gene transfer methods in...
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Gene transfer methods in plants
Gene transfer or uptake of DNA refers to the process that moves a specific piece of DNA into cell.
The directed desirable gene transfer from one organism to another and the subsequent stable integration & expression of foreign gene into the genome is referred as genetic transformation.
The transferred gene is known as transgene and the organism that develop after a successful gene transfer is known as transgenic.
Transgenic plant are the plant that carry the stably integrated foreign genes.
These plants may also be called transformed plants.
The transferred DNA may be expressed
for only short period of time following the
DNA transfer process and this is called
transient expression.
Stable transformation occur when DNA is
integrated into the plant nuclear genome
expression occurs in regenerated plant and
is inherited in subsequent generations.
STEPS IN TRANSFORMATION Identification of useful genes : Desirable genes located in
wild species, unrelated plant species, unrelated organism and animals.
Designing gene for insertion: The gene of interest is isolated from the donor source and cloned in the laboratory. The cloning is done generally using plasmid.
Insertion of gene into target plant: The cloned gene i.emultiple copies of the gene of interest are inserted into host plant or the recipient plant.
Identification of transgenic cells: Transformed cells are identified using selectable marker and are regenerated into whole plant in nutrient medium.
Regenerate plant compared with plant variety. It should look like parent variety except gene of interest.
GENE TRANSFER METHODS
1. Vector Mediated Gene Transfer
2. Vectorless or Direct Gene Transfer
Methods
VECTOR MEDIATED GENE TRANSFER
Plant gene vectors being exploited for transfer
of genes are plasmids of Agrobacterium ,
viruses & transposable elements.
Vectors: Small circular DNA molecule
occurring in bacteria, which can exchange
between different cells under natural condition.
Plasmids: Plasmids are the extrachromosomal
self replicating & double stranded, closed &
circular DNA molecule present in the bacterial
cell.
AGROBACTERIUM MEDIATED TRANSFORMATION
Agrobacterium , system historically first successful plant transformation system, breakthrough in 1983.
Breakthrough in gene manipulation in plants came by characterizing and exploiting plasmids carried by the bacterial plant pathogens Agrobacterium tumefaciens & Agrobacteriumrhizogenes .
Bacteria of the genus Agrobacterium – gene vectors for plant cells.
Agrobacterium – gram negative rods belong to bacterial family Rhizobiaceae
Agrobacterium – near soil level at junction of plant stem & root.
Agrobacterium tumefaciens : Induces crown gall disease
Agrobacterium rhizogenes : Induces hairy root disease
Agrobacterium radiobacter : An avirulent strain
Large Plasmids in theses agrobacteria are called Tumour inducing plasmids (Ti) and root inducing plasmid (Ri)
Diseases result from transfer and functional integration of particular set of Ti or Ri plasmid in plant chromosome.
On infection of the plant cell by A. tumefaciens
, a part of Ti plasmid called as the T-DNA
integrates into the host genome.
The result is uncontrolled growth of the plant
cells, which is directed by the genes of the “T-
DNA”
The disease thus caused is called ‘Crown gall’
Ti plasmid can be grouped into three types viz.,
octopine, nopaline and agropine
A recombinant Ti plasmid with target gene
inserted in the T-DNA can be integrated into
the plant DNA and made to be expressed.
Features of Ti plasmid which make them
attractive gene vector
Ti plasmid integrates into plant genome
and stably transmitted through division of
mitosis and meiosis.
Genes like nopaline synthase encoded by T-
DNA possess promoters that function in
plant cells.
Foreign gene/DNA inserted into ‘T-DNA’
region is integrated into plant genome.
Agrobacterium has broad host range hence
the gene of interest in the “T-DNA” can be
transferred to wide range of plants.
They possess site for insertion of foreign
gene which needs to be introduced.
They possess selectable marker i.e genes
which help in selecting the transformed
cells.
Transformation Technique Using Agrobacterium
Important requirement for Agrobacteriummediated gene transfer in plants:
Plant explants must produce acetosyringone /
Agrobacterium may be preinduced with synthetic acetosyringone .
Agrobacteria should have access to cell that are competent for transformation.
Transformation competent cells/tissue should be able to regenerate into whole plant.
