Tissue Culture Techniques
The general tissue culture techniques are listed below-
Preparing the culture medium
Plant tissue culture requires preparation of a medium onto which the plant organs are to be cultured or grown. The medium should have the following in optimum quantities-
- Inorganic Salts -Various macro- and microsalts
- Iron source
- Vitamins- Pyridoxine, Thiamine etc.
- Amino acids- Glycine, Leusine etc.
- Growth regulators or plant hormones- Auxins, Cytokinins, synthetic compounds.
- Carbohydrate supply- Sucrose.
Since a bruised or cut organ is to callus and grow, its susceptibily to infections becomes very high. To avoid death of the growing organs due to infections, it is important to sterilize all the articles to be used-
- Non-living articles- Autoclaved under high temperature and pressure
- Plant material - Washed in detergent such as Teepol, alcohol, mercuric chloride and finally with distilled water.
To maintain aseptic conditions, the entire experimentation is conducted in a laminar hood. All the articles are put in laminar flow which blows bacteria-free air over the working surface. Inoculation ( introduction of the plant organ into culture tubes) is conducted over a flame and each dissection tool is sterilized with acohol before use. The culture tubes are always kept corked tightly with a cotton plug.
Incubation of cultures
Incubation refers to allowing the growth of the excised organ and this is carried out in culture rooms, where temperature, light and humidity are controlled. The factors may need to be altered time again depending upon the response of the culture to these factors.
Culturing and Sub-culturing
The first step is the culturing of seeds of interest under aseptic conditions. Once plantlets are formed from these, organs from this aseptic plant are used for further culturing. When these plantlets reach an optimum growth, a tissue of interest, either stem, leaf etc., is excised. It is now technically called the explant . The exlant is then cultured onto the culture medium and this is process is called sub-culturing. After a few days of incubation, an unorganized mass of cells, now called the callus, forms. Gradually, the callus forms a plantlet, a process called differentiation, which can later be grown in gardens. (For pictures on callus differentiation, please see my userpage