Liquid Culture Systems for in vitro Plant Propagation

156 Annette Hohe & Ralf Reski
projects have been started in order to analyse gene functions on a large scale
(Nishiyama et al., 2001; Egener et al., 2002). Additionally, Physcomitrella is
an ideal organism for physiological analyses because of its clearly separated
and well-defined developmental stages (e.g. Bhatla et al., 2002). Moreover,
it is used also in applied plant biotechnology as a production system for
heterologous proteins (Reutter and Reski, 1996).
Thus, bioreactor cultures of Physcomitrella protonema suspensions have
been established for several reasons: i) protonema suspensions are used for
protoplast isolation for large scale transformation (Hohe et al., 2001; Hohe
and Reski, 2002). ii) Bioreactor cultures represent a ‘contained environment’
for the production of heterologous compounds (Reutter and Reski, 1996). iii)
Bioreactor culture offers a unique possibility for studying the effect of
environmental conditions on growth and development (Hohe et al., 2002).
This, in turn, is the basis for physiological studies as well as for the
optimisation of culture conditions for the production of heterologous
compounds.
In this paper we report on the effect of physical and chemical growth
conditions, i.e. gas atmosphere, light intensity and pH, on the growth and
development of bioreactor cultures of Physcomitrella protonema.
2. Materials and methods
Protonema suspension cultures of Physcomitrella patens were grown in a
modified Knop medium containing 4.24 mmol Ca(NO3)2, 1.02 mmol KCl,
1.84 mmol KH2PO4, 3.36 mmol MgSO4 and 0.045 mmol FeSO4, pH 5.8
(Reski and Abel, 1985). For batch cultures in bioreactors the medium was
additionally supplemented with 2.5 mmol ammonium tartrate.
Bioreactor cultures were done in glass vessels with a working volume of
either 5 or 10 litres, respectively (Applikon, Schiedam, The Netherlands).
Cultures were aerated with 0.3 vvm air or air supplemented with 2 % (v/v)
CO2 and stirred with a marine impeller running with 400 (10-litre vessel) or
500 rpm (5-litre vessel). Light intensity was 190 (10-litre vessel) or
120 µmol s -1 m -2 (5-litre vessel) provided by fluorescent tubes (Philips
TLD 25) either continuously or with a light/dark periodicity of 16/8 hours.
Control of pH was by automatic titration of 0.5 N KOH or HCl (ADI 1030
control device, Applikon, Schiedam, The Netherlands).
Growth was determined by measuring the dry weight of the cultures; two
50 ml samples were taken per day and the cell material dried to constant
weight at 105°C for 2 hours.
Culture was either done as a batch culture starting with approximately
30 mg l -1 dry weight, or as semi continuous culture, i.e. in a daily rhythm a