This document summarizes the process of plant genetic transformation using Agrobacterium tumefaciens. It describes how A. tumefaciens transfers T-DNA from its Ti plasmid into plant cells, integrating the T-DNA into the plant genome and expressing genes that cause crown gall disease. The document also outlines the key steps in the process, from gene transfer to the plant cell through regeneration of a transformed whole plant and methods to detect successful transformation events. Common genes inserted into transgenic crops are also listed, including genes for herbicide and insect resistance.

1. Presented by
Nishanth S
2016601106

2. • Transformation – the process of obtaining
transgenic plants.
• Transgenic plant – a plant with a foreign gene
from another plant that is incorporated into its
chromosome.
• 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.

4. Source Tissues for
Plant Gene
Transfer
Gene to be
transferred
Gene Transfer
Methods
Regeneration of
whole
Transformed Plant

5. • 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

7. • 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. • There are three kinds of Agrobacterium
species :--
• Dicots – Leaf discs
• Monocots – embryonic callus
Agrobacterium tumefaciens Crown gall disease
Agrobacterium rhizogenes Hairy root disease
Agrobacterium radiobacter Avirulent strain

9. Kingdom Bacteria
Phylum Proteobacteria
Class Alproteobacthaperia
Order Rhizobiales
Family Rhizobiaceae
Genus Agrobacterium
Species A. tumefaciens

10. • 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. • 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.

12. • 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.

13. 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.

14. 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. • Regeneration:
for shoot organogenesis, cytokinin (lower amounts of
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. • 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