COMPONENTS OF A PLANT TISSUE CULTURE MEDIUM

The components of a plant tissue culture medium can be classified into

1.Macronutrients

2.Micronutrients

3.Vitamins

4.Sucrose

5.Growth regulators

6.Undefined components

7.Agar

1.Macronutrients These are nutrients required in large quantities. They include nitrogen, phosphorus, potassium, sulphur, calcium, magnesium, iron, carbon, hydrogen and oxygen

2.Micronutrients These are nutrients required in minute quantities.They are also called trace elements.They include Boron, Molybdenum, Manganese, Cobalt, Zinc, Copper and Iodine

3. Vitamins Mostly B complex vitamins such as thiamine, nicotinic acid, pyridoxine, pantothenic acid and inositol are used

4.Sucrose It is used as the principal energy source. It is used at a concentration of 3%.

5.Growth regulators Two classes of plant growth regulators are used viz. auxins and cytokinins

Auxins The natural auxin Indole acetic acid (IAA) is not used since it is photolabile. The primary role of auxins is in producing cell division and cell enlargement resulting in callus ( a mass of unorganized tissue) from explants. For this purpose  2,4 dichlorophenoxyacetic acid (2,4-D) is used in the medium. 2,4-D is also used in making a suspension culture ( liquid medium containing thousands of plant cells). The second role of auxins is in the promotion of rooting in Stage III of micropropagation.For this purpose either Indole butyric acid (IBA) or Naphthalene acetic acid (NAA) is used

Cytokinins They have two roles that are important in tissue culture. They stimulate cell division resulting in callus growth. They also promote proliferation of shoot buds.The natural cytokinins zeatin and Isopentenyl adenine are very expensive. Hence they are not usually used. The synthetic cytokinins usually used are Kinetin (6-furfuryl aminopurine) and Benyl aminopurine (BAP)

6.Undefined components Their composition varies from source to source They include

a) Coconut milk from tender coconuts used at a concentration of 10%

b) Casein hydrolysate ( a mixture of amino acids produced by the hydrolysis of casein, the milk protein)

c) Yeast extract ( a source of vitamins)

d) Activated charcoal which is used  to promote rooting since it adsorbs ethylene

7.Agar It is used to solidify the medium.It is a polysaccharide derived from red algae It is used at a  concentration of 0.8%

MEDIA FORMULATIONS

Through trial and error scientists have discovered various chemical combinations (formulas) for specific purposes. The best known standard formula is that developed by Murashige and Skoog in 1962. It is primarly designed for micropropagation of herbaceous plants. A formula developed by Brent McGown and Grey Lloyd is called the Woody Plant medium (WPM) meant for woody plants. 

Other commonly used media include Gamborgs (B-5) medium (primarly used for suspension cultures), Nitsch and Nitsch medium (used for anther culture), White's medium ( for root culture), Lin and Staba medium (for production of secondary metabolites) and Vacin and Went medium (for orchid seed culture.)

Tissue culture techniques continued

Inoculation of the explant

This is done in the inoculation room in the sterile atmosphere of a laminar flow hood. The sterile air gently streaming through the hood allows you to freely open sterile culture tubes/ bottles and do the inoculation. 

 

 If the hood has ultraviolet germicidal lamp it should be turned on at least 15 minutes before working in the hood. Turn it off while working in the hood to avoid exposure of eyes and skin to the harmful uv rays. Except for the filter the hood should be wiped down gently with a disinfectant such as 70% alcohol or isopropanol. The air flow should be turned on 15 minutes before using the hood. Wash your hands thoroughly with soap and water and wipe them with 70% ethanol or isopropanol. wear a lab coat if possible. 

Tools used for inoculation such as forceps and scalpels are first wrapped in aluminium  foil and sterilized in an autoclave  Later they are sterilized by flaming them periodically over a Bunsen burner or a spirit lamp. Alternately they can be sterilized by keeping them in a glass bead sterilizer in between use

                                                                  Glass bead sterilizer

Points to remember while using the laminar flow hood

1.Avoid any obstruction of the laminar air flow because it will change the air flow pattern and so cause contamination  Do not have items such as beakers or racks of test tubes between the HEPA (high efficiency particulate) filter and the exposed cultures

2.Work at arms length as far back in the hood as it is practical

3.Do not make wide sweeping arm movements over the work area since contaminants may drop from the arms or sleeves

The more careful you are the lesser the chance of contamination