Production of secondary metabolites in berry cell cultures

In this diploma thesis the scale up of arctic bramble suspension cultures from 30 mL to 1 L was carried out in shake flasks. Growth conditions were optimized, especially the optimal concentration of inoculated cells and shaking rate were evaluated. During the optimisation of the 200 mL arctic bramble suspension cultures in shake flasks maximally a specific growth rate of 0.0080 h-1 and a dry weight of 22 g L-1 were achieved. A concentration of the inoculum of 100 g L-1 and a shaking rate of 103 rpm were evaluated as the optimal cultivation condition. The 1 L suspension cultures were grown under the optimised conditions and achieved a specific growth rate of 0.0066 h-1 and a dry weight of 19.7 g L-1. During the growth of the 1 L culture dissolved oxygen was measured to be under 5 % and a limitation of oxygen was assumed. The growth of arctic bramble suspension cultures in the Wave bioreactor was investigated. The growth was monitored as usually by determining the fresh and dry weight, pH value, conductivity and sugar concentration and the dissolved oxygen. The 1 L arctic bramble suspension cultures grew well in the Wave bioreactor and achieved maximally a specific growth rate of 0.0109 h-1 and a dry weight of 21.3 g L-1. The dissolved oxygen supply in the Wave bioreactor was good and no limitation was observed. Finally the elicitation of suspension cultures performed with s-ABA to produce new phenolic compounds was studied. The suspension cultures in the bioreactor were elicitated with s-ABA at different growth phase points and the phenolic compounds were analysed by HPLC. The time point of the elicitation did not cause any changes to phenolic profiles. Three phenolic compounds kaempferol, ferulic acid and sinapic acid were identified in non-elicitated as well as in elicitated cultures according to retention times and characteristic maxima in the spectra. In elicitated cultures the concentration of the compounds decreased after the elicitation. However, kaempferol hexoside was detected only in s¬ABA elicitated cultures eight hours after the elicitation and its concentration decreased until the end of the cultivation. Additionally the cryopreservation and the recovery of the arctic bramble suspension culture KAS 341/8 were studied. Three different cryopreservation methods were applied and two of them seemed to be suitable for arctic bramble suspension cultures. In one method glycerol and sucrose in high concentrations were added and then the culture was frozen by two-step directly. In the other method the culture was first pre-treated with sorbitol for two days and then DMSO was added prior to two-step freezing. After two subcultivations the recovered cultures were transferred to liquid media and they grew even better than non cryopreserved cultures by achieving a growth index of 4.21. The induction and the cultivation of callus from wild bilberry plants were investigated. Bilberry callus was induced from stem plantlets and the cultivation of the non friable callus culture was established. A modified Gamborg media seemed to be suitable for the induction of callus. On a Woody Plant media a strong reddish pigmentation was induced and the alteration to friable callus. The friable callus would provide the material for the establishment of a bilberry suspension culture.