Genetic Transformation Methods

There are a variety of methods available

for moving genes into recepient cells.
This method involves formation of
recombinant DNA molecule.
These methods are microinjection, electro
and chemical poration, and biolistics,
apart natural Agrobacterium- mediated
transformation.

Methods of gene transfer

Dr. Y. Venkateswarlu
Lecturer in Biotechnology,
Govt College (M),
Anantapur.

Methods of gene transfer

1.
2.
3.
4.

Agrobacteriumtumefaciens
Biolistics
Protoplasts(electroporation)
ProtoplastsuptakeofprecipitatedDNA(PEG,
polyamines,dextrans,calciumphosphate)
5. UptakeofDNAencapsulatedinliposomesintoprotoplasts
6. Microinjectionintosinglecells,protoplasts,pollen,zygotic
embryos,somaticandmicrosporeembryos
7. Virusvectors
8. IncubationofdryseedsandembryoswithDNA
9. Microlaser
10. Liposomeinjection..

Plant Transformation Methods

Physical
Microinjection
Pressure
Biolistics - gene gun/
particle bombardment
Electroporation
Microinjection
Silica/carbon fibers
Lazer mediated
SAT

Chemical
PEG
DEAE-dextran
Calcium phosphate
Artificial lipids
Proteins

Biological In planta
A. Tumefaciens
A. Rhizogenes
Virus

Two types of delivery systems

Naked DNA
Cell wall is the
primary resistance to
DNA uptake
Biolistics
SiC fibres
Protoplasts
Electroporation
Pollen

Vectored
Agrobacterium
Viruses

Microinjection of GOI

Naked DNA transfer

Electroporation
Protoplasts are cells
without a cell wall.
Produced by enzymic
degradation of the cell
wall.
DNA uptake enhanced
by electroporation or
treatments to change
plasmalemma charge
This electroporator is for low-current applications (Polyethylene Glycol).
such as those using small electrodes

PhysicalDNAdelivery
Biolistics / Gene Gun

Particle delivery system

Principle of Biolistics

Biolistics
It is Projectile method utilizing gold/ tungsten metal
particles to deliver genes to the cell.
Very small metal particles, much smaller than the cell
diameter, are coated in genetic material.
The coated particles are propelled into the cell using a
shot gun.
A metal plate placed before the cells stops the shell
cartridge, but allows the particles to pass through and
into the cells.
Once inside the cell, the genetic material is taken to the
nucleus and incorporated into the recipient's DNA

Particle Gun
DNA coated on pellets
is forced down the
barrel of a Particle Gun
by an explosive charge
The particles are forced
through the cell wall
where the DNA is
released

Petri Dish
with cultures

Explosive
Charge

Projectile
DNA coated
pellets
Barrel
Vent
Stop plate

10

The Gene Gun

DNA vector is coated onto gold
or tungsten particles
Particles are accelerated at high
speeds by the gun
Particles enter plant tissue DNA
enters the nucleus and
incorporates into chromosome
Integration with DNA

Sonication & SAAT

In Planta Transformation

Meristem transformation - Floral dip method

Pollen transformation

Chloroplast transformation - Horizontal gene transfer

Biological Transformation

Agrobacterium tumefaciens

&

Agrobacterium rhizogenes
The Gram-negative soil bacterium- as pathogen causes diseases in
plants called crown gall tumors & hairy root in many

dicotyledonous plants
Possible plant compounds, that initiate Agrobacterium to infect
plant cells - Acetosyringone, ferulic acid, gallic acid,
Hydroxybenzoic acid, pyrogallic acid, vanillin etc.
T-DNA, a part of the bacterial tumor-inducing (Ti) plasmid/ Root
inducing (Ri) plasmid, is transferred from the bacterium to the
plant cell with the aid of a number of Virulence (Vir) proteins
encoded by the megaplasmids of Ti-plasmid/ Ri-plasmid in
virulent strains.
In monocot not efficient - Transformation frequency very less

Crown Gall Plant tumor

Gall on
stem
Galls can be Huge

Gall on
leaf

Agrobacterium - A natural DNA delivery system

A plant pathogen found in nature - Infects many plant species Delivers its DNA and incorporates into plant chromosome and
expresses plant hormone genes and form galls at infection site.

Agrobacterium tumefaciens causes crown gall disease by first

transferring part of its DNA into an opening in the plant. The DNA
then integrates itself into the plant's genome and causes the
formation of the gall.

Natures Genetic Engineering

A model for the Agrobacteriummediated genetic transformation

Recognition and attachment

Vir genes expression by host signals
T-strand produce
T-complex export into host
Transport through cytoplasm and
nuclear
T-DNA uncoating and integration.

Cellular process of Agrobacteriumhost interaction

Transformation with Agrobacterium

Transferred DNA (T-DNA) modified by

Bacterial
Ti Plasmid chromosome

Removing phytohormone genes

Retaining essential transfer sequences
Adding cloning site for gene of interest

Used Right and Left borders of T-DNA (IS)

Agrobacterium contains a circle of DNA

(Ti plasmid) that carries the desired
genes
Co-cultivation of the Agrobacterium with
plant pieces transfers the DNA
Petri dish with leaf pieces
plus Agrobacterium

Delivering the Gene to the Plant

Transformation cassettes are developed in the lab
They are then introduced into a plant
Mostly two major delivery methods:
Agrobacterium
Gene Gun

Tissue culture
required to generate
transgenic plants

Production of transgenic plants

Isolate and clone gene of interest
Add DNA segments to initiate or
enhance gene expression
Add selectable markers
Introduce gene construct into plant
cells (transformation)
Select transformed cells or tissues
Regenerate whole plants

General transformation protocol

O/N A.r culture
Sterile explants
with dividing cells

Transformation

Wash

Inoculate (mins-hrs)
(bacterial attachment)

Co-cultivate (days)
Transfer of t-DNA

Recovery of transgenic plants

Transfer to
regeneration
medium plus
selective
antibiotics
Regeneration
of transgenic
plants

Transfer to medium
with bactericidal
antibiotics plus
selective antibiotics
(months)
Kill off Agrobacterium
and select transgenic
cells

Transfer to medium
with bactericidal
antibiotics (days)
Kill off Agrobacterium

Analysis of T0 plants
Morphology
Physiology
Yield characters
GUS expression
Gene expression
Confirmation with selectable marker &
Screen able marker

Gene expression
Resistance & Stress tolerance in plants:
Resistance:
- able to break-down or
- metabolize foreign molecules or
- introduction a new enzyme to metabolize
Tolerance:
- able to grow -foreign molecules
- either the target enzyme or
- altered form of enzyme

Agro biotech Revolution

THIRD WAVE
New crop of transgenic
plant technologies
-Amylase in Nicotiana

SECOND WAVE
Alteration in metabolic
pathway for quality
products

FIRST WAVE

Reducing post harvest

losses

Pest & Viral resistance

Development of value
added food products

Herbicide resistance

Improving oil quality

Abiotic stress resistance

Monoclonal Anti-bodies
in Soybean
Vaccines in Banana
Petro alternatives from
corn - Polylactide (PLA)