1. EXPLANT (Decapitated
Seedlings/Cells/ Protoplast/Leaf Discs) +
ACETOSYRINGONE+ WOUNDING+
AGROBACTERIA
2. Cocultivation to allow infection
3.Transformed & non-transformed tissue
Antibiotics to kill bacteria (Carbencilin,
cefataxime etc.)
4. Transformed & non-transformed
tissue
5. Selective media to kill non-
transformed tissue
6. Transformed tissue/callus
7.Transformed shoots
8. Rooted Shoots
9. Adult Plant
Infection of wounded plant
Seedlings decapitated and freshly cut
surface wound is inoculated with overnight
culture of Agrobacterium
Tumour produced excised out and grown
as callus culture.
Transforming callus are picked off &
regenerated.
Cocultivation
Protoplast isolated during cell reformation
stage
Incubated for 24-40 hrs in a suspension of
Agrobacteria at about 100 bacteria per
protoplast.
Transformation occur during subsequent
few days of cocultivation and exposure to
selective agent.
Leaf disc method This procedure can be performed with any tissue
explant- provided good source for initiation of plant differentiation.
Newly emerged cotyledon – useful material
In disc/explant method- tissue segment excised & tissue allowed to incubate in Agrobacteriumsuspension for few hrs to 3 days & then cultured on a media for bacterial growth to take place.
Tissue/explant- media containing a bacteriostaticagent –eliminates bacteria.
Explants- media selection of transformed plant cells & which contains antibiotics.
ADVANTAGES
Natural means of transfer.
Agrobacterium capable of infecting intact plant cells, tissue and organs.
Agrobacterium is capable of transferring large fragments of DNA very efficiently without substantial rearrangements.
Integration of T-DNA is relatively precise process.
Stability of gene transferred is excellent.
DISADVANTAGES
Host range limitation.
Cells in a tissue that are able to regenerate are difficult to transform.
VIRUS MEDIATED GENE TRANSFER
Vectors based on virus desirable – high efficiency of gene transfer can be obtained by infection and amplification of transferred genes that occurs via viral genome replication.
Viral infection of cell result in addition of new genetic material which is expressed in the host.
Additional genetic material incorporated in the genome of plant virus might be replicated and expressed in the plant cell along with viral genome.
Replicating genomes of plant viruses are non-integrative vectors as compared to those vectors based on the T-DNA of Agrobacteriumtumaefaciens which are integrative gene vectors.
Non-integrative vectors as plant virus vector do not integrate into the host genome; rather they spread systematically within a plant and accumulate to high copy numbers in their respective target cells.
Vectors for transferring genes into plant are based on DNA/RNA molecule that naturally express their genetic information in plant cells.
VECTORLESS or DIRECT GENE
TRANSFER
1. Physical Gene Transfer
2. Chemical Gene Transfer
3. DNA imbibitions by cell, tissue &
organ
PHYSICAL GENE TRANSFER
Direct delivery of naked DNA to
the plant cell.
DNA mediated gene transfer
ELECTROPORATION
Use of short electric impulse of high field strength.
Electric impulses increases the permeability of protoplast and allow entry of DNA molecule into the cells, if DNA is in direct contact with the membrane.
If host cell has cell walls, enzymes are used to dissolve the walls, leaving only theses protoplast and the foreign DNA is introduced via electroporation.
Electroporation pulse generated by discharging a capacitor across the electrodes in a specially designed electroporator chamber.
Protoplast exposed to short electric pulse
which open the transient membrane
channels through which DNA can pass
target cells & then cultured in vitro on
appropriate media.
Protoplast in ionic solution containing the
vector DNA are suspended between
electrodes & due to high voltage, pores are
made on the walls of protoplast which
facilitate the entry of DNA.
PARTICLE BOMBARDMENT/BIOLISTIC/PARTICLE GUN METHOD
Biolistic is process of bombarding cells with microscopic projectile coated with DNA .
Shot at high velocity from particle gun into cells/tissue.
Promising for plant which can not be infected by Agrobacterium .
DNA delivery to plant cell made possible when heavy microparticle or microcarrier (tungsten/gold) coated with the DNA of interest are accelerated to very high initial velocity are made to bombard the living plant cell.
