Cii o

1:1 chelate

Fig. 3.5 Copper chelated with amino acid, glycine.

Iron chelates. A key property of iron is its capacity to be oxidized easily from the ferrous [Fe(II)] to the ferric [Fe(III)] state, and for ferric compounds to be readily reduced back to the ferrous form. In plants, iron is primarily used in the chloroplasts, mitochondria and peroxisomes of plants for effecting oxidation/reduction (redox) reactions. The element is required for the formation of amino laevulinic acid and protoporphyrinogen (which are respectively early and late precursors of chlorophyll) and deficiency leads to marked leaf chlorosis. Iron is also a component of ferredoxin proteins, which function as electron carriers in photosynthesis.

Iron is therefore an essential micronutrient for plant tissue culture media and can be provided from either ferrous or ferric salts. In early experiments, ferrous sulphate or ferric citrate or tartrate were used in media as a source of the element. Citric and and tartaric acids can act as chelating agents for some divalent metals (Bobtelsky and Jordan, 1945), but are not very efficient at keeping iron in solution (Fig 3.6). If Fe2+ and Fe3+

ions escape from the chelating agent, they are liable to be precipitated as iron phosphate. The iron may then not be available to plant cells, unless the pH of the medium falls sufficiently to bring free ions back into solution. The problem of precipitation is more severe in aerated media and where the pH of the medium drifts towards alkalinity. Under these conditions Fe2+ (ferrous) ions are oxidized to Fe3+ (ferric) ions and unchelated ferric ions may then also be converted to insoluble Fe(OH)3. For plant hydroponic culture, the advantages of adding iron to nutrient solutions in the form of a chelate with EDTA was first recognised in the 1950's (Jacobson, 1951; Weinstein et al., 1951). Street et al., (1952) soon found that iron in this form was less toxic and could be utilised by in vitro cultures of isolated tomato roots over a wider pH range than ferric citrate. Klein and Manos (1960) showed that callus cultures of several species grew more rapidly on White (1954) medium if Fe3+ ions from Fe2(SO4)3 were chelated with EDTA, rather than added to the medium from the pure compound, and Doerschug and Miller (1967), that 0.036 mM Fe from NaFeEDTA was as effective as 0.067 mM Fe as ferric citrate, in promoting shoot bud initiation on lettuce cotyledons. Iron presented as ferric sulphate (0.025 mM Fe) was much less effective than either chelated form.

Skoog and co-workers began to use EDTA in media for tobacco callus cultures in 1956 and discussed their findings in the same paper that describes MS medium (Murashige and Skoog, 1962). The addition of an iron (Fe)-EDTA chelate once again greatly improved the availability of the element. Following this publication, (Fe)-EDTA complexes were rapidly recognised to give generally improved growth of all types of plant cultures (Nitsch, 1969). EDTA has now become almost a standard medium component and is generally preferred to other alternative chelating agents (Table 3.8).

Preparation and use. (Fe)-EDTA chelates for tissue cultures are prepared in either of two ways.

• A ferric or ferrous salt is dissolved in water with EDTA and the solution is heated;

• A ready-prepared salt of iron salt of EDTA is dissolved and heated.

Heating can take place during the preparation of chelate stock solutions, or during the autoclaving of a medium.

The form of iron complexed is invariably Fe(III). If iron has been provided from ferrous salts, it is oxidised during heating in aerated solutions. The rate of oxidation of the ferrous ion is enhanced in some complexes and retarded in others (Albert, 1958). That of Fe2+-EDTA is extremely rapid (Kolthoff and Auerbach (1952). Only a small proportion of Fe2+ is likely to remain: its chelate with EDTA is much less stable than the Fe(III) complex. Iron is however thought to be absorbed into plants in the ferrous form. Uptake of iron from EDTA probably occurs when molecules of Fe(III)-chelate bind to the outer plasma membrane (the plasmalemma) of the cytoplasm, where Fe(III) is reduced to Fe(II) and freed from the chelate (Chaney et al., 1972; Romheld and Marschner, 1983).

