The composition of medium for the tissue culture is the most important key factor in the successful culture of plant cells. The medium should be accurately defined of inorganic and organic chemical additives so as to provide i) the nutrients for the survival of the plant cells, tissues and organs under culture and ii) the optimal physical condition of pH, osmotic pressure, etc.
In the culture of plant cells formulating optimum type of medium favorable for in vitro culture was achieved many years ago. The Knop's (1865) mineral solution was the widely used medium by early investigators. Gautherat (1939) developed callus culture medium from Uspenski and Uspenskaia (1925) nutrient solution. A systematic study of mineral reqiurements of plant tissue and organs in culture was made by Murashige and Skoog (1962) followed by the scientists Linsmaier and Skoog (1965), Vasil and Hildebrant (1966) and Nitsch and Nitsch (1969) resulting in several media to suit particular needs.
A standard basal medium consists of a balanced mixture of macronutrients and micronutrients (usually salts of chlorides, nitrates, sulphates, phosphates and iodides of Ca, Mg, K, Na, Fe, Zn and B, a carbon source, vitamins, phytohormones and organic additives. Among the above mentioned nutrients some are essential and some are optional. The essential components include inorganic nutrients and organic nutrients like carbohydrates besides phytohormones and vitamins, organic additives like natural extract and liquid endosperm are optional.
Inorganic nutrients of a plant cell culture are those required by the normal plants. The optimum concentration of each nutrient for achieving maximum growth rates varies considerably. The major elements are N, P, K, S, Mg and Ca. Other nutrients such as Co, Fe, B, Zn, Mo, Cu, I are microelements.
Of all the mineral nutrients N plays a vital role in growth and differentiation of cultured tissues. The range of inorganic nitrogen varies from 25 mM to 60 mM according to the requirements. Nitrogen is generally supplied in the form of NH4 along with NO3.
Ammonium ion as nitrogen source is usually unsuitable, probably because under such conditions the pH of the medium has a tendency to fall below 5 during culture, resulting in reduced availability of nitrogen. Cells can be grown with NH4 as the sole N source when the medium is provided with organic acids such as malate, succinate, citrate or fumerate. Further, the concentration of NH4-N should not exceed 8 mM. Generally NO3-N can be used as a sole N source but often there is a beneficial effect if the media contains NH4 -N. Phosphorus
Phosphorus is usually supplied in the form of phosphates. It is the primary buffering constituent in tissue culture media. Phosphorus levels greater than 2mM are often inhibitory to growth of tissues.
The optimum concentration of K needed is 20 mM. At low nitrogen concentration presence of potassium enhances the formation of somatic embryos. The medium supplemented with potassium nitrate produces more embryos than the medium with ammonium nitrate. Sulphur
Sulphur is provided in the form of sulphates. Besides, the sulphur containing amino acids like L-cysteine, L-methionine and glutathione are satisfactory sources for sulphur. Calcium and Magnesium
The optimal concentration of Ca required is 3mM. An antagonism between Ca and Mg has been demonstrated and it was found that an increase in the concentration of one element increased the requirement for the other.
The microelements viz., Fe, Mn, B, Zn, Mo, Cu, I and Co have a profound effect on growth of tissue in vitro. The availability of the iron is reduced at high pH due to precipitation. To avoid this, Fe is supplied as chelated EDTA complex. These elements produce toxic effect, if they are applied at higher level. A good growth of tissue can be achieved when the concentration of...
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