Liquid Culture Systems for in vitro Plant Propagation

56 Anne Kathrine Hvoslef-Eide et al.
in connection to sample withdrawal, due to an eventual drift in the electrode
during an experiment. The measurement must be made immediately after
sample withdrawal in case of high CO2 concentrations in the sample. If the
concentration of CO2 in the sample is higher than in the air, CO2 -equilibrium
will appear and pH value of the sample will rise rapidly (Cazzulino et al.,
1991).
5.4 Stirring speed and stirrer direction
Agitation by one impeller, with two slightly pitched narrow blades seems
to be a functional construction for propagation of plant cells and embryos.
So far, minimum or no damage from shear forces is recognized. Because of
the two-directional mixing (10 seconds each direction), a slow rotational
speed (30 rpm) is sufficient to avoid quiet zones. Together with the silicone
tubing, which provides bubble-free gas supply, the slow speed gives no foam
formation, preventing the need for a foam control system.
5.5 Other recommended elements
CO2 measurements are a good indicator of growth, and may serve as an
on-line inspection. The bioreactors described do not have equipment for
analysing CO2 content. This will be the first priority when the bioreactors are
to be upgraded. It would enable measuring the gas concentration in
headspace or dissolved in the medium. The CO2 sensor can also be situated
outside the aseptic barrier, and so permit the measurement of CO2 partial
pressure in the exhaust from the bioreactor. Continuously connecting an
infrared sensor to the lid may also perform measurements of the gas
concentrations, so that samples can be withdrawn at regular intervals.
Sensors for biomass, as optical density sensors, may also be used as
indicators of growth. Application of image analysis technology is another
alternative for measuring cell density (Cazzulino et al., 1991; Pépin et al.,
1999). As well as measuring biomass concentration, these tools give
information about aggregate size and distribution, pigmentation and
morphology of the culture, which is a great advantage when growing
embryos.
To be able to keep low oxygen concentrations, a nitrogen supply is
required. If factorial experiments with both high and low oxygen
concentrations are to be run, both oxygen and nitrogen are needed as
enrichment gases. The best way of adding these gases, with regard to
regulation and control, is to use three flow regulators, one for air, another for
oxygen and another for nitrogen.