Didier Courtois

The influence of salicylic acid elicitation of shoots, callus, and cell suspension cultures on production of naphtodianthrones and phenylpropanoids in Hypericum perforatum L.

Hypericum perforatum is a well known medicinal plant. The main pharmacological properties are due to the presence of naphtodianthrones such as hypericin and pseudohypericin. Unfortunately the levels of these compounds vary under different environmental conditions. Elicitation of in vitro cultures is a useful approach to enhance and extend production of desirable products. Therefore, the effects of salicylic acid were characterized on different explants of H. perforatum L. (cells, calli and shoots) cultured in vitro. It appears at first that salicylic acid did not affect growth and development of these explants. In addition, the production of both hypericin and pseudohypericin has doubled in elicited cell suspension cultures but not in the two other cultures. Furthermore, phenylpropanoids that are among the most frequently observed metabolites affected upon treatment of in vitro culture material with elicitors, were produced and the enzymatic activities of phenylalanine ammonia lyase and of chalcone isomerase were stimulated upon elicitation. These effects were dependant of the type of in vitro culture, the concentration of salicylic acid and the duration post-elicitation. The H. perforatum cells were globally more sensitive to salicylic acid elicitation when maintained in an undifferentiated state and particularly in cell suspension cultures. In the absence of glands considered as the sites of naphtodianthrones biosynthesis, cells and calli were capable of producing these compounds. This implies that salicylic acid could act at biosynthesis level but not for the accumulation of both hypericin and pseudohypericin. Consequently, the regulation of this process is more complex than cited in the literature involving the responsibility of only Hyp-1 gene, encoding a hypericin biosynthetic enzyme, cloned and characterized from H. perforatum.

High isoflavone content and estrogenic activity of 25 year-old Glycine max tissue cultures

Soy isoflavones are phytoestrogens which have been associated with several health benefits. In the present study, we report the production of isoflavones in a collection of 40 strains of soya cell cultures established in 1975. A large variability in the isoflavone composition was observed and high-producing strains, with an isoflavone content of up to 46.3 mg g(-1) dry wt., were found. In comparison with soybeans, many callus strains had a higher isoflavone concentration (10-40 times) and a different ratio of genistin to daidzin forms. The highest producing strain was transferred to liquid medium in an Erlenmeyer flask and in a 10 l stirred-tank bioreactor where high isoflavone content (7% dry wt.), concentration (880 mg l(-1)) and a maximum productivity estimated to 60 mg l(-1) d(-1) were obtained. We further studied the estrogenic activity of pure compounds compared to plant cell culture extracts in the estrogen-responsive human endometrial Ishikawa cell line. Estrogen was confirmed to be 1000-10,000 times more active than isoflavones. The estrogenic activity of the extracts correlated to their isoflavone content. The activity of the malonyl isoflavones, assessed here for the first time, was lower than the aglycones. Taken together, these results suggest that soya cell cultures can be used as an alternative source to soybeans to provide high concentrations of bioactive isoflavones.

Reduction of α,β-unsaturated ketones by plant suspension cultures

Abstract α,β-Unsaturated ketones were reduced by various plant cells grown under different cultural conditions. The stereochemistry of the reduction of (-)-carvone by Medicago sativa was found to be identical to that obtained with other organisms.

Expression of a carrot invertase gene in tobacco suspension cells cultivated in batch and continuous culture conditions

Plant cells (Nicotiana tabacum) were genetically modified to produce an heterologous protein, the acidic invertase from carrot, and invertase production from suspension tobacco cells was investigated. Suspension cultures were grown in shake flasks and stirred bioreactor. Total invertase activity was growth related. A 75 d continuous culture in 10 l bioreactor was performed. Our study demonstrates the high potential of plant cell cultures for long term production of heterologous protein.

Conversion of tryptamine to serotonin by cell suspension cultures of Peganum harmala

Abstract Biotransformation of tryptamine to serotonin by cell cultures of Peganum harmala was performed in 250 ml conical flaskes or 10 l bioreactor with high serotonin yields (2.5 g/l of culture and 20% of the biomass dry weight). The specific biotransformation rate reached more than 100 mg/g dry weight/day. The influence of pH, auxin concentration, and temperature were studied. Phenobarbital stimulated the reaction. Immobilized cells showed a lower biotransformation rate than cell suspensions. The stability of the cell line after cryostorage (growth and biotransformation capability) was established.

Expression of the human milk protein sCD14 in tobacco plant cell culture

The human milk protein sCD14 was expressed in tobacco plant cell cultures. Tobacco cells were transformed with a modified cd14 cDNA minus the GPI-tail and either the native human signal peptide (SP) or a plant SP, under the control of the CaMV-35S promoter. Transformants were screened using PCR and Southern blot analysis. The functionality of the inserted cDNA was checked by northern blot analysis for the presence of recombinant sCD14 mRNA. The detection of the protein has been observed by western blot analysis at an estimated level of 5 µg l−1 in a non-soluble fraction of the culture medium.

Cloning and Cell Sorter

The ability of plant cell cultures to produce useful compounds (pharmaceutical, food additives, cosmetics) with large-scale production has been investigated by numerous authors from scientific and economical points of view. (For reviews see: Zenk and Deus 1982, Kurz and Constabel 1983, Rosevear 1984. Yamada and Hashimoto 1984, Brodelius 1985, Dougall 1985, Sahai and Knuth 1985, Collinge 1986, Fowler 1986a, Fujita and Tabata 1986, Mac Laren 1986.)

Rapid production of irones by maturation of orris rhizomes with two bacterial strains

Abstract Two bacterial strains, Serratia liquefaciens and Pseudomonas maltophilia , were isolated from orris rhizomes ( Iris pallida ) which were cultu