Question

Discuss electroporation and microballistics (gene Gun) as di rect DNA transfer methodology

Answer 1

Electroporation

Electroporation basically involves the use of high field strength electrical impulses to reversibly permeabilize the cell membranes for the uptake of DNA. This technique can be used for the delivery of DNA into intact plant cells and protoplasts.

The plant material is incubated in a buffer solution containing the desired foreign/target DNA, and subjected to high voltage electrical impulses. This results in the formation of pores in the plasma membrane through which DNA enters and gets integrated into the host cell genome.

In the early years, only protoplasts were used for gene transfer by electroporation. Now a days, intact cells, callus cultures and immature embryos can be used with suitable pre- and post-electroporation treatments. Electroporation has been successfully used for the production of transgenic plants of many cereals e.g. rice, wheat, maize.

Particle Bombardment (Biolistics):

Particle (or micro projectile) bombardment is the most effective method for gene transfer, and creation of transgenic plants. This method is versatile due to the fact that it can be successfully used for the DNA transfer in mammalian cells and microorganisms.

The micro projectile bombardment method was initially named as biolistics by its inventor Sanford (1988). Biolistics is a combination of biological and ballistics. There are other names for this technique- particle gun, gene gun, bio blaster.

Micro carriers (micro projectiles), the tungsten or gold particles coated with DNA, are carried by macro carriers (macro projectiles). These macro-carriers are inserted into the apparatus and pushed downward by rupturing the disc.

The stopping plate does not permit the movement of macro carrier while the micro carriers (with DNA) are propelled at a high speed into the plant material. Here the DNA segments are released which enter the plant cells and integrate with the genome.