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Genetic transformation in plants

1. Introduction
Term genetic transformation refers the ability to move DNA into an organism and thereby alter its
genotype or genetic makeup. This technique play central role to both basic and applied molecular biology.
The application these conventional or classical techniques has produced significant achievements of major
food crops and also overproduction of valuable plant secondary metabolites
In nature, many strains of bacteria exchange genetic information during a process known as
conjugation, and the new genetic information is passed on to subsequent generations. The advantage of
using bacteria relates to the single-celled nature of the organisms. Only one cell needs to be changed in
order to integrate the new genetic information and allow for its transmission to the next generation. This
natural exchange of genetic information allows the organism an increased capacity to adapt to its
environment.
Transformation is usually more difficult with multicellular organisms, however, and can be
particularly challenging with some important plant species like cereals. The multicellular nature of most
plants introduces the complication of transforming each cell of the plant in order to fully integrate the new
information. Often the approach taken in the transformation of higher organisms, such as plants, is to
transform an individual plant cell and then regenerate it into a whole organism. This step calls upon the
pluripotent nature of plant cells. That is, a whole plant can grow from a single cell.

Genetic transformation of plants and other organisms occurs naturally. Bacteria carry this out
routinely. It has been observed that viruses are also able to move DNA (or RNA) into an organism and
cause significant changes in genetic organization in plant cells. Soil bacteria, such as Agrobacterium
tumefaciens (tumor inducing bacterial sp.) and Agrobacterium rhizogenes, (root inducing bacteria) are great
examples of natural transformation systems, causing crown gall disease and ‘hairy root syndrome’
respectively. Agrobacterium can transfer a section of its own DNA, known as the T-DNA or ‘transfer
DNA’ into plant cells. For A. tumefaciens this normally occurs at wound sites and the transferred DNA
causes the plant to develop crown gall disease. The story does not end there however. Along with causing a
gall, the Agrobacterium harnesses the plant’s machinery to produce unique sugars called opines that the
bacterium uses as a nutrient supply.
2. Common plant transformation techniques
Plant Genetic Transformation is carried out by Physical, Chemical & Biological agents. Out of
these some important plant Genetic Transformation techniques are listed below.

Agrobacterium mediated gene transfer - transfer of DNA from bacteria to plants.


Biolistics - rapidly propelled tungsten or gold micro projectiles coated with DNA are blasted into cells.
Electroporation - Electrical impulses are used to increase membrane and cell wall permeability to DNA
contained in the surrounding solution.
Microinjection - Injection of DNA directly into the cell nucleus using an ultrafine needle.
Poly-ethylene glycol mediated gene transfer- plant cell protoplasts treated with PEG are momentarily
permeable, allowing uptake of DNA from the surrounding solution.
2.1 Agrobacterium mediated gene transfer

The newest and most promising method technology for transformation of Bt corn is Agrobacterium
tumefaciens mediated transformation using a bacterial plasmid as a vector for the Bt gene. This method of
transformation is the most widely used to introduce foreign genes into plant cells. A. tumefaciens contains a
Ti plasmid (tumor-inducing) which normally infects dicotyledonous plant cells, making the bacteria an
excellent vector for the transfer of foreign DNA. By removing the tumor inducing genes and replacing
them with the genes of interest, efficient transformation can occu

Plant transformation mediated by the soil bacteria Agrobacterium tumefaciens has become the most used
method for plant transformation. A. tumefaciens naturally infects the wound sites in dicotyledonous plant
causing the formation of the crown gall tumors. Since the discovery of the bacterial origin of these
neoplastic diseases, a large number of researches have focused on the study of this process, firstly with the
hope to understand the mechanisms of oncogenesis in general and applied it to study of cancer disease in
animals and humans. Later this hypothesis was discarded and the interest on crown gall disease largely
decreased until it was evident that A. tumefaciens is capable to transfer a particular DNA segment (T-DNA)
of the tumor-inducing (Ti) plasmid into the nucleus of infected cells where it is subsequently stable
integrated into the host genome and transcribed, causing the crown gall disease. The T-DNA contains two
types of genes: the oncogenic genes, encoding for enzymes involved in the synthesis of auxins and
cytokines and responsible for tumor formation; and the genes encoding for the synthesis of opines, a
product resulted from condensation between amino acids and sugars, which are produced and excreted by
the crown gall cells and consume by A. tumefaciens as carbon and nitrogen sources. Outside the T-DNA,
are located the genes for the opine catabolism, the genes involved in the process of T-DNA transfer from
the bacterium to the plant cell and for the bacterium-bacterium plasmid conjugative transfer genes.

2.3 Biolistics
Some cells, tissues and intracellular organelles are impermeable to foreign DNA,
especially plant cells. biolistics, including particle bombardment, is a commonly used
method for genetic transformation of plants and other organisms. To resolve this problem
in gene transfer, the gene gun machine is used for the transfer of desired gene
The gene gun is part of the gene transfer method called the biolistic method. In this
method, DNA or RNA adheres to biological inert particles i.e. gold or tungsten. By this
method, DNA-particle complex is put on the top location of target tissue in a vacuum
condition and accelerated by powerful shot to the tissue, then DNA will be effectively
introduce into the target cells. Uncoated metal particles could also be shot through a
solution containing DNA surrounding the cell thus picking up the genetic material and
proceeding into the living cells.

Electroporation

Electroporation is the process where cells are mixed with a DNA construct and then briefly exposed to
pulses of high electrical voltage. The cell membrane of the host cell is penetrable thereby allowing foreign
DNA to enter the host cell. Some of these cells will incorporate the new DNA and express the desired gene.
While direct introduction of DNA into monocots by electroporation has proven successful, the
electroporation technique is used in a relatively small proportion of cases in comparison to other methods.

