Liquid Culture Systems for in vitro Plant Propagation

Bioreactor design for propagation 51
bioreactor and multiply the cells in culture, before inoculating all bioreactors
with the same large-scale cell culture.
4. Results and Discussion
4.1 Comparing Cyclamen cell growth in Erlenmeyer flasks and
in our bioreactor
We have performed comparisons using identical Cyclamen persicum Mill
cultures simultaneously, in both Erlenmeyer flasks and our bioreactors. We
started the experiment with the same inoculum for both culture systems and
using the same initial cell density and the same proliferation medium
(Schwenkel and Winkelmann, 1998; Winkelmann et al., 1998), following
the procedures described in Hvoslef-Eide and Munster (1998; 2001). The
results during the first two-week growth period in four bioreactors showed
significantly better growth in bioreactors; by fresh weight, dry weight,
packed cell volume (PCV) and cluster viability measured by FDA staining
(Widholm, 1972) of living cells (Figure 4). The standard deviation is less in
the bioreactors than in flasks, indicating a more uniform and predictable
culture in bioreactors in our system. Erlenmeyer flasks are probably more
variable because of the inability to control the various important
environmental parameters. Preil and co-workers (Preil et al., 1988; Preil,
1991) have shown the same improved growth pattern for poinsettia cell
cultures using Braun Biostat bioreactors compared with Erlenmeyer flasks
when the bioreactors were depleted of excess CO2 that may build up in a
closed (recirculated gas) silicone tube system. Hohe et al. (1999a) found
better growth in Erlenmeyer flasks of Cyclamen persicum cells compared to
Applikon bioreactors (2 l) when they allowed the CO2 to build-up in the
headspace. However, in later experiments, Hohe et al. (1999b; 2001)
depleted CO2 from the circulating gas mixture and obtained improved
growth in the bioreactors. This measure is not necessary in our bioreactors,
since the aeration system is exhaustive and not in a closed circuit. We have
investigated the gas permeability of the silicone tubes (Hvoslef-Eide and
Munster, 1997) and found that the permeability could vary between tubes of
different bioreactors, with build-up of CO2 and differences in growth of cells
between identical bioreactors as a consequence. We concluded that the
tubing age and number of autoclave cycles to which they have been subject,
could affect their permeability and now always change the tubes in all six
bioreactors at the same time after this.