Liquid Culture Systems for in vitro Plant Propagation

516 E. Gontier et al.
3.3 Establishment of bubble flask cultures
The above results were promising but the heterogeneity was quite
important and it was not possible to carry out many experiments in parallel
with a single bioreactor. It was then decided to build a simplified bioreactor
in which oxygen pressure and pH were not controlled. This system consisted
of a 3-litre flask in which the shoot culture was performed (Figure 12 C and
Figure 2, Bioreactor 2). This system was coupled with a 2.5-litre bottle that
allowed draining of the culture medium for weighting the biomass.
The first growth experiments led to good results in terms of growth and
reproducibility (Figure 5). In these conditions, furanocoumarin levels were
equivalent to those measured in Erlenmeyer flasks or Setric bioreactor
experiments. The mean biomass production within 42 days was 0.4 g DW
per day of culture corresponding to a calculated productivity of 0.97 mg FC
per day of culture and per gram DW of inoculum in 1.5 litres (3.2 mg FCs
day -1 l -1 ). On this basis, it was demonstrated that lowering the sucrose
concentration in the medium (10 g l -1 instead of 30 g l -1 ) improved the fresh
weight to nearly double (Figure 6). This led to values of: 1.8 mg FC per day
of culture and per gram DW of inoculum in 1.5 litres. Because it was easy to
build up many copies of the bioreactor (low technology and low cost), it was
possible to run many experiments (and replicates) in parallel (Figure 12 D).
3.4 Effect of temporary immersion
The permanent immersion regime (in bubble flasks, Figure 2, Bioreactor 2)
and temporary immersion one (Figure 2, Bioreactor 3) were compared
(Figure 7). Our results show that, over a 44 day culture, temporary
immersion is beneficial for growth (a 17% increase). It seems also to be
positive for shoot quality, as no vitrification occurred whereas it happened
very often in the permanent immersion regime. No significant differences
appeared in FCs contents. The calculated productivity was 2.6 mg FCs
produced per day of culture and per gram DW of inoculum in 1.5 litres
(1.7 mg FCs day -1 l -1 ).
In order to try to improve the culture process, we performed some cultures in
temporary immersion and replaced the culture medium by a fresh one, each
time the growth slowed down (Figure 8). Then, very high densities of
biomass were obtained and productivity was steadily high over a long period
(140 days). The mean biomass production and calculated FCs productivity
were 0.6 g DW per day and 3.8 mg of FCs per day and per gram DW of
inoculum (5 mg FCs day -1 l -1 ). Furthermore, when reaching such densities,
shoots were found to be deeply coloured green all around the mass, more
yellow-green inside and yellow-white in the centre of the culture (Figure 12 E).