Optimisation of a bioreactor culture of the moss Physcomitrella patens for mass production of protoplasts

One prerequisite for plant functional genomic projects is the development of a high-throughput transformation platform. As the moss Physcomitrella patens can be transformed via PEG-mediated DNA-uptake into protoplasts, a semi-continuous bioreactor culture of this plant was optimised regarding protoplast isolation efficiencies. Under standard conditions protoplast yields were 2.8 x 10(4)/mg dry weight. This yield was increased sixfold by supplementation of the medium with 460 mg/l ammonium tartrate. The same effect was achieved by controlling the pH-value in the bioreactor culture with a setpoint of 4.5. In contrast, pH control with a setpoint of 7.5 reduced the protoplast yield compared to a culture without pH control to 11%. A semi-continuous culture of Physcomitrella in a 5-1 bioreactor grown at pH 4.5 yielded sufficient cell material for more than 100 transformations per day. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.