To SEMINAR

On

AGROBACTERIUM MEDIATE
TRANSFORMATION ON HORTICULTURE CROPS

DNA and Genes

A gene = Is a segment of DNA

Guanine (G)

Cytosine (C)

Adenine (A)

Thymine (T)

bases
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DNA

gene

What is Gene transfer

Gene transfer is to transfer a gene from one DNA molecule to another DNA molecule.

(Arat, 2001)

Different methods for gene transfer

Method Particle delivery system

The gene gun was created as a new way of proceeding with gene transformation of grains in response to the difficulty of getting bacteria across grain cell walls. It was designed by John Sanford at Cornell University in 1987 and made introducing new genetic material into plant Cells. 4

Pressure gauge

Disk with DNAcoated particles

Vacuum line

Gas line

Vacuum chamber

Helium pressure

Gene Gun
Model from BioRad: Biorad's Helios Gene Gun

Microinjection
The microinjection method uses a fine needle to inject a solution of DNA into a cell.

Rapid transient expression assays using Agrobacterium infiltrations

Electroporation Technique
Power supply

Plant cell

Duracell

Protoplast

DNA containing the gene of interest

DNA inside the plant cell

The plant cell with the new gene

Agrobacterium

Agrobacterium
Agrobacterium is soil-borne. It is the dominant technology used for the production of genetically modified transgenic plants.

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(Tzfira and Citovsky, 2006)

Historical land mark in Agrobacterium biology

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Historical land mark in Agrobacterium biology contd.

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Advantages of Agrobacterium mediated Transformation

It is a natural means of transfer. It is capable of infecting intact plant cells, tissue & organs. Integration of T-DNA is a relatively precise process. The stability of gene transferred is excellent. Disadvantages It has the limitation of host range; some important food crops cannot be infected with Agrobacterium. Sometimes, cells in a tissue that are able to regenerate are difficult to transform. 13

Why Transgenic

World Population

Drought

Pests and Diseases'

Nature Calamity

Result

We need transgenic for

To meet the worlds need of food To increase yield To improve quality To cope better with climatic change Nutritional improvement Increased shelf life Improved taste and texture Stress resistance: drought, heat, cold, salt tolerance To reduce loss during transportation and storage Herbicide resistance Insect resistance Virus resistance

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How we do Transformations?

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Isolating The Gene

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Isolation Agrobacterium

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Cloning
Preparing genes to be inserted into
Agrobacterium (Cloning

Gene Cloning
When DNA is extracted from an organism, all its genes are obtained. In gene (DNA) cloning aparticular gene is copied (cloned).
Morley, 2002

How is DNA cloned?

DNA is extracted
Restriction enzymes, e.g. EcoRI, HindIII, etc., cut the DNA into small pieces Different DNA pieces cut with the same enzyme can join, or recombine.
Restriction enzymes

DNA

Roberts 2005 ,

Restriction Enzymes
Danna and Nathans, 1971

Dan Nathans (left) and Hamilton Smith (right), 1978.

Roberts, 2005

Restriction Enzymes
Over 3000 Restriction Enzymes have been studied in detail, and more than 600 of these are available commercially and are routinely used for DNA modification and manipulation in laboratories.

Roberts, 2005

Roberts and Kenneth Murray

How Restriction Enzymes are Named

Derivation of the EcoRI Name

Abbreviation Meaning
Escherichia coli RY13 First identified

Description
genus species strain order of identification in the bacterium

E co R I

Roberts, 2005

RESTRECTION ENZYME sem\seminar\Rest.swf

DNA Cloning
Bacterial plasmids are cut with the same restriction enzyme A chunk of DNA can thus be inserted into the plasmid DNA to form arecombinant

Cloning sem\seminar\clo.swf

Plant Genetic Engineering Process

Cell Plant cell

Extracted DNA

A single gene

Transformation Transgenic plant

Cell division

Agrobacterium sem\seminar\Agro.swf

Application of Agrobacterium in Horticulture crops

GMO VEGETABLES
Potato: pest tolerance and high protein content Tomato: viral, fungal and pest tolerance Brinjal: pest tolerance Cauliflower: pest tolerance Cabbage: pest tolerance Okra: viral and pest tolerance Blue : Resistant to blackspot disease

Case study

Worlds first blue Rose

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Three steps to make blue Rose

Switch off the production of red pigment Open the door to production of blue pigment; and then Produce blue pigment.

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CSIRO, 2005

One gene involved in flower colour, is the Dihydroflavonol Reductase (DFR) gene.
In roses the DFR gene is very good at producing red pigment and hence the range of commonly seen rose colours. However, the rose DFR gene is particularly bad at producing blue pigment, hence the difficulty in breeding a blue rose.
52 CSIRO, 2005

Preventing red pigment

The first critical step in producing a blue

rose was to stop the rose DFR gene making red pigment. Florigene and Suntory used (CSIROs) gene silencing technology to turn off the activity of the rose DFR gene so that it didnt produce red pigment.

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CSIRO, 2005

The second step towards a blue rose is to open the door to allow for blue pigment to be produced.

Florigene and Suntory inserted a gene commonly called a

delphinidin gene from pansy

that opened the door to the production of blue pigment in the rose flowers.

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CSIRO, 2005

The first critical step in producing a blue rose was to stop the rose DFR gene making red pigment.

52 CSIRO, 2005

Know we can enjoy by blue rose

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Question?
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Are the Genetically Modified Foods: (GMO) Harmful or

Helpful

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Debate
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Altered Crops Draw Protests, Pro and Con

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Conclusion
In more than 20 years, the use of Agrobacterium to genetically transform plants has advanced from a dream to a reality. The use A. tumefaciens to create transgenic plants has become routine for many crops. GMO have the potential to solve many of the worlds hunger and malnutrition problems. GMO Help to protect and preserve the environment by increasing yield and reducing reliance upon chemical pesticides and herbicides.