Liquid Culture Systems for in vitro Plant Propagation

Control of growth and differentiation 157
certain amount of suspension was harvested and replaced by fresh medium
in order to maintain a certain density of the suspension (determined by
measurement of the dry weight). Here, the average dilution rate was 0.18 -
0.2 d -1 .
All experiments were executed in parallel bioreactor runs using identical
inoculum and repeated at least twice.
3. Results
The effect of three environmental parameters on bioreactor cultures of
Physcomitrella was investigated for two purposes - optimisation of culture
growth, as well as analysing the effect of these parameters on moss
development.
In 10-litre batch cultures the effect of light and CO2 on the
photoautotrophically growing moss cultures was studied. Cultures grown
under standard conditions with 16 h light per day and aerated with air grew
with a doubling time (td) of 2.3 d (Figure 1). By using continuous
illumination td was reduced to 1.7 d. Supplementation of the aeration gas
with 2 % (v/v) CO2 resulted in a reduction of td to 1.3 d.
Using both, continuous illumination and CO2-enriched aeration gas, the
doubling time was reduced to 1.2 d (Figure 1): the growth rate was doubled
compared to the control. The pH of the culture medium decreased
throughout the culture period, probably due to ammonium uptake (Figure 1).
This pH decrease was especially pronounced in cultures showing a high
growth rate, thus reflecting higher nitrogen uptake of the more vigorously
growing cultures.
Growing suspension cultures without pH-control in ammonium-free
medium resulted in an average pH of 5.8 throughout a semi continuous
bioreactor culture in 5-litre vessels (Figure 2). Control of the pH with set
points of 7.0 and 4.5, respectively, had a pronounced effect on the growth
rate during exponential growth (day 0 – 3) before the start of the semi
continuous culture mode (Figure 2). Here the doubling time was 3 d for the
culture without pH control and 2.8 d at pH 4.5, but reduced to 1.7 d at
pH 7.0. However, the pH did not affect culture growth during the semi
continuous culture mode: here the dilution rate was between 0.18 and 0.2 d -1
for all cultures corresponding to a doubling time of 3.9 to 3.5 d. In contrast,
pH control markedly affected protonema differentiation: At pH 4.5 the
cultures predominantly developed chloronema, whereas at pH 7.0 caulonema
development was increased.