In most recent plant tissue culture work, EDTA has been added to media at an equimolar concentration with iron, where it will theoretically form a chelate with all the iron in solution. However, it has been found in practice that the Fe(III)-EDTA chelate, although stable at pH 2-3, is liable to lose some of its bound iron in culture media at higher pH levels; the displaced iron may form insoluble ferric hydroxides and iron phosphate (Dalton et al., 1983). If this occurs, free EDTA will tend to form chelates with other metal ions in solution. Some micronutrients complexed with EDTA may then not be available to the plant tissues. Re-complexing may also happen if the EDTA to Fe ratio is increased by decreasing the amount of iron added to the medium (as has been proposed to solve the precipitation problem, see Chapter 4). It is not possible to add very much more than 0.1 mM EDTA to culture media because the chelating agent can become toxic to some plants (see below).

Hill-Cottingham and Lloyd-Jones (1961) showed that tomato plants absorbed iron from FeEDTA more rapidly than they absorbed EDTA itself, but concluded that both Fe and Fe-chelate were probably taken up. They postulated that EDTA liberated by the absorption of Fe, would chelate other metals in the nutrient solution in the order given at the beginning of Section 3.6.. Teasdale (1987) calculated that in many media, nearly all the copper and zinc, and some manganese ions might be secondarily chelated, but it is unclear whether micronutrients in this form are freely available to plant tissues. One presumes they are, for deficiency symptoms are not reported from in vitro cultures.

Ambiguous descriptions. In many early papers on plant tissue culture, the authors of scientific papers have failed to describe which form of EDTA was used in experiments, or have ascribed weights to EDTA, which should refer to its hydrated sodium salts. Singh and Krikorian (1980) drew attention to this lack of precision. They assumed that in papers where Na2EDTA is described as a medium constituent, it indicates the use of the anhydrous salt (which would give 11 mol/l excess of EDTA to iron, with unknown consequences). However, the disodium salt of EDTA is generally made as the dihydrate (Beilstein's Handbuch der Organischen Chemie) and this is the form which will almost invariably have been used, Na2EDTA merely being a shorthand way of indicating the hydrated salt without being intended as a precise chemical formula.

Further confusion has arisen through workers using ready-prepared iron-EDTA salts in media without specifying the weight or molar concentration of actual Fe used. Mono-, di-, tri-, and tetra-sodium salts of EDTA are possible, each with different (and sometimes alternative) hydrates, so that when a research report states only that a certain weight of 'FeEDTA' was used, it is impossible to calculate the concentration of iron that was employed with any certainty.

The compound 'monosodium ferric EDTA' with the formula NaFeEDTA (no water of hydration) exists, and is nowadays commonly selected as a source of chelated iron. However in some papers 'NaEDTA' has been used as an abbreviation for some other form of iron-EDTA salt. For example the paper of Eeuwens (1976) describing Y3 medium, says that to incorporate 0.05 mM iron, 32.5 mg/l 'sodium ferric EDTA' was used. The weight required using a compound with the strict molecular formula NaFeEDTA would be 18.35 mg/l. Hackett (1970) employed 'Na4FeEDTA'. Gamborg and Shyluk (1981) and Gamborg (1982) said that to prepare B5 or MS medium with 0.1 mM Fe, 43 mg/l of 'ferric EDTA' or 'Fe-versenate' (EDTA) should be weighed. The compound recommended in these papers was probably the Na2FeEDTA.2H2O chelate (theoretical mol. Wt. 428.2) as was the 'FeEDTA' (13% iron) employed by Davis et al., (1977). It should be noted that NaFeEDTA is the only source of Na in MS medium apart from the contamination in the gelling agents.

Alternatives to EDTA. A few other chelating agents have been used in culture media in place of EDTA. The B5 medium of Gamborg et al., (1968) was originally formulated with 28 mg/l of the iron chelate 'Sequestrene 330 Fe'. According to HeberleBors (1980), 'Sequestrene 330 Fe' is FeDTPA (Table 3.9), containing 10% iron (Anon, 1978). This means that the concentration of Fe in B5 medium was originally 0.05 mM. Gamborg and Shyluk (1981) have proposed more recently that the level of Fe should be increased to 0.1 mM. B5 medium is now often used with 0.1 mM FeEDTA, but some researchers still prefer FeDTPA, for example (Garton and Moses, 1986) used it in place of FeEDTA in Lloyd and McCown (1981) WPM medium for shoot culture of several woody plants.

Fig. 3.6 Chemical structures of some chelating agents and iron chelates.

Growth regulatory effects of chelating agents.

