Liquid Culture Systems for in vitro Plant Propagation

Temporary immersion system: a new concept 181
Vitis vinifera L. that longer shoots were obtained in larger containers.
Grapevine explant growth was so strong in liquid medium using tilting
machines, that it soon became necessary to use larger containers such as 910
ml square wide-mouth Mason jars, as opposed to 125 ml flasks to avoid
crowding of the cultures, benefiting from a larger aperture to remove
material, and larger volumes of medium to prevent early deficiencies in
certain constituents of the medium (Harris and Mason, 1983). Using larger
containers means that larger volumes of media can be used, which can have
a positive effect on plant material proliferation and growth.
4.4 Oxygenation and forced ventilation
Work by Alvard et al. (1993) on banana meristem propagation clearly
showed that a lack of oxygen in the liquid culture medium was a major
limiting factor for small explant growth. In their experiment, the absence of
liquid medium stirring led to explant asphyxia. Bubble aeration of the
explant or medium encouraged growth but partial immersion of the explant
did not provide sufficient oxygen. Temporary immersion clearly proved to
be the most effective culture system. However, for other systems, the
positive effect of aeration has not been proved. Aitken-Christie and Jones
(1987) showed that aeration alone did not stimulate shoot growth in radiata
pine and was not a contributing factor to the increased growth found with
nutrient replenishment. As early as 1952, Stewart et al. attributed better
shoot growth to an improved oxygen supply through alternating immersion.
Yet, the pliofilm seal on their recipient vessel had no negative effect on
growth after culturing for two months.
In systems using pneumatic propulsion of the nutrient medium, this type
of operation was found to cause forced ventilation leading to complete
renewal of the culture atmosphere on each immersion. According to Teisson
and Alvard (1995), gas exchanges in such a system primarily occur during
immersion and are caused indirectly by movement of the liquid and directly
by the air pump under the most frequently used culture conditions that
correspond to complete renewal of the culture atmosphere after 5 minutes of
immersion for a 1-litre bioreactor. Such forced ventilation with air
containing the gas concentrations and relative humidity of the culture room
probably has positive effects (Krueger et al., 1991). The relative humidity
resulting from forced ventilation may stimulate transpiration in the plants,
which will then be more effectively adapted to ex vitro conditions (Wardle et
al., 1983).