Liquid Culture Systems for in vitro Plant Propagation

Comparison of secondary plant metabolite production 527
2. Materials and methods
2.1 General in vitro culture conditions
All in vitro cultures were performed on MS basal medium containing
macroelements, microelements and vitamins according to Murashige and
Skoog (1962) in microcontainers (250 ml polypropylene vessels). For callus
and suspension cultures of Fabiana, Hypericum and Lavandula the MS
medium was additionally supplemented with 1 mg l -1 nicotinic acid, 1 mg l -1
pyridoxine HCl, 10 mg l -1 thiamine HCl, 0.25 mg l -1 NAA, 0.25 mg l -1 2,4-D,
0.25 mg l -1 kinetin and 30 g l -1 sucrose. For shoot multiplication of
Hypericum and Lavandula the medium was supplemented with 0.5 mg l -1
nicotinic acid, 0.5 mg l -1 pyridoxine HCl, 9.9 mg l -1 thiamine HCl, 1 mg l -1
BA and 30 g l -1 sucrose. For shoot multiplication of Fabiana half
concentrated MS macro- and microelements were used supplemented with
100 mg l -1 myo-inositol, 1 g l -1 peptone, 2 mg l -1 glycine, 0.5 mg l -1 nicotinic
acid, 0.5 mg l -1 pyridoxine HCl, 0.1 mg l -1 thiamine HCl, 1 mg l -1 NAA,
1 mg l -1 gibberellic acid A3 and 20 g l -1 sucrose. For Cymbopogon also MS
macro- and microelements as well as vitamins were used. For callus and
suspension cultures the medium was supplemented with 0.9 mg l -1 thiamine
HCl, 5 mg l -1 2,4-D, 0.8 mg l -1 kinetin and 20 g l -1 sucrose. For shoot
multiplication of Cymbopogon the additional medium supplements were
0.3 mg l -1 BAP and 30 g l -1 sucrose. Solid medium was prepared by addition
of 2.5 g l -1 Phytagel, the pH was adjusted to 5.7 before autoclaving. The
culture temperature was 26°C, the light intensity was 50 µmol s -1 m -2 for 16
hours per day. Callus cultures were kept in the dark.
2.2 Suspension and bioreactor culture
Cell suspensions were initiated by culturing 3 g callus in 20 ml liquid
medium in 100 ml Erlenmeyer flasks. The cultures were kept in the dark on
an orbital shaker (100 rpm) at 24°C. Obtained cell suspensions were
subcultured every week by inoculating 60 ml medium in 300 ml Erlenmeyer
flasks with 8 g cell fresh weight.
The bioreactor culture of Hypericum was performed in a 2-litre stirred
tank bioreactor (Chemap A.S., Switzerland). The bioreactor was equipped
with a bubble free aeration system using silicone tubes, the setpoint of
dissolved oxygen was 80 % of air saturation. Stirring was performed by
using a large blade stirrer running with 40 rpm. For Lavandula a 5-litre
bioreactor (Braun Biotech, Germany) was equipped with a bubble aeration
(setpoint of dissolved oxygen concentration: 60 %) and a marine impeller