Microparticle penetrate the cell wall & get integrated into the plant genome.
High frequency of integration of multicopy insertion; no regeneration protocol necessary.
DNA coating is sophisticated technology & requires precise preparation of DNA coated gold/tungsten particle.
Gold- uniform size & shape, less toxic to cells.
Coating of micropellet with DNA by precipitation is important step.
1.25 to 18 mg microparticles are mixed with 0.5 to 70 µg of plasmid DNA in CaCl2 (0.25 –2.5 M) and spermidine (0.1 M) solution.
Mixture continuously vortexed to ensure uniform coating.
After DNA precipitation, micropellets palcedon macrocarrier membranes & allowed to dry and immediately used.
ADVANTAGES
It is clean and safe.
Transformation of organized tissue
Universal delivery system.
Transformation of recalcitrant species
Study of basic plant development process. DISADVANTAGES
In plant , gene transfer leads to non-homologous integration into chromosome and is characterized by multiple copies and some degree of rearrangement.
Emergence of chiameral plant.
Lack of control over the velocity of bombardment, which often lead to substantial damage to the target cell.
MICROINJECTION
Direct mechanical introduction of DNA under microscopical control in specific target.
Microinjection is able to penetrate intact cell wall.
Host range independent and does not require a protoplast regeneration system.
Cells/protoplast-glass micropipette of 0.5-10.0 µm diameter tips are used for transfer of macromolecule into the cytoplasm/nucleus of recipient cell/protoplast.
Recipient cells can be immobilized by using methods such as agarose embedding, agar embedding, poly-lysine treated glass surface & suction holding pipette.
MICROINJECTION
Once injection achieved, the injected cell
must be cultured properly to ensure its
continued growth and development.
Disadvantage- production of chimeric plant
with only a part of plant is transformed.
Process slow, expensive, requires highly
skilled and experienced personnel.
MACROINJECTION
Injection of DNA solution (5-10 µl) by
micropipettes into the developing floral
side shoot (tillers) of plant.
Within the floral tillers are achesporial cells
that give rise to pollen in the developing sac
by two meiotic cell division.
Such cells might also be able to take up
large molecules such as DNA.
LIPOSOME MEDIATED GENE TRANSFER
Liposome are small lipid bags, in which large number of plasmids are prepared artificially.
They can be induced to fuse with protoplast using devices like PEG, therefore have been used for gene transfer.
Liposome mediated transformation has been achieved by including positively charged agent such as cations in the mixture or using the cationic liposome preparation.
Advantage: Protection of DNA/RNA from nuclease digestion.
Low cell toxicity.
Stability and storage of nucleic acid due to encapsulation in liposome.
High degree of reproducibility.
Applicable to wider range of cell type
SILICON CARBIDE MEDIATED GENE TRANSFER
Silicon Carbide Fibers (SCF) average 0.6 µm in diameter and 10-80 µm in length.
These fibers have capacity deliver DNA into plant cells.
Methods involve vortexing mixture of plasmid DNA encoding a selectable/ screenable marker gene, SCF & the explants tissue in eppendorf tube.
SCF has great intrinsic hardness with sharp cutting edges.
DNA delivery in this system is presumably due to cell wall penetration by DNA coated SCF During vortexing of SCF with explant, DNA adhering to fibers might enter the cells.
Silicon Carbide Fibers (SCF) function as numerous needle facilitating DNA delivery into the cells.
During mixing process, DNA penetrates the cell to become stably integrated into the nuclear genome.
Advantage: Ability to transform walled cells thus avoiding protoplast isolation, relative ease of the procedure & very low equipment cost.
Disadvantage: SCF has some carcinogenic properties.
ULTRASOUND MEDIATED GENE TRANSFER
Ultrasound-stimulating uptake of foreign DNA
by plant protoplast & leaf segment.
Procedure involve immersion of explant
(leaves/protoplast) in sonication buffer
containing plasmid DNA & sonication with an
ultrasonic pulse generator at an acoustic
intensity of 0.5W/cm 2 for 30 min.
Samples rinsed in buffer solution and then
cultured for growth and differentiation.
Advantage: Technique simple, inexpensive &
multifunctional.
