Iodine is not recognised as an essential element for the nutrition of plants (Rains, 1976), although it may be necessary for the growth of some algae, and small amounts do accumulate in higher plants (ca. 12 and 3 mol/kg dry weight in terrestrial and aquatic plants respectively - Raven, 1986). However, the iodide ion has been added to many tissue culture media (e.g. to 65% of micronutrient formulations).
The practice of including iodide in plant culture media began with the report by White (1938) that it improved the growth of tomato roots cultured in vitro. Hannay (1956) obtained similar results and found that root growth declined in the absence of iodine which could be supplied not only from potassium iodide, but also from iodoacetate or methylene iodide, compounds which would only provide iodide ions very slowly in solution by hydrolysis. Street (1966) thought that these results indicated that iodine could be an essential nutrient element, but an alternative hypothesis is that any beneficial effect may result from the ability of iodide ions to act as a reducing agent (George et al., 1988). Oxidants convert iodide ions to free iodine. Eeuwens (1976) introduced potassium iodide into his Y3 medium at 0.05 mM (ten times the level used by Murashige and Skoog), as it prevented the browning of coconut palm tissue cultures. The presence of 0.06 ^M potassium iodide slightly improved the survival and growth of cultured Prunus meristems (Quoirin and Lepoivre, 1977).
Although Gautheret (1942) and White (1943) had recommended the addition of iodine to media for callus culture, Hildebrandt et al., (1946) obtained no statistically significant benefit from adding potassium iodide to tumour callus cultures of tobacco and sunflower. However, as the average weight of tobacco callus was 11% less without it, the compound was included (at different levels) in both of the media they devised. Once again iodine also had no appreciable effect on tobacco callus yield in the experiments of Murashige and Skoog (1962), but was nevertheless included in their final medium. Other workers have omitted iodine from MS medium (e.g. Roest and Bokelmann, 1975; Perinet et al., 1988; Gamborg, 1991) or from new media formulations without any apparent ill effects. However, Teasdale et al., (1986); Teasdale, (1987) reported a definite requirement of Pinus taeda suspensions for 25 mM
KI when they were grown on Litvay et al., (1981) LM medium.
There seems, at least in some plants, to be an interaction between iodine and light. Eriksson (1965) left KI out of his modification of MS medium, finding that it was toxic to Haplopappus gracilis cells cultured in darkness: shoot production in Vitis shoot cultures kept in blue light was reduced when iodine was present in the medium (Chee, 1986), but the growth of roots on rooted shoots was increased. Chee thought that these results supported the hypothesis that iodine enhanced the destruction and/or the lateral transport of IAA auxin. This seems to be inconsistent with the suggestion that I- acts mainly as a reducing agent.
Silicon (Si) is the second most abundant element on the surface of the earth. Si has been demonstrated to be beneficial for the growth of plants and to alleviate biotic and abiotic stress (Epstein, 1971). The silicate ion is not normally added to tissue culture media, although it is likely to be present in low concentrations. Deliberate addition to the medium might, however, improve the growth of some plants. Adatia and Besford (1986) found that cucumber plants depleted silicate from a hydroponic solution and in consequence their leaves were more rigid, had a higher fresh weight per unit area and a higher chlorophyll content than the controls. The resistance of the plants to powdery mildew was also much increased.
Chelating agents. Some organic compounds are capable of forming complexes with metal cations, in which the metal is held with fairly tight chemical bonds. The complexes formed may be linear or ring-shaped, in which case the complex is called a chelate (from the Greek word meaning a crab's claw). Metals can be bound (or sequestered) by a chelating agent and held in solution under conditions where free ions would react with anions to form insoluble compounds, and some complexes can be more chemically reactive than the metals themselves. For example, Cu2+ complexed with amino acids is more active biologically than the free ion (Cruickshank et al., 1987). Chelating agents vary in their sequestering capacity (or avidity) according to chemical structure and their degree of ionisation, which changes with the pH of the solution. Copper is chelated by amino acids at relatively high pH, but in conditions of greater acidity, it is more liable to be complexed with organic acid ligands (White et al., 1981). The higher the stability of a complex, the higher the avidity of the complexing agent. One, and in many cases, two or three molecules of a complexing agent may associate with one metal ion, depending on its valency.
Despite tight bonding, there is always an equilibrium between different chelate complexes and between ions in solution. Complexing agents also associate with some metal ions more readily than with others. In general Fe3+ (for agents able to complex with trivalent ions) complexes have a higher stability than those of Cu2+, then (in descending order), Ni2+, Al3+ (where possible), Zn2+, Co2+, Fe2+, Mn2+ and Ca2+ (Albert, 1958; Reilley and Schmid, 1958). For a chelated metal ion to be utilised by a plant there must be some mechanism whereby the complex can be broken. This could occur if it is absorbed directly and the ion displaced by another more avid binding agent, or if the complex is biochemically denatured. Metals in very stable complexes can be unavailable to plants; copper in EDTA chelates may be an example (Coombes et al., 1977). High concentrations of avid chelating agents are phytotoxic, probably because they competitively withdraw essential elements from enzymes.
Naturally-occurring compounds act as chelating agents. Within the plant very many constituents such as proteins, peptides, porphyrins, carboxylic acids and amino acids have this property (Albert, 1958; Martin, 1979): some of those with high avidity are metal-containing enzymes. Amino acids are able to complex with divalent metals (Fig. 3.5). Grasses are thought to secrete a chelating agent from their roots to assist the uptake of iron (Romheld and Marschner, 1986). There are also synthetic chelating agents with high avidities (stability constants) for divalent and trivalent ions. Some are listed in Table 3.9, and the structure of those most commonly used in plant culture media is illustrated in Fig 3.6. The application of synthetic chelating agents and chelated micronutrients to the roots of some plants growing in alkaline soils can improve growth by supplying essential metals such as iron and zinc which are otherwise unavailable. The addition of such compounds to tissue culture media can help to make macro- and micro-nutrients more accessible to plant cells.
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