Although most iron, previously complexed to chelating agents such as EDTA, EDDHA and DTPA (Table 3.9) is absorbed as uncomplexed ions by plant roots, there is evidence that the chelating agents themselves can be taken up into plant tissues (Weinstein et al., 1951; Tiffin et al, 1960; Tiffin and Brown, 1961). Chelating compounds such as EDTA, in low concentrations, exert growth effects on plants, which are similar to those produced by auxins. The effects include elongation of oat coleoptiles (Heath and Clark, 1956a.b), and etiolated lupin hypocotyls (Weinstein et al, 1956), the promotion of leaf epinasty (Weinstein et al., 1956) and the inhibition of root growth (Burstrom, 1961, 1963). Hypotheses put forward to explain these observations have included that:

• chelating agents act as auxin synergists by sequestering Ca from the cell wall (Thimann and Takahashi, 1958);

• the biological properties of the natural auxin IAA may be related to an ability to chelate ions; other chelating agents therefore mimic its action (Heath and Clark, 1960).

Burstrom (1960) noted that EDTA inhibited root growth in darkness (not in light) but that the growth inhibition could be overcome by addition of Fe3+ or several other metal ions (Burstrom, 1961). He recognised that reversal of EDTA action by a metal does not mean that the metal is physiologically active but that it might only release another cation, which had previously been made unavailable to the tissue by chelation.

Effects in tissue culture. Growth and morphogenesis in tissue cultures have been noted on several occasions to be influenced by chelating agents other than EDTA. It has not always been clear whether the observed effects were caused by the chelation of metal ions, or by the chelating agent per se.

The growth rate of potato shoot tips was increased by 0.01-0.3 mg/l 8-hydroxyquinoline (8-HQ) when cultured on a medium which also contained EDTA (Goodwin, 1966), and more callus cultures of a haploid tobacco variety formed shoots in the absence of growth regulators when DHPTA was added to Kasperbauer and Reinert (1967) medium which normally contains 22.4 mg/l EDTA. The DHPTA appears to have been used in addition to the EDTA, not as a replacement, and was not effective on callus of a diploid tobacco (Kochhar et al., 1970). In the same experimental system, Fe-DHPTA and Fe-EDDHA were more effective in promoting shoot formation from the haploid-derived tissue than Fe with CDTA, citric acid or tartaric acid (Kochhar et al., 1971).

The inclusion of EDTA into a liquid nutrient medium caused the small aquatic plant Lemna perpusilla to flower only in short day conditions whereas normally the plants were day-neutral (Hillman, 1959, 1961). In the related species Wolffia microscopica, plants did not flower unless EDTA was present in the medium, and then did so in response to short days (Maheshwari and Chauhan, 1963). When, however, Maheshwari and Seth (1966) substituted Fe-EDDHA for EDTA and ferric citrate, they found that plants not only flowered more freely under short days, but also did so under long days. The physiological effect of EDTA and EDDHA as chelating agents was thus clearly different. This was again shown by Chopra and Rashid (1969) who found that the moss Anoectangium thomsonii did not form buds as other mosses do, when grown on a simple medium containing ferric citrate or Fe-EDTA, but did so when 5-20 mg/l Fe-EDDHA was added to the medium instead. An optimum concentration was between 5 and 8 mg/l. Rashid also discovered that haploid embryoids developed more freely from in vitro cultures of Atropa belladonna pollen microspores when Fe-EDDHA was incorporated into the medium, rather than Fe-EDTA (Rashid and Street, 1973). Heberle-Bors (1980) did not obtain the same result, and found that FeEDTA was superior to FeEDDHA for the production of pollen plants from anthers of this species and of two Nicotianas. In tobacco, the production of haploid plants was greatest with FeEDTA, next best with FeDTPA, FeEGTA, FeEDDHA, and poorest with Fe citrate. Each complex was tested at or about the same iron concentration. Heberle-Bors also showed that che-lating agents are differentially absorbed by activated charcoal (see Chapter 7). In tissue culture of rose (Van Der Salm, 1994), Prunus (Mallosiotis et al., 2003), citrus (Dimassi et al., 2003) and red raspberry (Zawadzka and Orlikowska, 2006), it is advantageous to use FeEDDHA rather than FeEDTA.

Toxicity caused by chelating agents. Although low concentrations of EDTA markedly stimulate the growth of whole plants in hydroponic cultures by making iron more readily available, the compound begins to be toxic at higher levels. By comparisons

Table 3.9 Some common chelating agents.

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