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

144 The Components of Plant Tissue Culture Media II
acid was an effective buffer and appeared to enhance
the effect of the glutamine and alanine which they
added to their medium. Vyskot and Bezdek (1984)
found that the buffering capacity of MS medium was
increased by adding either 1.25 mM sodium citrate or
1.97 mM citric acid plus 6.07 mM dibasic sodium
phosphate. Citric acid and some other organic acids
have been noted to enhance the growth of Citrus
callus when added to the medium (presumably that of
Murashige and Tucker, 1969) (Erner and Reuveni,
1981). For the propagation of various cacti from
axillary buds, Vyskot and Jara (1984) added sodium
citrate to MS medium to increase its buffering
capacity.
Recognized biological buffers: Unlike organic
acids, conventional buffers are not metabolised by the
plant, but can poise pH levels very effectively.
Compounds which have been used in plant culture
media for critical purposes such as protoplast
isolation and culture, and culture of cells at very low
inoculation densities, include:
• TRIS, Tris(hydroxymethyl)aminomethane;
• Tricine, N-tris(hydroxymethyl)methylglycine;
• MES, 2-(N-morpholino)ethanesulphonic acid;
• HEPES, 4-(2-hydroxyethyl)-1-piperazine(2ethanesulphonic
acid); and
• CAPS, 3-cyclohexylamino-1-propanesulphonic
acid.
Such compounds may have biological effects
which are unrelated to their buffering capacity.
Depending on the plant species, they have been
known to kill protoplasts or greatly increase the rate
of cell division and/or plant regeneration (Conrad
et al., 1981). The ‘biological’ buffers MES and
HEPES have been developed for biological research
(Good et al., 1966).
MES: MES is one of the few highly effective and
commercially available buffers with significant
buffering capacity in the pH range 5-6 to which plant
culture media are usually adjusted and has only a low
capacity to complex with micronutrients. It is not
toxic to most plants, although there are some which
are sensitive. Ramage and Williams (2002) report
that shoot regeneration from tobacco leaf discs was
not affected by MES when increasing the
concentration up to 100 mM. De Klerk et al (2007),
though, observed a decrease of rooting from apple
stem slices with increasing MES concentration (see
below; Fig. 4.4). This effect of MES was not
understood. It only occurred during the first days of
the rooting process and was not observed during the
outgrowth phase after the meristems had been
formed.
During the culture of thin cell layers of Nicotiana,
Tiburcio et al. (1989) found that the pH of LS
medium could be kept close to 5.8 for 28 days by
adding 50 mM MES, whereas without the buffer pH
gradually decreased to 5.25. Regulating pH with
MES alters the type of morphogenesis which occur in
this (and other) tissues (see below).
Parfitt et al. (1988) found 10 mM MES to be an
effective buffer in four different media used for
tobacco, carrot and tomato callus cultures, and peach
and carnation shoot cultures, although stabilizing pH
did not result in superior growth. The tobacco, peach
and carnation cultures were damaged by 50 mM
MES. Tris was toxic at all concentrations tested,
although Klein and Manos (1960) had found that the
addition of only 0.5 mM Tris effectively increased
the fresh weight of callus which could be grown on
White (1954) medium when iron was chelated with
EDTA.
MES has also been used successfully to buffer
many cultures initiated from single protoplasts
(Müller et al., 1983) and its inclusion in the culture
medium can be essential for the survival of individual
cells and their division to form callus colonies (e.g.
those of Datura innoxia - Koop et al., 1983). MES
was found to be somewhat toxic to single protoplasts
of Brassica napus, but ‘Polybuffer 74’ (PB-74, a
mixture of polyaminosulphonates), allowed excellent
microcolony growth in the pH range 5.5-7.0
(Spangenberg et al., 1986). Used at 1/100 of the
commercially available solution, it has a buffering
capacity of a 1.3 mM buffer at its pK value (Koop
et al., 1983).
Banana homogenate is widely used in orchid
micropropagation media. Ernst (1974) noted that it
appeared to buffer the medium in which slipper
orchid seedlings were being grown.
5.1.2. The uptake of ions and molecules
The pH of the medium has an effect on the
availability of many minerals (Scholten and Pierik,
1998). In general, the uptake of negatively charged
ions (anions) is favoured at acid pH, while that of
cations (positively charged) is best when the pH is
increased. As mentioned before, the relative uptake of
nutrient cations and anions will alter the pH of the
medium. The release of hydroxyl ions from the plant
in exchange for nitrate ions results in media
becoming more alkaline; when ammonium ions are