Plant Tissue Culture ~ University Notes

Meaning of Plant Tissue Culture:

Plant tissue culture is the maintenance and growth of plant cells, tissues and organs on a suitable culture medium in vitro, e.g., in a test tube or any other suitable vessel. Plant tissue cultures are often classified according to the type of in vitro growth, such as callus and suspension cultures, or the explants used for culture initiation, e.g. embryo culture, another culture, etc.

An explants is the part of a plant that is excised from its original location and used for initiating a culture.

Surface sterilisation and sterilisation. It is essential that the explants, glassware, culture containers or vessels, media and the instruments used for plant tissue culture must be free from microbes. Hence the explants are treated with specific anti-microbial chemicals, and the process is called surface sterilisation.

Suitable sized plant material (explant) is sterilised as follows:

(i) Bring the explant to be sterilised in well sterilised laboratory and prepare pieces for sterilisation.

(ii) Clean the working area and hands with alcohol, put on mask and cap, and light the spirit lamp.

(iii) Keep 3 or 4 petridishes in a line, add disinfectant (e.g., mecuric chloride 0.01 to 0.1% aqueous solution, or 20% sodium hydrochloride) in first plate and autoclaved distilled water in subsequent plates.

(iv) Place plant pieces in first plate and immerse the material with the help of sterilised forceps for 5-10 minutes depending upon the disinfectant used.

(v) Transfer material from first to second petriplate, rinse gently and pass to third and fourth plates, one by one with thorough rinsing.

(vi) Finally drain the distilled water, and prepare suitable sized explants.

A quick dip in 70% ethanol (15-30 seconds), is always advantageous, before surface sterilisation with disinfectant.

The vessels, media and instruments are also suitably treated with steam, dry heat, alcohol or subjected to filtration to make them free from microbes.

Generally, autoclave is used to sterilise medium, glassware and tools for the purpose of plant tissue culture. Sterilisation of material is carried out by increasing moist heat (121°C) due to increased pressure inside the vessel (15-22 psi, i.e., pounds per square inch) for 15 minutes for routine sterilisation. Moist heat kills the microorganisms and makes the material free from microbes.

Surface strerilisation of explants and their transfer to culture media must be done under aseptic conditions.

Nutrient media could be prepared in a separate room where sufficient space is available for keeping and weighing chemicals, and putting glassware.

Vitamins and growth hormones are carefully weighed.

Stock solutions of chemicals are kept in refrigerator to avoid contamination.

The technique of plant tissue culture enables us to study the cells, tissue or organs by isolating them from the plant body and growing aseptically, in suitable containers, on an artificial nutrient medium, under controlled environmental conditions.

Thus (i) Nutrient medium (ii) Aseptic conditions and (iii) Aeration of the tissue are important aspects of the technique of in vitro culture.