Liquid Culture Systems for in vitro Plant Propagation

496 Han Bouman & Annemiek Tiekstra
material used for adaptations and improvements of hydroponic media for
many plants (Anonymous, 1999). For olive, Rugini (1984) used the
composition of developing shoots and embryos, which led to rather high P
and much lower Ca concentrations. In preliminary experiments, tissuecultured
and young greenhouse-grown Gerbera reacted unfavourably to
mineral adaptations derived from analyses of young plants, which had
similar P and Ca concentrations as in Rugini’s media.
A second choice concerns how to obtain the mineral concentrations for
the essential macronutrients without adding Na and Cl. The very low
concentrations of these two elements, necessary for growth, are invariably
present in other salts: high concentrations of Na and Cl can be detrimental
for growth (George, 1993). For the final modified formulations, usually
compromises had to be made to obtain a suitable composition for
satisfactory plant growth.
The third choice concerned the data obtained from the tissue analyses:
results were in mmol per g DW or FW for the various elements, but for the
media, the basis of the concentrations (mmol per litre) had to be decided
upon. We decided to start from a nitrogen concentration similar to that used
in conventional protocols and then to calculate the concentrations of the
other elements in relation to the concentration value for nitrogen.
Fourthly, it had to be decided in which molecular form the nitrogen
would be supplied, viz., how much ammonium and how much nitrate. Whilst
it is recognised that the ammonium-nitrate ratio is important for growth and
development (Avila et al., 1998), this ratio is not dealt with in the research
reported here. Experiments showed that the ratio had some influence,
especially for Gerbera, but provided that the ratio was not extreme, its effect
was not as profound as the mineral adaptations.
A general problem, which often occurred during the preparation of the
modified media for Gerbera (but also for other species) was the precipitation
of white insoluble salts mostly composed of Ca and P. When mixing the
salts, no precipitate was noticed, but the endothermic reaction was started at
high temperatures during autoclaving, resulting in precipitates after
autoclaving. Because of this precipitation, infections became more difficult
to detect, and a loss of specific minerals occurred and the pH was reduced
because H + was released from both HPO4 2- and H2PO4 - . The analysis of the
medium after such precipitations showed that the amount of Ca and P that
had precipitated was low and reduced the medium-concentration of P by less
than 10%. However, because of the pH reduction, there was softening of the
agar gel: in some cases the agar did not gel sufficiently. DKW medium was
opaque and showed a yellow precipitation before autoclaving. This
precipitate contained Fe, probably linked to phosphate, with some Ca3(PO4)2.
Schenk et al. (1991) also reported these medium-precipitates after