T-DNA mediated Gene Transfer

in Plants (T-DNA 는 식물에서 유전자 이동을 중재했다 )

Sathiyaraj Srinivasan
사티야라지 스리니바산
Dept. of Oriental Medicinal
materials and Processing
Kyung Hee University,
Republic of Korea – 449 701
Gene Transfer Technique

To transfer a gene from one DNA molecule to another DNA molecule.

Gene Transfer includes isolation of gene, manipulation (target gene

insertion ) and reintroduction of DNA into the cells or model organisms.

It used to make a crop to resistant to a particular herbicide, pest, weeds

or introducing a novel trait, or producing a new protein or enzyme.

The gene transfer methods normally include three categories:

1. transduction by biochemical methods,

2. transduction by physical methods,
3. virus-mediated transduction.

 The gene transfer results can be transient and stable transduction.

Transfection Types:

Transient transfection

In transient transfection, the transfected DNA is not integrated into

host chromosome. DNA is transferred into a recipient cell in order to obtain a
temporary but high level of expression of the target gene.

Fast, flexible, not influenced by position effects, can provide large

amounts of protein for detailed characterization, does not require regeneration
system, gene expression limited to specific plant tissues.

Stable transfection

Stable transfection is also called permanent transfection. By the stable

transfection, the transferred DNA is integrated (inserted) into chromosomal
DNA and the genetics of recipient cells is permanent changed.

Time consuming, labor intensive, influenced by position effects, large

production volumes, requires regeneration system, large amounts of
glasshouse space, gene expression in all plant tissues
Transformation Methods:

Generally, there are 9 ways for gene transfer: (as published by

Sambrook, 2001).

(1) Lipid-mediated method,

(2) Calcium-phosphate mediated,
(3) DEAE-dextran-mediated,
(4) Electroporation,
(5) Biolistics,
(6) Viral vectors,
(7) Polybrene,
(8) Laser transfection,
(9) Gene transfection enhanced by elevated temperature.
Introduction to Agrobacterium tumefaciens

Agrobacterium tumefaciens is a ubiquitous soil borne pathogen

responsible for Crown Gall disease, affecting many higher species of plant.

It’s a Gram negative, motile, rod shaped bacterium which is non

sporing, and is closely related to the N-fixing rhizobium bacteria which form root
nodules on leguminous plants.

The bacterium is surrounded by a small number of peritricious flagella.

Virulent bacteria contain one or more large plasmids, one of which

carries the genes for tumour induction and is known as the Ti (tumour inducing)
plasmid.
 Crown Gall Disease

Agrobacterium tumefaciens
Mechanism of infection of Bacterium in plants
Ti Plasmid :
The tumor inducing plasmid (pTi) contains a portion that is
transferred to the plant cell is Transfer DNA (T-DNA)

Ti plasmids can be classified according to the opines produced

1. Nopaline plasmids: carry gene for synthesizing nopaline in the plant

and for utilization (catabolism) in the bacteria. Tumors can differentiate
into shooty masses (teratomas).

2. Octopine plasmids: carry genes (3 required) to synthesize octopine

in the plant and catabolism in the bacteria. Tumors do not differentiate,
but remain as callus tissue.
3. Agropine plasmids: carry genes for agropine synthesis and
catabolism. Tumors do not differentiate and die out.

4. Ri plasmids: induce hairy root disease on some plants and crown

gall on others; have agropine-type genes and may have segments from
both nopaline and octopine plasmids
 Vir Genes and their Functions
Vir Gene Function
Vir A, Sense phenolic compounds from wounded plant cells and induce
expression of other virulence genes
Vir G
VirD2 Endonuclease; cuts T-DNA at right border to initiate T-strand synthesis

Vir D1 Topiosomerase; Helps Vir D2 to recognise and cleave within the 25bp
border sequence
Vir D2 Covalently attaches to the 5I end of the T-strand, thus forming the T-DNA
Complex. Also guides the T-DNA complex through the nuclear pores
Vir C Binds to the 'overdrive' region to promote high efficiency T-strand
Synthesis
Vir E2 Binds to T-strand protecting it from nuclease attack, and intercalates with
lipids to form channels in the plant membranes through which the
T-complex passes
Vir E1 Acts as a chaperone which stabilises Vir E2 in the Agrobacterium

Vir B & Vir Assemble into a secretion system which spans the inner and outer
bacterial membranes. Required for Export of the T-complex and Vir E2
D4
into the plant cell
 Important genes encoded by
Ti plasmid
1. Cytokinins
(plant hormone for cell plant division and tumorous growth)

2. Enzymes for indoleacetic acid (auxin) synthesis

Another plant hormone (inducing stem and leaf elongation, inducing parthenocarpy and
preventing aging)

3. Enzymes for synthesis and release of novel plant

metabolites:
the opines (uniques amino acid derivatives)
the agrocinopines (phosphorylated sugar derivatives) .
Nopaline
Opines and agrocinopines are NUTRIENTS for A.tumefacies.
They can not be used by other bacterial species
It provides unique niche for A.tumefaciens
Ti plasmids and the bacterial chromosome act in concert
to transform the plant

1. Agrobacterium tumefaciens chromosomal genes: chvA, chvB, pscA

required for initial binding of the bacterium to the plant cell and code
for polysaccharide on bacterial cell surface.