2.4 Microinjection

Microinjection is the direct mechanical introduction of DNA under microscopical control .A target can be a
defined cell within a multicellular structure such as embryo, ovule and meristematic cells or a defined
compartment of a cell. By examination with a microscope, a cell is held in place with gentle suction while
being manipulated with the use of a blunt capillary. A fine pipet is then used to insert the DNA into the
cytoplasm or nucleus. This technique is effective with plant protoplasts and tissues.
mong the several methods of plant transformation, four have yielded the best results:
Agrobacterium species-mediated transformation, microprojectile bombardment,
microinjection, and direct transformation. Each of these methods has merits and
limitations and is used in specific situations. At this time there is no single technique that
is suitable for all species.

Agrobacterium Mediated Transfer


Tumors and uncontrolled cellular growth in plants can occur due to genetic factors or
bacterial and viral infections. An example is crown gall in plants, where tumors are
caused by bacteria that causes uncontrolled growth on the stem of the infected plants.
This problem is caused by Agrobacterium tumefaciens, a soil bacterium that infects some
plants because of a wound on the plant. Plasmids present in the bacteria are responsible
for tumor growth after infection by A. tumefaciens. The bacteria are able to recognize
wounds on the plant, and this induces the transfer of the bacterial plasmid into the plant.
The plasmids are capable of integrating into the DNA of the host plant, causing
uncontrolled plant growth and the formation of tumors. The ability of A. tumefaciens to
efficiently transfer plasmid DNA into the host has made it important in early studies in
genetic transformation.

Agrobacterium tumefaciens was the first vector used for introduction of foreign DNA in
plant cells. Although Agrobacterium has only been used to infect dicot plant species,
such as soybean, tomato, pea, and cotton, the protocol has been modified to allow the
bacteria to infect some monocot (grass) species as well. Many research groups working
with plants have found this to be the preferred transformation approach. Another soil
bacteria, Agrobacterium rhizogenes, causes the growth of secondary roots after infection.
This bacterial species has also been used for plant transformation.

The basis of this transformation method is the bacterial plasmid, which contains the
genetic sequence that is integrated into the host genome. One of the most important parts
of a plasmid is the region responsible for the translocation of its DNA into the host plant
genome. This is called transfer DNA (T-DNA), and this area of DNA is key to the tumor
growth in infected plants. The region is located between the right border and left border
(RB and LB) of the plasmid. Plasmids also contain other important DNA sequences;
some of them control the production of auxin and cytokinin, two important plant
hormones involved in tumor formation. With the use of the restriction enzymes, a
transgene can be introduced between the right border and left border of the plasmid,
allowing the bacteria to transfer novel genes into the recipient plant.

One of the techniques used for transformation mediated by A. tumefaciens uses leaf
disks. Leaf disks of about 6 mm in diameter are cultured on a tissue-culture media
containing A. tumefaciens with plasmids containing the transgene. After approximately a
month of incubation in the tissue culture medium, seedlings start to develop on the leaf
disks. Through selection methods, transgenic seedlings are identified for whole
plantregeneration.

Microparticle Bombardment
This technique has also been called microprojectile acceleration or biolistics, but
microparticle bombardment is the formal name for the machine called a gene gun. This
method, developed at Cornell University, was designated biolistic (biologic + ballistics =
biolistic), because high-speed microscopic projectiles (microprojectiles) are accelerated
into the cells to be transformed.
This transformation method consists of the acceleration of a macroprojectile loaded with
millions of tungsten or gold microspheres about 1 µm in diameter (microparticle). The
microspheres are coated with the transgene, or DNA of the gene of interest. Microspheres
have a high specific mass, allowing them to acquire the needed momentum to penetrate
the target cells. The macroparticle is propelled in the direction of the cells at high speed,
but it is retained, after a small distance, on a steel mesh so that the microparticles
continue in the direction of the target cells. Helium gas at high pressure is used to propel
the macroparticle, and the acceleration chamber operates under a partial vacuum, which
allows for improved microsphere movement. Once inside the target cells, the DNA
coating the microspheres is released and can be integrated into the plant's genome.

Many of the commercial transgenic crop varieties on the market today were developed
using the gene gun. However, due to its cost and the complex integration patterns
resulting from this method, several research groups are reducing its use.

Microinjection
This method was developed for animal transformation but has also been extended to
plants. Although very difficult and laborious, DNA microinjection has yielded positive
results and has been used in several laboratories.

In this technique, microcapillary needles are used to introduce DNA directly into cells.
Each cell to be transformed must be manipulated individually. One of the advantages of
this method is that the optimum amount of DNA can be injected into the target cells,
which helps to ensure optimal integration. Positive results have already been obtained in
several crop species such as corn, wheat, soybean, tobacco, and rice, and in animals like
salmon, cattle, and swine.

Direct Transformation
Transformation using direct methods was accomplished soon after the first
Agrobacterium-mediated transformation. These methods use protoplasts (cells after the
removal of the cellular wall) as targets for transformation. This is a simple method that
consists of adding great amounts of transgenic plasmids to a protoplast culture, which
guarantees that a small proportion of the protoplasts will be taken up (assimilated) by the
plasmids. The assimilation rate can be increased with the addition of polyethylene glycol
(PEG) or the use of an electric discharge (electroporation). No barrier to direct
transformation has been detected, indicating that this method can be used with virtually
any species. The problem with this method lies in the difficulty of regenerating a whole
plant starting from protoplasts. Therefore, it has not been used as widely as the other
methods.