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Plant Growth Regulators III
habituated non-organogenic sugarbeet callus produces less ethylene and more polyamines than its hormone-dependent counterpart. There are also marked differences between habituated and nonhabituated lines in terms of general metabolism. Thus, both Poder et al., (1998) and Bisbis et al., (2000b) showed that cyanide-resistant respiration was much higher in habituated than in normal callus (both organogenic and non-organogenic). Likewise Hagege et al., (1992) demonstrated that levels of antioxidant enzymes (superoxide dismutase, glutathione reductase, monodehydroascorbate reductase, dehydroascorbate reductase and ascorbate reductase) were much higher in habituated sugarbeet
callus than in the normal form and has suggested (Hagege, 1996) that in general, habituated cells have very efficient oxidant scavenging systems. On the basis of changed metabolic patterns in habituated calluses including the high activity of antioxidant enzymes, accumulation of proline and polyamines reduction in tetrapyrroles and lipid peroxidation. Ledily et al., (1993) proposed that such systems were in effect under permanent stress and they drew comparisons with animal cancer cells and vitrified tissue and later suggested (Gaspar et al., 1998) that the changes serve to lock the cells in their habituated state.
18. THE TIMING AND DURATION OF GROWTH REGULATOR TREATMENTS 18.1. GROWTH REGULATOR EXPOSURE
It is common practice to vary the growth regulators to which tissues are exposed in vitro; for instance, callus is often initiated on a medium with one set of growth regulators and then moved to a second medium with different regulants for the induction of morphogenesis. Both the nature and the duration of the first and second treatments can be important and, because it is inconvenient to move explants frequently between one medium and another, recommended protocols are often a compromise between the most effective timings and durations of growth regulator exposure, and what is practical. Experimentation is complicated by the difficulty of determining growth regulator carry-over from one stage to another (for example, exogenouslyapplied auxins can become conjugated to other molecules from which they may be released when auxin is withdrawn from the medium), and the difficulty in determining the level of endogenous growth substances, (particularly cytokinins) which may be complementing, or antagonising exogenous treatments (Phillips, 1987). The effect of the nature of the growth regulators in an induction medium, and the combined effects of concentration and timing, are well illustrated by some of the results of Behki and Lesley (1980). Tomato leaf disc explants were cultured for varying lengths of time with growth regulators, which induced callus formation. Small pieces of the callus (or the explant itself when transfer was effected after 1 day) were then moved to a medium containing other regulants known to be capable of inducing shoot formation. The results are illustrated in Fig. 7.6.
Whether there was continued callus growth, or the formation of adventitious shoots or roots, depended on the nature of the induction medium and the time at which transfer took place. Auxin is necessary early in the culture period to induce cell proliferation (Phillips, 1987). C
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18.2. PULSE TREATMENTS
When cultured material is exposed to a substance for only a short period of time, it is frequently said to have been given a pulse of that substance or regulant. Sometimes a pulse of a growth regulator at a relatively high rate can be as effective as a lower concentration, which is present continuously (Thomas and Tran-Van, 1982). Cotyledonary node explants of Calocedrus decurrens, for example, produced axillary shoots if exposed to 7-10 μM BA in the proliferation medium, or to 125 μM BA for 3 h (after which they were moved to a regulant-free medium) (Jelaska, 1987). Adventitious buds of Picea alba developed faster after a pulse of 250 μM BA for 2 h than if placed continually on a medium containing 5 μM BA (Von Arnold and Eriksson, 1985). By supplying pulses of a growth regulator for progressively longer periods, it is possible to monitor the gaining of competence and the onset of determination in tissues. These processes can be further studied by the experimental application of metabolic inhibitors at specific times. Explants require to be exposed to a growth regulator for a minimum length of time for a given morphogenic event to be initiated. Hussey (1977) showed that morphogenesis in Freezia could be determined by the duration of