HAU16008-02-7 - Nutrient Media for Plant Tissue Cultures...

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Nutrient Media for Plant Tissue Cultures One of the first decisions that must be made when developing a tissue culture system is what medium to use. Nutrient media for plant tissue culture are designed to allow plant tissues to be maintained in a totally artificial environment. Many different tissue culture media have been developed, but only a few have found wide-spread use, e.g. MS (Murashige and Skoog, 1962). SH (Shenck and Hildebrandt), and Gamborg's B5. One of the most successful media, devised by Murashige and Skoog (Murashige and Skoog, 1962) was formulated by analyzing the inorganic components in tobacco plants and then adding them to medium in amounts similar to those found in the plants. Not only did they find that the ions themselves were important, but the forms in which the ions were supplied were critical as well. In addition to mineral elements, the macro- and micronutrients that are similar to what is found in fertilizers, nutrient media also contain organic compounds such as vitamins, plant growth regulators, and a carbon source. I. Mineral elements A. Macroelements consist of N, K, P, Ca, Mg , and S . 1. Nitrogen (N) - Nitrogen is essential for plant growth. Most inorganic nitrogen is converted to amino acids and then to proteins. Nitrogen is typically added to plant nutrient media as the nitrate ion (NO 3- , oxidized) and/or the ammonium ion (NH 4+ , reduced), which are added as inorganic salts. Inorganic nitrogen generally ranges from 25-60 mM in nutrient media. In devising media, both the total amount of nitrogen as well as the relative amounts of NO 3- and NH 4+ are important. There are usually lower levels of NH 4+ than NO 3- in medium; nitrate is usually added at concentrations between 25 and 20 mM and ammonium at concentrations between 2 and 20 mM. For example, the amount of NH 4+ in MS medium is less than half that of NO 3- and in other media the NH 4+ concentration is lower still. Cultures of some species can proliferate on medium containing nitrates alone, and some can grow on a medium with ammonium as the sole inorganic nitrogen source if one or more of the TCA cycle acids (citrate, succinate, malate) are included in the medium at concentrations of about 10 mM. In poorly buffered media, the use of both nitrogen forms helps maintain pH. Also, many plant species appear to respond best if they are given both forms, although the reason for this is not known. Nitrogen may also be added to medium in an organic form, as amino acids such as proline or glutamine, hydrolysates (such as casein hydrolysate), or, as above, as organic acids. Organic nitrogen is already reduced, i.e. in the form in which most nitrogen exists in the plant, and so may be taken up more readily than inorganic nitrogen. The organic forms are often added to media that do not contain ammonium. However, almost always, some inorganic nitrogen is present.
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