DNA TRANSFER via POLLEN
Pollen has been suggested as vector for
gene transfer by various workers.
It has been reported that introduction of
DNA into gamete followed by fertilization
& zygotic embryogenesis will result in gene
transfer.
This kind of approach would be simpler,
faster & cheaper than the in vitro methods.
Main problem presence of cell wall &
action of nucleases on the DNA.
CHEMICAL GENE TRANSFER METHODS
Involves plasma membrane destabilizing
&/or precipitating agents.
Protoplast are mainly incubated with DNA
in buffer containing PEG, Poly L-ornithine,
polyvinyl alcohol or divalent ions.
PEG MEDIATED GENE TRANSFER
First conclusive demonstration of uptake & integration of isolated Ti plasmid DNA into plant protoplast was reported in Petunia & tobacco in the presence of PEG/Poly L-ornithine .
Protoplast are isolated- particular concentration of protoplast suspension taken in tube- followed by addition of plasmid DNA.
To this 40% PEG 4000 (w/v) dissolved in mannitol & calcium nitrate solution added slowly because of high viscosity & mixture incubated for few minutes.
Advantage: form of the DNA applied to
the protoplasts is controlled entirely by
the experimenter and not by an
intermediate biological vector.
Disadvantage: system requires protoplast
& a functional system for regeneration of
these protoplast to calluses & whole
plant.
CALCIUM PHOSPHATE COPRECIPITATION
DNA mixed with CaCl 2 solution & isotonic
phosphate buffer to form a DNA CaPO 4
precipitate.
Precipitate is allowed to react with actively
dividing cells to several hours, washed and then
incubated in fresh culture medium.
Physiological shock with DMSO can increase
the transformation efficiency to certain extent.
Relative success depends on high DNA
concentration and its apparent protection in the
precipitate.
POLY CATION DMSO TECHNIQUE
Involves the use of polycation, polybrene to
increase the absorption of DNA to the
surface followed by a brief treatment with
25-35% DMSO to increase the membrane
permeability & enhance the uptake.
Advantage of polybrene is that it is less toxic
than other polycation & high transformation
efficiency require very small quantities of
plasmid DNA to be used
DEAE DEXTRAN METHOD
Transformation of cell with DNA
complexed to high molecular weight
polymer diethyl amino ethyl (DEAE)
dextran is used to obtain efficient transient
expression.
Efficiency increases to 80% when DMSO
shock is given.
This technique does not produce stable
transformant
DNA IMBIBITION BY CELLS , TISSUES,
EMBRYO & SEEDS
Incubation of cells, tissues & organ with
DNA for transformation has met with little
or no success and hence has not resulted in
any proven case of integrative
transformation.
Advantages of Transgenic Plants
Improvement in Yield: Gene technology plays important role in increasing the productivity of food, fiber and vegetable crop ensuring food security.
Transgenes generally are not yield enhancing genes. The increase in yield or productivity is achieved by controlling losses caused by various insects, diseases and abiotic stresses.
Herbicide resistance -Gene technology has been used to develop herbicide resistant cultivars in cotton, maize , wheat, tobacco, potato etc.
In theses crops, cultivars resistant to glyphosate, gluphosinate and some other herbicide has been developed.
Improvement in Quality: Quality is
adjudged by three ways viz., nutritional
quality, market quality and industrial quality.
Gene technology has helped in improving
all these three types of quality in different
crops.
Insect resistance -Gene technology has
played a key role in developing insect
resistant cultivar in several crops.
Resistance to abiotic stresses: Gene
technology can also be used for developing
cultivars tolerant to environmental
stresses such as drought, sol salinity, acidity,
cold frost etc.
Industrial Products -Gene technology has
great potential for the production of
biodegradable plastics, obtaining
therapeutic proteins, pharmaceuticals and
edible vaccines from transgenic plants
Rapid and Accurate Technique: Gene technology rapid and highly accurate method of crop improvement.
Development of cultivar by this techniques takes 4-5 years against 10-12 yrs taken by conventional method.
No barrier for gene transfer
Gene technology permits transfer of genes between any two individual whether related or unrelated.
The gene of interest can be transferred from micro-organism to higher plants and even from animals to plants.
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