot-Resistant Tomato”Anti-sense gene complementary to

polygalacturonase (PG)PG = pectinase accelerates plant decay/rotting

Flav’r Sav’r tomato

Normal rice

“Golden” rice

Golden 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

Phytoene

Phytoene synthase

Gernayl Gernayl diphosphate (GGPP)

Phytoene desaturase

Lycopene

Lycopene cyclase

Beta carotene

Complete biochemical pathway in the rice for production of beta-carotene, a precursor for

vitamin A.

Edible VaccinesEdible 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 E.coli was produced in potato. Ingestion of this transgenic potato resulted in satisfactory vaccinations and no adverse effects.

Approved Transgenic Approved Transgenic plantsplants

SoybeanSoybean Corn Corn CottonCotton Oil Seed rapeOil Seed rape SugarbeetSugarbeet SquashSquash TomatoTomato TobaccoTobacco

CarnationCarnationss

PotatoPotato FlaxFlax PapayaPapaya ChicoryChicory RiceRice MelonMelon