Phosphorus is a vital element in plant biochemistry. It occurs in numerous macromolecules such as nucleic acids, phospholipids and co-enzymes. It functions in energy transfer via the pyrophophate bond in ATP. Phosphate groups attached to different sugars provide energy in respiration and photosynthesis and phosphate bound to proteins regulates their activity. Phosphorus is absorbed into plants in the form of the primary or secondary orthophosphate anions H2PO4- and HPO42- by an active process, which requires the expenditure of respiratory energy. Phosphate, in contrast to nitrate and sulphate, is not reduced in plants, but remains in the highly oxidized form. It is used in plants as the fully oxidized orthophosphate (PO43-) form.
In culture media the element is provided as soluble potassium mono- and di-hydrogen phosphates. The di- and mono-valent phosphate anions respectively provided by these chemicals are interconvertible in solution depending on pH. Monovalent H2PO4- predominates at pH values below 7, characteristic of most tissue culture media, and it is this ion, which is most readily absorbed into plants (Devlin, 1975). Conversion of H2PO4- into divalent HPO42- begins to occur as solutions become more alkaline. The divalent ion is said to be only sparingly available to plants but Hagen and Hopkins (1955) and Jacobsen et al., (1958) thought that its absorption could be significant, because even though the ion is normally at a relatively low concentration in nutrient solutions, its affinity with the site of absorption is greater than that of the mono-valent form. Trivalent PO43-, which appears in alkaline solutions, is not generally absorbed by plants.
In some early tissue culture media, all (e.g. Bouharmont, 1961), or part (e.g. Vacin and Went, 1949) of the phosphorus was supplied as sparingly-soluble phosphates. A slow rate of phosphorus availability seems to be possible from such compounds. The optimum rate of uptake of phosphate (HPO42-) into cultured Petunia cells occurred at pH 4 (Chin and Miller, 1982) but Zink and Veliky (1979) did not observe any decline in the absorption of phosphate by Ipomoea suspension cultures at pH 6.5, when HPO42- and H2PO4- were present in approximately equal concentrations. Plant tissue cultures secrete phosphatase enzymes into the medium (Ciarrocchi et al., 1981), which could release phosphate ions from organic phosphates.
In the cytoplasm, phosphate is maintained at a constant concentration of 5-10 mM, more or less independent of the external concentration. Phosphate in the vacuole fluctuates according to the external concentration but does not increase above 25 mM (Schachtman et al., 1998). When there is a high supply of phosphate and it is taken up at rates that exceed the demand, a number of processes act to prevent toxic phosphate concentrations, among others storage into inorganic compounds such as phytic acid. High concentrations of dissolved phosphate can depress growth, possibly because calcium and some microelements are precipitated from solution and/or their uptake reduced. In Arabidopsis thaliana, four different phophate transporter genes have been isolated (APT1-4). In vivo, the genes are predominantly expressed in the roots and their expression is constitutive or induced by phosphate starvation. Overexpression of APT1 gene in tobacco cell cultures increased the rate of phosphate uptake (Mitsukawa et al., 1997).
Although the concentration of phosphate introduced into plant culture media has been as high as 19.8 mM, the average level is 1.7 mM and most media contain about 1.3 mM. However many reports indicate that such typical levels may be too low for some purposes. When phosphate is depleted from MS medium, there is an increase in free amino acids in Catharanthus roseus cells, because protein synthesis has ceased and degradation of proteins is occurring (Ukaji and Ashihara, 1987). Phosphate (starting concentration 2.64 mM) and sucrose were the only nutrients completely depleted in Catharanthus roseus batch suspension cultures, and the period of growth could be prolonged by increasing the levels of both (MacCarthy et al., 1980). MS medium contains only 1.25 mM phosphate which may be insufficient for suspension cultures of some plants. The phosphate in MS medium is insufficient for Cardamine pratensis suspension cultures, all having been absorbed in 5 days: it is however adequate for Silene alba suspensions (Bister-Miel et al., 1985).
The phosphate in MS medium is also inadequate for static cultures of some plants, or where a large amount of tissue or organs are supported on a small amount of medium (for example where many separate shoots are explanted together in a static shoot culture). The concentration of the ion is then likely to be reduced almost to zero over several weeks (Barroso et al., 1985; Singha et al., 1987; Lumsden et al., 1990). Insufficient levels of phosphate were present from MS during culture of Hemerocallis, Iris and Delphinium (Leiffert et al., 1995). Although growth can continue for a short while after the medium is depleted of phosphate, for some purposes it has been found to be beneficial to increase the phosphate concentration of MS to 1.86 mM (Jones and Murashige, 1974), 2.48 mM (Murashige et al., 1972; Murashige, 1974; Jakobek et al., 1986), 3.1 mM (Miller and Murashige, 1976) or 3.71 mM (Thorpe and Murashige, 1968, 1970), for example, to induce adventitious shoot formation from callus, or to increase the rate of shoot multiplication in shoot cultures. It should be noted that there is in vivo a significant retranslocation of phosphate from older leaves to the growing shoot (Schachtman et al., 1998). Retranslocation also occurs in tissue culture. In Dahlia culture in liquid medium, phosphate is almost completely taken up after 2 weeks (Fig. 3.4a). In spite of this, the concentration in tissues formed after the exhaustion is 'normal' (Fig. 3.4b). The depletion of phosphate early during culture has also a major effect on the pH of tissue culture media in which added phosphate is the major buffering component. When phosphate levels are increased to obtain a more rapid rate of growth of a culture, it can be advisable to investigate the simultaneous enhancement of the level of myo-inositol in the medium
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