Production of Transgenic Cell by the Help of Transformation Technique

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BACKGROUND

Transgenesis is the process of introducing transgene into living organism, as a result of which the organism will exhibit a new thing and transmit that thing to its offspring.¹A transgene is a genetic material that is transferred naturally or by a no.of genetic engineering methods from an organism to another one. The transgene holds the potential to change the phenotype of an organism. Transgenic plants are a type of genetically modified plants where foreign genes are inserted into desired plants.² The generation of transgenic plants is the method of transformation and thus the technique is known as Transformation Technique.

So the basic objective is Production of Transgenic Cell by the Help of Transformation Technique.

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REQUIREMENTS

Materials:       Plant material

Chemicals:     Buffer solution

        40% PEG (w/v)

    Mannitol

    Calcium nitrate

Instruments:  Incubator

        Pipette

        Glasswares

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PROCEDURE

Types of transformation methods

Physical methods

Electroporation

This method involves the use of high field strength electrical impulses to reversibly permeabilize the cell membranes for the uptake of DNA.

The delivery of DNA into intact plant cells and protoplasts is done.

Incubate the plant material in a buffer that contains the foreign DNA & then put through high voltage electrical impulses.

As a result pores formation takes place in the plasma membrane via which DNA enters and gets homogenized into the host cell genome.³

This method has been highly used for production of transgenic plants of various cereals; for example: rice, wheat, maize.

Particle bombardment (biolistics)

This method was initially named as Biolistics by its investor Sanford (1988). A combination of biological and ballistic is known as Biolistics. The other names of this technique are gene gun, particle gun and bio blaster.

The foreign genes containing the genes which are to be transferred are coated on the surface of small tungsten/gold particles & then bombarded into the target tissue or cells by using a particle gun.

Two types of plant tissue are basically used for this method that are the primary explants and the proliferating embryonic tissues.⁴ This method is used for the transformation of many cereals such as rice, wheat, maize.

Microinjection⁴

It is a method that involves manual insertion of the DNA into a target cell.

The target cell may be that identified from protoplasts, intact cells, embryos.

This technique involves transfer of gene into the nucleus of a plant cell or protoplast through a micropipette (0.5-10.0 pm tip).⁵

The recipient cells are kept in agarose hence by a pipette (holding pipette).

The transformed cell is then cultured and grown thus developing into a transgenic plant

Liposome mediated transformation:

Liposomes are the lipid vesicles that are artificially created. These lipid vesicles contain phospholipids membrane.

These are employed to fuse with protoplasts in order to transfer the genes.

This method involves mixing of liposomes to the protoplast surface, then fusion at the site of attachment and then release of plasmids inside the cell.

This method is successfully used in mammalian cells for the delivery of protein, drugs.

Silicon carbide fibre-mediated transformation

The silicon carbide fibres are 10-100 pm in length and 0.3-0.6 pm in diameter.

These fibres have the ability to penetrate the cell wall and plasma membrane and then can be delivere DNA into the cells.

These fibres are mixed with plant material.⁶

During the mixing, the DNA adhering to the fibres, enters the cells and gets stably mixed with the host genome.

Chemical Methods

Polyethylene glycol (peg)-mediated transfer

PEG weakens the plasma membrane of protoplasts and makes it absorbent to naked DNA. DNA break into the nucleus of the protoplasts & gets mixed with the genome.⁷ This method involves the separation of protoplasts & their suspension, then addition of plasmid DNA and then followed by a slow addition of 40% PEG (w/v) (dissolved in mannitol and calcium nitrate solution). After incubation of this mixture, protoplast gets transformed.

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CONCLUSION

The transgenic plant was produced by using different transformation methods.

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REFERENCES

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4. Gilbert N.,”Case studies: A hard look at GM crops”.2013: 497(7447): 24-26.
   
5. Kristin L., Mercer, Joel D., “Gene Flow from transgenic maize to landraces in Mexico”: Agriculture, Ecosystems & Environment. 2008: 123(1-3): 109-115.
   
6. Arias D.M., Rieseberg L.H., “Gene flow between cultivated and wild sunflowers”. Theoretical and Applied Genetics, 1994:89(6): 655-60.
   
7. Burkhardt P.K., “Transgene Rice (oryza sativa) endosperm expressing daffodil( Narcissus Pseudonarcissus) Ohytoene Synthase accumulates phytoene, a key intermediate of provitamin A biosynthesis” Plant Journal: 1997: 1071-1078.