In this Presentation i have collected some basic information about that how Bacteria causes diseases in plants.... crown gall disease is discussed. Contact 0311 9469029

1. AGROBACTERIUM MEDIATED
TRANSFORMATION
BY
ASNAD KHAN
ROLL NO 19TH
SEMESTER 6TH

2. OUTLINES
• Introduction To Transgenic Plants
• Discovery
• Requirements
• Introduction To Agrobacterium
• Agrobacterium Species
• Taxonomic Classification
• Ti-plasmid & Its Structure
• Crown Gall Disease
• Regeneration , Selection And Detection

3. TRANSGENIC PLANTS
• TRANSFORMATION
The process of
obtaining transgenic plants.
• TRANSGENIC PLANT
A plant with a foreign gene
from another plant that is
incorporated into its chromosome.

4. DISCOVERY
• Marc Van Montagu and Jeff
Schell, discovered the gene
transfer mechanism between
Agrobacterium and plants,
which resulted in the
development of methods to
alter the bacterium into an
efficient delivery system for
genetic engineering in plants.

5. REQUIRMENTS FOR PLANTS
GENETIC TRANSFORMATION
• SourceTissuesforPlant GeneTransfer
• Genetobetransferred
• GeneTransferMethods
• Regenerationof whole Transformed
Plants TransformedPlants

6. CONTINUED
• Most common genes (and traits)
in transgenic or biotech crops
are
 Herbicide resistance
 Insecticide resistance
 Bt genes in field corn (maize)
 Virus-resistance (protein coat)
genes
 99% are herbicide traits
 1% are other traits

7. AGROBACTERIUM
• Discovered by Smith and Townsend
(1907).
• A soil-born gram negative
bacterium. It is a rod shaped and
motile and belongs to the
bacterial family of Rhizobiaceae.
• It is a Phytopathogen, and it is
regarded as
• Nature’s most effective plant genetic
engineer. It is the natural expert of
inter-kingdom gene transfer.

8. TYPES OF AGROBACTERIUM
There are three kinds of Agrobacterium
species
Agrobacterium
tumefaciens
Crown Gall Diseases
Agrobacterium
rhizogenes
Hairy Root Diseases
Agrobacterium
radiobacter
Avirulent Strain

9. TAXONOMIC CLASSIFICATION
KINGDOM BACTERIA
PHYLUM PROTEOBACTERIA
CLASS ALPHAPROTEOBACTERIA
ORDER RHIZOBIALES
FAMILY RHIZOBIACEAE
GENUS AGROBACTERIUM
SPECIES A. TUMEFACIENS

10. TI-PLASMID AND VIRULENCE GENES
• A Ti Plasmid (200kb) Is A Circular Piece Of DNA Found In
Almost All Agrobacteria.
• Three Main Regions:
1. T-DNA Region – Between Right & Left DNA Border (24
Kb Each) Oncogene Opine
2. Virulence Region
3. Opine Catabolism Region

11. STRUCTURE
OF Ti-
PLASMID

12. CROWN GALL DISEASE
BY A.TUMEFACIENS
• Infects wounded or damaged part of
plants causing plant tumor called
crown gall.
• The entry of the bacterium is faciliated by
the release of phenolic compounds like
Acetosyringone and Hydroxysyringone.
• Crown gall occurs when the
bacterium releases its Ti-plasmid
into the plant cell cytoplasm. The
T-DNA is transferred to the host
cell via wounded section.

13. CROWN GALL
• The T-DNA carries genes that
code for proteins involved in
the biosynthesis of growth
hormones (auxin and
cytokinin) and novel plant
metabolites namely- opines
and agropines.
• Growth hormones – plant cell
proliferation
• Opines & agropines – source of C
and energy

14. TRANSFORMATION OF CELLS BY
A.tumefaciens
1. Signal induction to Agrobacterium – by the phenolic
compounds and some sugars, induces biochemical changes
that help in T-DNA transfer.
2. Attachment of Agrobacterium to plant cells –
Agrobacterium attaches to cell through polysaccarides
and cellulose fibres.
3. Production of virulence protein – signal induction causes
virulence protein to form Vir A, which induces Vir G, this
then induces the production of rest of the virulence proteins
like Vir D1/D2, Vir E, Vir B.

15. 4. Production of single stranded T-DNA – this is recognised
by vir D and thus carried forward to the host by Vir D2.
5. Transfer of T-DNA out of Agrobacterium – the DNA
strand is carried out by Vir D2. Through a channel made
by Vir B.
6. Transfer of T-DNA into plant cells and integration –
integration of the DNA into the host cell is helped by Vir
E2, which protects the DNA from degradation by host
cell restriction modification system, while Vir D2 helps to
navigate the DNA to the nucleus.
This process is called Illegimate Recombination, since it
does not depend on the sequence similarity..

16. REGENARATION,SELECTION,DETECTION
• REGENERATION:
For Shoot Organogenesis,Cytokinin (LowerAmountsOf Auxin)Are
Required.
• SELECTION:
(Two Antibiotics Are Required)
1. An Antibiotic To Kill The Agrobacterium, While Not
Affecting The Plant’s Cell Growth And Division.
2. A Second Antibiotic Allows Growth Of
Transformed Shoots But Inhibits Growth Of
Untransformed Plant Cells.

17. CONTD
• Detection of the “trait” gene:
1. PCR methods can detect the presence of the trait DNA.
2. Protein detection methods are used where a gene product is
produced that defines the trait.
3. Verification of the corporation of the trait gene into the
plant’s chromosome.
By southern hybridization
By demonstrating transfer of the trait to the original
transformant’s progeny

18. THANK YOU
ASNAD KHAN