PLANT BIOTECHNOLOGY

PLANT TRANSFORMATION
METHODS
PROF. V. PALANICHAMY

BY:
INSIYA TANKIWALA
13BBT0201

Direct Methods:
1. Particle Bombardment
A gene gun or a biolistic particle delivery system, originally designed
for plant transformation, is a device for injecting cells with genetic
information; the inserted genetic material are termed transgenes. The
payload is an elemental particle of a heavy metal coated with
plasmid DNA. This technique is often simply referred to
as bioballistics or biolistics.
This device is able to transform almost any type of cell, including
plants, and is not limited to genetic material of the nucleus: it can also
transform organelles, including plastids.

A gene gun is used for injecting cells with genetic information, it is also
known as biolistic particle delivery system. Gene guns can be used
effectively on most cells but are mainly used on plant cells.
The following are the steps to use a Gene Gun:
Step 1: The gene gun apparatus is ready to fire.
Step 2: When the gun is turned on and the helium flows through.
Step 3: The helium moving the disk with DNA coated particles toward the
screen.
Step 4: The helium having pushed the particles moving through the screen
and moving to the target cells to transform the cells.
The target of a gene gun is often a callus of undifferentiated plant cells
growing on gel medium in a Petri dish. After the gold particles have impacted

the dish, the gel and callus are largely disrupted. However, some cells were
not obliterated in the impact, and have successfully enveloped a DNA coated
gold particle, whose DNA eventually migrates to and integrates into a
plant chromosome.
Cells from the entire Petri dish can be re-collected and selected for
successful integration and expression of new DNA using modern biochemical
techniques, such as a using a tandem selectable gene and northern blots.
Selected single cells from the callus can be treated with a series of plant
hormones, such as auxins and gibberellins, and each may divide and
differentiate into the organized, specialized, tissue cells of an entire plant.
This capability of total re-generation is called totipotency. The new plant that
originated from a successfully shot cell may have new genetic (heritable)
traits.
2. Microinjection
Here the DNA is directly injected into plant protoplasts or cells
(specifically into the nucleus or cytoplasm) using fine tipped (0.5 - 1.0
micrometer diameter) glass needle or micropipette. This method of
gene transfer is used to introduce DNA into large cells such as oocytes,
eggs, and the cells of early embryo.

3. Electroporation
Electroporation, or electropermeabilization, is a microbiology technique
in which an electrical field is applied to cells in order to increase the
permeability of the cell membrane, allowing chemicals, drugs,

or DNA to be introduced into the cell. In microbiology, the process of

electroporation is often used to transform bacteria, yeast,
or plant protoplasts by introducing new coding DNA. If bacteria
and plasmids are mixed together, the plasmids can be transferred into
the bacteria after electroporation. Several hundred volts across a
distance of several millimeters are typically used in this process.
Afterwards, the cells have to be handled carefully until they have had a
chance to divide, producing new cells that contain reproduced
plasmids. This process is approximately ten times more effective than
chemical transformation.

High concentration of plasmid DNA containing the gene of interest is added

to a suspension of protoplast and the mixture is given a shock with an
electric field of 200-600 V/cm. The protoplasts are then grown in tissue
culture for a period of one or two weeks. The selection pressure is then
applied to select the transformed one. Both maize and rice protoplast have
been successfully transformed with efficiencies of between 0.1 and
1%.Introduction and expression of transgenes in plant protoplasts were also

reported. Moreover transient expression of fluorescent fusion proteins in

protoplasts of suspension cultured cells were also explained

4. Silicon Carbide Fibres

Silicon carbide mediated method is also one of the transformation method

used to transform plants. This method is least complicated. In this technique
fibres are used which are single crystals of silica organic minerals like silicon
carbide which possess an elongated shape, having a diameter of 0.6 mm and
a length of 10 80 mm. Moreover, it also exhibits a high resistance to
expandability. In this method silicon carbide fibers are added to

a suspension containing plasmid DNA and plant tissue (immature embryos,

callus, cell cluster). It is then mixed in commercial shakers or in vortex. Fibres
coated with DNA penetrate the plant cell wall in the presence of small holes
produced at the time of collision between fibres and plant cells. The factors
on which the efficiency of transformation depends are the plant material,
fiber size, parameters of vortexing, shape of the vessels used, and the
characteristics of the plant cells, especially the thickness of the cell wall. This
process is easy and quick. It is not so expensive and useful for various plant
materials. The main drawback of this technique is low transformation
efficiency, damage to cells negatively influencing their further regeneration
capability, and the need of following extraordinarily rigorous precaution
protocols during laboratory work, as breathing the fibers in, especially
asbestos ones, can lead to serious sicknesses. Silicon carbide whiskermediated embryogenic callus transformation of cotton (Gossypium hirsutum
L.) and regeneration of salt tolerant plants were also reported.
5. Polethylene Glycol/ Protoplast Fusion
This technology is applicable for protoplast only. The chemical used is
polyethylene glycol. It stimulates endocytosis and thereby causing the
uptake of DNA. In this method protoplast are kept in polyethylene glycol
(PEG) solution. The concentration of PEG used is 15% having 8000 dalton
molecular weight. After exposure of protoplasts to exogenous DNA in
presence of PEG and other chemicals, PEG is removed and intact protoplast
are then cultured to form cells with walls and colonies inturn. Selection
pressure is then applied to get the transformants. The transfer of gene
across the protoplast membrane can be initiated by a number of chemicals
of which polyethylene glycol is the most important. It has become the most
widely used due to the availability of simple transformation protocol. Method
was developed using calcium alginate micro beads to immobilize DNA
molecules in combination with polyethylene glycol treatment also.

6. Liposome Mediated Gene Transfer

Liposomes are circular lipid molecules with an aqueous interior that can carry
nucleic acids. It encapsulates the DNA fragments and then adheres to the
cell membranes and fuse with them to transfer DNA fragments. Thus, the
DNA enters the cell and then to the nucleus. It is a very efficient technique
used to transfer genes in bacterial, animal and plant cells. Various reports on
the integration of genes introduced by means of liposomes followed by
transgenic plant regeneration for tobacco and wheat have been published so
far.

Indirect Methods:
1. Agrobacterium mediated
Among the various vectors used in plant transformation, the Ti plasmid
of Agrobacterium tumefaciens has been widely used. This bacteria is
known as natural genetic engineer of plants because these bacteria
have natural ability to transfer T-DNA of their plasmids into plant
genome upon infection of cells at the wound site and cause an
unorganized growth of a cell mass known as crown gall. Ti plasmids are
used as gene vectors for delivering useful foreign genes into target
plant cells and tissues. The foreign gene is cloned in the T-DNA region
of Ti-plasmid in place of unwanted sequences.

To transform plants, leaf discs (in case of dicots) or embryogenic callus

(in case of monocots) are collected and infected
with Agrobacterium carrying recombinant disarmed Ti-plasmid vector.
The infected tissue is then cultured (co-cultivation) on shoot
regeneration medium for 2-3 days during which time the transfer of TDNA along with foreign genes takes place. After this, the transformed
tissues (leaf discs/calli) are transferred onto selection cum plant
regeneration medium supplemented with usually lethal concentration
of an antibiotic to selectively eliminate non-transformed tissues. After
3-5 weeks, the regenerated shoots (from leaf discs) are transferred to
root-inducing medium, and after another 3-4 weeks, complete plants
are transferred to soil following the hardening (acclimatization) of
regenerated plants. The molecular techniques like PCR and southern
hybridization are used to detect the presence of foreign genes in the
transgenic plants.