As a major cation, calcium helps to balance anions within the plant, but unlike potassium and magnesium, it is not readily mobile. Because of its capacity to link biological molecules together with coordinate bonds, the element is involved in the structure and physiological properties of cell membranes and the middle lamella of cell walls. The enzyme P-(1^3)-glucan synthase depends on calcium ions, and cellulose synthesis by cultured cells does not occur unless there are at least micro-molar quantities of Ca2+ in the medium. Many other plant enzymes are also calcium-dependent and calcium is a cofactor in the enzymes responsible for the hydrolysis of ATP.
Although calcium can be present in millimolar concentrations within the plant as a whole, calcium ions are pumped out of the cytoplasm of cells. Ca2+ is sequestered in the vacuole, complexes with calcium-binding proteins and may precipitate into calcium oxalate crystals to maintain the concentration at around only 0.1 mM. The active removal of Ca2+ is necessary to prevent the precipitation of phosphate (and the consequent disruption of phosphate-dependent metabolism) and interference with the function of Mg2+. The uniquely low intra-cellular concentration of Ca2+ allows plants to use calcium as a chemical 'second messenger' (Hepler and Wayne, 1985; Sanders et al., 1999). Regulatory mechanisms are initiated when Ca2+ binds with the protein calmodulin, which is thus enabled to modify enzyme activities. A temporary increase in Ca2+concentration to 1 or 10 mM does not significantly alter the ionic environment within the cell, but is yet sufficient to trigger fundamental cell processes such as polarized growth (for example that of embryos - Shelton et al., 1981), response to gravity and plant growth substances, cytoplasmic streaming, and mitosis (Ferguson and Drbak, 1988; Poovaiah, 1988). Physiological and developmental processes, which are initiated through the action of phytochrome are also dependent on the presence of Ca2+ (Shacklock et al., 1992). A short-term increase in cytosolic free Ca2+ has been observed for osmoadaptation (Taylor et al., 1996), phytoalexin synthesis (Knight et al., 1991), thermotolerance (Gong et al., 1998) and induction of free-radical scavengers (Price et al., 1994).
Large quantities of calcium can be deposited outside the protoplast, in cell vacuoles and in cell walls. Calcification strengthens plant cell walls and is thought to increase the resistance of a plant to infection. By forming insoluble salts with organic acids, calcium immobilises some potentially damaging by-products. The element gives protection against the effects of heavy metals and conveys some resistance to excessively saline conditions and low pH. 2+
The Ca2+ ion is involved in in vitro morphogenesis and is required for many of the responses induced by plant growth substances, particularly auxins and cytokinins. In the moss Funaria, cytokinin causes an increase in membrane-associated Ca2+ specifically in those areas which are undergoing differentiation to become a bud (Saunders and Hepler, 1981). Protocorm formation from callus of Dendrobium fibriatum was poor on Mitra et al., (1976) A medium when calcium was omitted (Mitra et al., 1976) and in Torenia stem segments, adventitious bud formation induced by cytokinin seems to be mediated, at least in part, by an increase in the level of Ca2+ within cells (Tanimoto and Harada, 1986). Exogenous Ca2+ enhanced the formation of meristemoids and the first phases of outgrowth into organs in tobacco pith explants (Capitani and Altamura, 2004). In carrot, somatic embryogenesis coincides with a rise of free cytosolic Ca2+ (Timmers et al., 1996) and applied Ca2+ increases the number of somatic embryos (Jansen et al., 1990).
Calcium deficiency in plants results in poor root growth and in the blackening and curling of the margins of apical leaves, often followed by a cessation of growth and death of the shoot tip. The latter symptoms are similar to aluminium toxicity
(Wyn Jones and Hunt, 1967). Tip necrosis has been especially observed in shoot cultures, sometimes associated with hyperhydricity. It often occurs after several subcultures have been accomplished (e.g. in Cercis canadensis - Yusnita et al., 1990). After death of the tip, shoots often produce lateral branches, and in extreme cases the tips of these will also die and branch again. The cause of tip necrosis has not always been determined [e.g. in Pistacia shoot cultures (Barghchi, 1986), where shoots showing symptoms may die after planting out (Martinelli, 1988)]. The occurrence of necrosis was reduced in Pistacia (Barghchi loc. cit.) and Prunus tenella (Alderson et al., 1987) by more frequent subculturing, but this is a costly and time-consuming practice. In Pictacia, calcium reduced necrosis (Barghchi and Alderson, 1996).
Tip necrosis was found in Psidium guajava shoot cultures after prolonged subculturing, if shoots were allowed to grow longer than 3 cm, and was common in rapidly growing cultures (Amin and Jaiswal, 1988); it occurred on Sequoiadendron giganteum shoots only when they were grown on relatively dilute media (Monteuuis et al., 1987). Necrosis of Rosa hybrida 'White Dream', was cured by adding 0.1 mg/l GA3 to the medium (Valle and Boxus, 1987).
Analysis of necrotic apices has shown them to be deficient in calcium (Debergh, 1988), and a shortage of this element has been associated with tip necrosis in Amelanchier, Betula, Populus, Sequoia, Ulmus, Cydonia and other woody plants, although the extent of damage is variable even between genotypes within a species (Sha et al., 1985; Singha et al., 1990). As calcium is not remobilised within plant tissues, actively growing shoots need a constant fresh supply of ions in the transpiration stream. An inadequate supply of calcium can result from limited uptake of the ion, and inadequate transport, the latter being caused by the absence of transpiration due to the high humidity in the culture vessel. A remedy can sometimes be obtained by reducing the culture temperature so that the rate of shoot growth matches calcium supply, using vessels which promote better gas exchange (thereby increasing the transpiration and xylem transport), or by increasing the concentration of calcium in the medium (McCown and Sellmar, 1987). The last two remedies can have drawbacks: the medium will dry out if there is too free gas exchange; adding extra calcium ions to the medium is not always effective (e.g. in cultures of Castanea sativa - Mullins, 1987); and can introduce undesirable anions. Chloride toxicity can result if too much calcium chloride is added to the medium (see below). To solve this difficulty, McCown et al., (Zeldin and McCown, 1986; Russell and McCown, 1988) added 6 mM calcium gluconate to Lloyd and McCown (1981) WPM medium to correct Ca2+ deficiency, without altering the concentrations of the customary anions. There is a limit to the concentration of calcium, which can be employed in tissue culture media because several of its salts have limited solubility.
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