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Methods of introducing foreign genes in cell are: 1. Electroporation 2. Particle Bombardment Gun 3. Microinjection.
Method # 1. Electroporation:
In this method an electric field is used to transfer the foreign DNA into the fragile cells. High voltage electricity is applied to the protoplast suspension containing recombinant or naked plasmids.
The electrical impulse creates large pores in the cell membrane. Through these pores foreign DNA enters the protoplast. The transformed protoplast is now cultured. This method is used to introduce DNA in rice, wheat, sorghum, petunia, maize and tobacco.
Method # 2. Particle Bombardment Gun:
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Prof. Stanford developed this in 1987. As the name indicates, it shoots foreign DNA into plant cells or tissues, at a very high speed (Fig. 5). Other names given to this technique are bombardment, biolistic process, micro projectile bombardment, and particle bombardment or particle acceleration. This method is used in cells, which do not show sufficient response to transfer gene through Agrobacterium. This is successfully used in rice, sorghum, chickpea, and pigeon-pea.
The apparatus consists of a chamber in which vacuum is created. The top a cylinder is temporarily sealed off from the rest of the chamber. This is done with the help of a rupture disc. Helium gas is passed into the cylinder. Close to the rupture disc is placed a plastic micro carrier, which carries DNA coated tungsten particles. These are microscopic pellets. The target tissue is placed in the apparatus.
Between the target cell and micro carrier, there is a stopping screen. Helium gas passes in the cylinder at a high voltage. Plastic disc gets ruptured when pressure of cylinder exceeds the bursting point. The helium gas propels plastic micro carrier containing the DNA coated micro pellet. The micro pellet passes through the stop screen and enters the target cells. Transformed cells are cultured on the culturing medium. These are then selected on the basis of gene expression.
Method # 3. Microinjection:
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This technique is used to deliver foreign DNA into a living cell, e.g. oocyte through a glass micropipette. One end of the glass pipette is heated till it becomes somewhat liquefied. This end is quickly stretched to form a very fine tip, with a diameter of 0.5mm, same as the injection needle.
Cells to be microinjected are placed in a container under a microscope. The holding pipette holds the target cell at the tip when gently sucked. The tip of micropipette is passed through the cell membrane. Contents of the needle are placed in the cytoplasm and the empty needle is pulled out.
