Inorganic Medium Components

Plant tissues and organs are grown in vitro on artificial media, which supply the nutrients necessary for growth. The success of plant tissue culture as a means of plant propagation is greatly influenced by the nature of the culture medium used. For healthy and vigorous growth, intact plants need to take up from the soil:

• relatively large amounts of some inorganic elements (the so-called major plant nutrients): ions of nitrogen (N), potassium (K), calcium (Ca), phosphorus (P), magnesium (Mg) and sulphur (S); and,

• small quantities of other elements (minor plant nutrients or trace elements): iron (Fe), nickel (Ni), chlorine (Cl), manganese (Mn), zinc (Zn), boron (B), copper (Cu), and molybdenum (Mo).

According to Epstein (1971), an element can be considered to be essential for plant growth if:

1. a plant fails to complete its life cycle without it;

2. its action is specific and cannot be replaced completely by any other element;

3. its effect on the organism is direct, not indirect on the environment;

4. it is a constituent of a molecule that is known to be essential.

The elements listed above are - together with carbon (C), oxygen (O) and hydrogen (H) - the 17 essential elements. Certain others, such as cobalt (Co), aluminium (Al), sodium (Na) and iodine (I), are essential or beneficial for some species but their widespread essentiality has still to be established.

The most commonly used medium is the formulation of Murashige and Skoog (1962). This medium was developed for optimal growth of tobacco callus and the development involved a large number of dose-response curves for the various essential minerals. Table 3.1 shows the composition of MS compared to the elementary composition of normal, well-growing plants. From this table, the relatively low levels of Ca, P and Mg in MS are evident. The most striking differences are the high levels of Cl and Mo and the low level of Cu. Each plant species has its own characteristic elementary composition which can be used to adapt the medium formulation. These media result often in a much improved growth (Rugini, 1984; El Badaoui et al., 1996; Pullman et al., 2003; Bouman and Tiekstra, 2005; Nas and Read, 2004; Gongalves et al., 2005). A major problem in changing the mineral composition of a medium is precipitation, which may often occur only after autoclaving because of the endothermic nature of the process.

Plant tissue culture media provide not only these inorganic nutrients, but usually a carbohydrate (sucrose is most common) to replace the carbon which the plant normally fixes from the atmosphere by photosynthesis. To improve growth, many media also include trace amounts of certain organic compounds, notably vitamins, and plant growth regulators.

In early media, 'undefined' components such as fruit juices, yeast extracts and protein hydrolysates, were frequently used in place of defined vitamins or amino acids, or even as further supplements. As it is important that a medium should be the same each time it is prepared, materials, which can vary in their composition are best avoided if at all possible, although improved results are sometimes obtained by their addition. Coconut milk, for instance, is still frequently used, and banana homogenate has been a popular addition to media for orchid culture.

Plant tissue culture media are therefore made up from solutions of the following components:

• macronutrients (always employed);

• micronutrients (nearly always employed but occasionally just one element, iron, has been used);

• sugar (nearly always added, but omitted for some specialised purposes);

• plant growth substances (nearly always added)

• vitamins (generally incorporated, although the actual number of compounds added, varies greatly);

• a solidifying agent (used when a semi-solid medium is required. Agar or a gellan gum are the most common choices).

• amino acids and other nitrogen supplements (usually omitted, but sometimes used with advantage);

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