• Virulence region (vir) carried on pTi, but not in the transferred region
(T-DNA). Genes code for proteins that prepare the T-DNA and the
bacterium for transfer.

3. T-DNA encodes genes for opine synthesis and for tumor production.

4. oc (opine catabolism) genes carried on the pTi and allows the

bacterium to utilize opines as nutrient.
 Agrobacterium chromosomal DNA

chvA pscA
chvB

T-DNA
tra bacterial conjugation
pTi
vir genes
opine catabolism
transfer to the plant
oriV inc pTi’s are in the same inc group.
 Ti Plasmid
T-DNA
region
DNA between
L and R borders is
transferred to plant
as ssDNA;
Tumor-
producing
genes
T-DNA encoded
Opine catabolism genes can be
substituted by
Virulence region target genes
ORI
 The basis of Agrobacterium-mediated genetic
engineering
 T-DNA of A. tumefaciens is excised and integrates into
the plant genome as part of the natural infection
process.
 Any foreign DNA inserted into the T-DNA will also be
integrated.
 Ti-plasmid based vectors

Binary systems Co-integrated vectors

Needs 2 vectors: Needs 3 vectors

Disarmed Ti plasmid
Disarmed Ti plasmid
capable for infection
with gene of interest Form
(no vir genes) co-integrated plasmid Intermediate vector
after homologous with T-region
recombination on T-DNA and gene of interest
Helper vector (transferred by conjugation)
for infection Helper vector
(with vir genes) for transfer of
intermediate plasmid into A.tum
 Co-integrated vectors
(hybrid ti-plasmids)

In a modern labs
rarely used

DISADVANTAGES:
1) Long homologies required between the Ti plasmid
and the E. coli plasmids (pBR322 based Intermediate vectors)
making them difficult to engineer and use

2) Relatively inefficient gene transfer compared to the binary vector

 Ti plasmid vector systems
are often working as binary vectors

DISADVANTAGE: Depending on the orientation, plasmids with two

different origins of replication may be unstable in E. coli.

ADVANTAGE: small vectors are used, which increases transfer efficiency

from E. coli to Agrobacterium. No intermolecular recombination is needed
 Promoters useful for expression
in transgenic plants
35S, cauliflower mosaic virus 35S promoter

CaMoV is a circular dsDNA genome virus

CaMoV 35S is a strong promoter

that is active
in essentially all dicot plant tissues.
 Vir Gene Vir D1 & D2 cut
expression T-DNA at right
induced and left borders. Formation of T-
complex

Phenolics AGROBACTERIUM
detected by the
VirA/VirG two T-DNA VirD2 attaches to
component exposed 5I end
sensor system.
Bacterial
Plasmid
Formation of T-Pilus
VirA VirG

PLANT CELL

Phenolics
Produced by VIP1 associates with
Wounded the complex to target
Plant cell it to the nucleus
VIP2 associates the
complex to

Gall Formation! transcriptionally

T-DNA integrates into plant DNA active DNA
and gall production is initiated.
 Procedure for creation a transgenic plant
1. Both plasmids are transfected into A.tumefaciens

2. Plant cell culture is infected with A.tumefaciens

3. Products of Vir genes excised gene of interest within T-DNA

and transfer it to plant chromosome
T-DNA Repeat Polylinker Kan-resistance gene T-DNA Repeat

Gene of interest
4. Plant cells are selected on kanamycine
5. Presence of transgene confirmed by PCR
6. Whole plant could be grown from transformed cells !!!
 Transgenic Plants

 Currently 16 countries permit the cultivation of transgenic crops

 99% of the total acreage is in 6 countries
 USA, China, Argentina, Canada, Brazil* & Australia
 Five crops are currently in release
 soybean, cotton, corn, canola, squash


tomato, potato and flax in past

 Since the first widespread release in 1996, land devoted to transgenic crops
has increased by 10 - 50% yearly
 90% of Canadian canola is GMO - mostly transgenic
 20+ countries suspected of growing transgenic crops without official
approval
 Global area of transgenic crops
(ISAA Brief. Global Review of Commercialised Transgenic crops: 1998 & 2001)

60

Millions of hectares
 Acreage of transgenic 50
crops has gone from 40
nothing in 1995 to 30
around 135 million
20
acres in 2001.
10
0
1995 1997 1999 2001
Approved Transgenic plants
 Soybean  Carnations
 Corn  Potato
 Cotton  Flax
 OilSeed rape  Papaya
 Sugarbeet  Chicory
 Squash  Rice
 Tomato  Melon
 Tobacco
 Types of GM crops (1998)

40
35
 Soybean and corn are the 30
major GM crops 25

area
20
* Large acreage
15
* Grown in the USA 10
* Can be regenerated 5
 Acreage of potatoes is small 0

Soybean

Cotton
Corn

Oil Seed
(<0.1 million hectares)
 Types of genetic modification

Millions of hectares
25
20
 >99% of all 15
transgenic crops 10
are either herbicide 5
or insect resistant 0

resistance
Herbicide

Others
Insect
 <1% have other
traits