Liquid Culture Systems for in vitro Plant Propagation

520 E. Gontier et al.
3.5 Effect of permeabilization
Based on our previous experience (Boitel-Conti et al., 1995-1997;
Gontier et al., 2002), we tested the potential effects of Tween20 as a
membrane permeabilizing agent for FCs release in Ruta shoot cultures.
Thus, after the growth phase (20 weeks, 4 parallel cultures corresponding to
figure 8) some of the shoots were permeabilized with Tween20 3 % (v/v)
with or without addition of umbelliferone (an FC precursor). The evolution
of FCs in the culture medium was measured as a function of time (Figure 10)
and the best results were obtained for the Tween treatment. Following
addition of umbelliferone, neither the shoot FC content (Figure 11) nor the
FC release (Figure 10) were improved. FCs levels in the biomass (Figure
11), measured on a sample before and after the treatment, significantly
decreased only when a high increase was observed for the FCs concentration
in the medium (i.e. after Tween treatment). Furthermore, the biomass could
be rinsed and cultivated again for weeks (at least) after being permeabilized,
showing that cell viability was not completely altered.
FCs concentration
in biomass (mg g-1 DW)
30
20
10
0
ns
ns s
before after before after before after before after
Control Umbelliferone 3% Tween20 3% Tween20 +
Treatment
s
5Mop
5,8Mop
8Mop
Pso
Umbelliferone
Figure 11: Ruta shoot furanocoumarin levels before and after the culture medium is replaced
by a fresh one (control) containing umbelliferone and/or Tween20 as precursor and
permeabilizing agent respectively. Experiments conducted in 3-litre bubble flasks (Figure 2,
Bioreactor 2) after 20 weeks of growth [ns: non significant difference; s: significant
difference; p