What is plant tissue culture

It is a technique by which a plant cell, tissue or an organ can be cultured in vitro under sterile conditions on a nutrient medium. The scientific basis of plant tissue culture is totipotency which is the ability of a plant cell to produce a whole plant. ( On the other hand animal cells show pluripotency which is the ability of an animal cell to differentiate into any type of tissue eg. stem cells). The concept of totipotency was put forward by Gottlieb. Haberlandt in 1902. Hence he is called the father of plant tissue culture.  

Gottlieb Haberlandt




Totipotency was later demonstrated in carrot cells by F.C. Steward in 1954. He initiated callus (a mass of unorganized tissue) from carrot phloem tissue using 2,4-D. From this a suspension culture (liquid medium containing thousands of plant cells aerated on a shaker) was produced. This was later transferred to a medium without 2,4-D when each carrot cell was transformed into a somatic embryo and finally a carrot 

                            
 F.C. Steward

 

  

Plant tissue culture techniques

Explant

It is a plant cell tissue or an organ which is used to initiate a culture. Practically any part of the plant can be used as an explant. Explants range in size from 0.1mm to 2 cm.

Points to remember in selecting an explant

1.The explants should be taken from the best plant

2.Select explants from plants grown in greenhouses. They have lesser chances of contamination

3.The bigger the explants the better the response

4.The younger the explants the better the response

Surface sterilization of the explants

The outer surface of the explant is infested with microbes. They have to be removed before explants can be placed on the culture medium. This process is called surface sterilization. Explants like inner cabbage leaves do not need surface sterilization. One of the following chemicals can be used.

1.70% ethanol or isopropanol

2.1-10% sodium or calcium hypochlorite

3.0.1-1% Mercuric chloride

From my personal experience sodium hypochlorite works best in temperate climate ( it worked well in my experiments done in USA) but in a tropical climate like that in India 0.1% mercuric chloride is a better choice. 

Because of the presence of a waxy layer on the explant a surfactant ( a chemical which removes surface tension of water) is used in the surface sterilizing solution. A drop of liquid soap or Tween 20 can be used.

Procedure for surface sterilization

1.Wash explants in running water for 30 minutes

2.Treat with 0.1% mercuric chloride containing surfactant

3.Wash thrice in sterile distilled  water

4.Trim on sterile filter paper in sterile petridish

5. Inoculate on the medium

Steps 2-5 should be done in a laminar flow hood / clean air work station

If the explant turns brown during treatment dip in an antioxidant solution before trimming. An antioxidant solution is made by dissolving 100mg of ascorbic acid and 150 mg of citric acid in a litre of water. Sterilize before use

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