The Components of Plant Tissue Culture Media II - Horticultural ...

128 The Components of Plant Tissue Culture Media II
the subject should be tempered with the knowledge
that some or all of the sucrose in the medium is liable
to be broken down into its constituent hexose sugars,
and that such inversion will also occur within plant
tissues, where reducing sugar levels of at least 0.5 per
cent are likely to occur (Helgeson et al., 1972).
3.3.1. Autoclaving.
A partial hydrolysis of sucrose takes place during
the autoclaving of media (Ball, 1953; Wolter and
Skoog, 1966) the extent being greater when the
compound is dissolved together with other medium
constituents than when it is autoclaved in pure
aqueous solution (Ferguson et al., 1958). In a fungal
medium, not dissimilar to a plant culture medium,
Bretzloff (1954) found that sucrose inversion during
autoclaving (15 min at 15 lbs/in 2 ) was dependent on
pH in the following way:
pH 3.0 100%
pH 3.4 75%
pH 3.8 40%
pH 4.2 25%
pH 4.7 12.5%
pH 5.0 10%
pH 6.0 0%
These results suggest that the proportion of
sucrose hydrolysed by autoclaving media at
conventional pH levels (5.5-5.8) should be negligible.
Most evidence suggests that this is not the case and
that 10-15% sucrose can be converted into glucose
and fructose. Cultures of some plants grow better in
media containing autoclaved (rather than filtersterilised)
sucrose (Ball, 1953; Guha and Johri, 1966;
Johri and Guha, 1963; Verma and Van Huystee,
1971; White, 1932) suggesting that the cells benefit
from the availability of glucose and/or fructose.
However, Nitsch and Nitsch (1956) noted that
glucose only supported the growth of Helianthus
callus if it had been autoclaved, and Romberger and
Tabor (1971) that the growth of Picea shoot apices
was less when the medium contained sucrose
autoclaved separately in water (or sucrose autoclaved
with only the organic constituents of the medium)
than when all the constituents had been autoclaved
together. They suggested that a stimulatory
substance might be released when sugars are
autoclaved with agar.
In other species there has been no difference
between the growth of cultures supplied with
autoclaved or filter sterilised sucrose (Mathes et al.,
1973) or growth has been less on a medium
containing autoclaved instead of filter sterilised
sucrose (Stehsel and Caplin, 1969).
3.3.2. Enzymatic breakdown.
Sucrose in the medium is also inverted into
monosaccharides during the in vitro culture of plant
material. This occurs by the action of invertase
located in the plant cell walls (Burstrom, 1957;
Yoshida et al., 1973) or by the release of extracellular
enzyme (King and Street, 1977). In most cultures,
inversion of sucrose into glucose and fructose takes
place in the medium; but, because the secretion of
invertase enzymes varies, the degree to which it
occurs differs from one kind of plant to another.
After 28 days on a medium containing either 3 or 4%
sucrose, single explants of Hemerocallis and
Delphinium had used 20-30% of the sugar. Of that
which remained in the medium which had supported
Hemerocallis, about 45% was sucrose, while only 5%
was sucrose in the media in which Delphinium had
been grown. In both cases the rest of the sucrose had
been inverted (Lumsden et al., 1990).
The sucrose-inverting capacity of tomato root
cultures was greatest in media of pH 3.6-4.7.
Activity sharply declined in less acid media (Weston
and Street, 1968). Helgeson et al. (1972) found that
omission of IAA auxin from the medium in which
tobacco callus was cultured, caused there to be a
marked rise in reducing sugar due to the progressive
hydrolysis of sucrose, both in the medium and in the
tissue. A temporary increase in reducing sugars also
occurred at the end of the lag phase when newly
transferred callus pieces started to grow rapidly. It is
interesting to note that cell wall invertase possessed
catalytic activity in situ, whether or not tobacco tissue
was grown on sucrose (Obata-Sasamoto and Thorpe,
1983).
In cultures of some species, uptake of sugar may
depend on the prior extracellular hydrolysis of sugar.
This is the case in sugar cane (Komor et al., 1981);
and possibly also in Dendrobium orchids (Hew et al.,
1988) and carrot (Kanabus et al., 1986). Nearly all
the sucrose in suspension cultures of sugar cane and
sugar beet was hydrolysed in 3 days (Zamski and
Wyse, 1985) and Daucus carota suspensions have
been reported to hydrolyse all of the sucrose in the
medium (Thorpe, 1982) into the constituent hexoses
within 3 days (Kanabus et al., 1986) or within 24 h
(Dijkema et al., 1990). However, most species can
take up sucrose directly as was shown through studies
with asymetrically labeled 14 C-sucrose (Parr and
Edelman, 1975), and metabolise it intracellularly.
Within the cell, soluble invertase, sucrose phosphate
synthetase and sucrose synthetase serve to hydrolyse
sucrose (Thorpe, 1982). Thus, in Acer (Copping and