with observations on animal tissues, Weinstein et al., (1951) suggested that toxicity arose through competition between EDTA and enzymes (and other physiologically-active complexes) in the plant, for metals essential to their activity. This will occur if the avidity of the chelating agent is greater than the metal binding capacity of proteins on the surface of cells (Albert, 1958).
Toxicity can also occur in in vitro cultures. Legrand (1975) found that an optimum rate of adventitious shoot initiation occurred in endive leaf segments when only 7.5 mg/l EDTA (one fifth the concentration used in MS medium) was employed. In these circumstances, higher levels of EDTA were clearly inhibitory and more than 55 mg/l prevented shoot formation. Dalton et al., (1983) found that 0.3 mM EDTA (compared to the 0.1 mM in MS medium) reduced the growth rate of Ocimum cell suspensions.
Flower buds of Begonia franconis died within a few days if cultured with a high level of FeEDTA (11.5 mM, i.e. 10-15 times the normal level) together with 0.4-1.6 mM H2PO4-. Berghoef and Bruinsma (1979a) thought that Fe3+ released from the FeEDTA complex, had precipitated the phosphate. Necrosis was avoided by increasing H2PO4- concentration to 6.4 mM.
Tissues may be damaged by culture in media containing synthetic chelating agents where the pH approaches neutrality, because at these pH levels, EDTA and EGTA have been shown to remove calcium ions from the membranes of mitochondria and this inhibits NAD(P)H oxidation and respiration (Moller and Palmer, 1981). Chelating agents have been found to inhibit the action of the growth substance ethylene (see Chapter 7) and are thought to do so by sequestering Cu ions within plant tissues, thereby interfering with the synthesis or action of a Cu-containing enzyme responsible for ethylene metabolism. EDTA can also inhibit the activity of plant polyphenol oxidase enzymes in vitro (Weinstein et al., 1951) and Smith (1968) thought that this might occur because EDTA made Cu ions less available for enzyme incorporation, when he found the chelating agent was able to prevent the blackening of freshly-isolated Carex flacca shoot tips. Several oxidative reactions are also biochemically catalysed by ions such as Cu2+, Co2+ and Zn2+, and where this is the case [e.g. the oxidation of glutathione - Martin (1979); catechol amine oxidation - Kiss and Gergely (1979)], chelating agents such as EDTA and CDTA are inhibitory.
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