Gene transfer methods in...

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.