Plant Tissue Culture: Nutrient Media
A plant that grows under normal conditions naturally has the capacity to make its own food. But, in plant tissue culture technique, the plants are grown in vitro and they have to be provided with the nutrients and physical conditions suitable for plant growth. The plant tissue culture nutrient medium used for differ plants differ in their composition. Each plant variety has a different nutrient requirement for its growth and propagation. There is no such plant tissue culture nutrient media that can be used the same for all plants and therefore it is necessary that the composition required for the desired growth be worked out individually. There are certain components which are inevitable in a culture medium and they have to be added in the right proportion to create a good growth media for the plants.
Essential Salts
The elements or mineral ions required for the growth of plants has been broadly classified into three categories: Macronutrients, micronutrients and Iron source.
Macronutrients
Elements which are required in huge amounts for the optimal growth of the plants are known as Macronutrients. Nitrogen, Phosphorus, Potassium, Calcium, Magnesium and Sulphur are the macronutrients used in plant tissue culture nutrient media.
Nitrate can by itself serve the requirement for nitrogen, but better results can be obtained in the presence of an ammonium source in the culture media. The nitrate requirement is between 25 – 20 mM and ammonium is 2 to 20 mM, depending upon the plant that is grown. Including a TCA cycle acid in the nutrient media enables the use of ammonium as the sole source of nitrogen.
Potassium is usually used in the plant tissue culture media in the nitrate or chloride form at 20-30mM. Phosphorus, Magnesium, Sulphur and Calcium is used at concentrations ranging from 1-3 mM.
Micronutrients
Micronutrients are elements like Manganese, cobalt, boron, iron, molybdenum, copper, iodine and zinc which are required in trace amounts for the growth and development of plants. Iron is one of the critical micronutrients as it plays a vital role in chlorophyll synthesis. It also carries out the oxidation/reduction reactions. The problem with providing iron in culture media is that it forms insoluble compounds and is found as precipitate. To clear this issue, chelating agents are used which bind with the unstable metal ions making them available to the plants. The most commonly used chelating agent is sodium or potassium EDTA.
Manganese and zinc plays their role in enzyme reactions and used in concentration of 5-30 mM. Boron is supplied as boric acid and is used up in lignin biosynthesis and phenolic acid metabolism. Copper is added in 0.1 mM concentration as cupric sulphate and is used in enzyme reactions. Iodine is not a mandatory ingredient in plant tissue culture nutrient media, yet is added to promote the growth of root and callus.
Carbon and Energy Source
Carbohydrate is the energy source for plants growth. Glucose and sucrose are the most effective while in some situations fructose may also be used. Sucrose is provided in a concentration of 2-3 percentage. It is essential that external carbohydrate be provided as only very few cell lines have the capability of making their own carbohydrate in culture medium.
Vitamins
Under normal conditions, plants synthesize the vitamins that are required for their growth. But, in vitro conditions usually plants are unable to synthesise the vitamins and adversely affect the plant growth. The most commonly supplied vitamins are thiamine (Vit B1), nicotinic acid, Vitamin B6 and myo-inositol. Thiamine forms a part of almost all plant culture mediums and are added in concentration of 0.1 to 10.0 mg/liter. Myo-inositol is actually a carbohydrate, but is added to most vitamin stock solutions for plant tissue culture nutrient media. It stimulates growth and is used at concentrations of 50-5000 mg/liter. There are many other vitamins which are used according to the need of the plant that is grown in vitro.
Other Organic Supplements
There are some organic substances like coconut milk, yeast extract, orange juice, protein hydrolase, tomato juice and malt extracts that have been used effectively to bring about tissue responses. Other than coconut milk and protein hydrolases, the others are only used under special conditions. Casein is usually used at 0.05-0.1% and coconut milk at 5-20%.
The addition of activated charcoal has also been found beneficial and helps in absorption of inhibitory compounds and growth regulators. Phytohormones get absorbed to AC and thus inhibit growth. Cell growth is promoted as AC binds to harmful phenols.
Gelling Agents
Agar due its specific properties is the most commonly used gelling agent in plant tissue culture nutrient media. Agar is used at 0.5 to 1.0% concentration. Agar has several advantages that make it the most favourable support system in in vitro culture:
è Agar simply mixed with water gels easily.
è This gel melts at 60 – 100 C and solidifies at 45 C. This makes it suitable for incubation temperature.
è Agar does not react with any of the culture media components.
è It is not broken down by plant enzymes.
Gelrite is another commonly used gelling agent which is a synthetic agent and gives a clear base making it easy to detect contamination. Other methods like cellophane paper, filter paper, polyurethane foam and fleece have also been used.
Plant Growth Regulators
Plant cell culture makes uses of five major growth regulators namely, auxins, cytokinins, giberellins, abscisic acid and ethylene. Auxins invoke cell division and growth and 2,4-Dichlorophenoxyacetic acid is the most commonly used auxin. Naturally occurring cytokinins
