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Dedifferentiated Taxus media cell cultures presenting the same genetic characteristics as the parent culture were established from transformed roots. Two transformed cell lines were studied: Rol C, carrying the T-DNA of A. rhizogenes 9,402 and TXS, carrying both the T-DNA of A. rhizogenes and the txs transgene of T. baccata under the control of the 35S CaMV promoter. In the second part of a previously optimized two-stage system, the transformed cell lines were cultured in a production medium supplemented with the elicitor methyl jasmonate. Taxane production in the transformed cultures was compared with an untransformed T. media cell line cultured in the same conditions. The highest taxane production was observed in the TXS cell line when cultured in the optimized production medium with methyl jasmonate, being 265% greater than in the untransformed control and 170% greater than in the Rol C cell line. However, txs expression and the activity of the enzyme taxadiene synthase in the TXS cells were lower than in the line carrying only the rol genes (Rol C). It is also noteworthy that the taxane production as well as the txs gene expression and TXS activity in all the cell lines, both transformed and untransformed, were clearly dependent on the elicitor action., ((c) 2010 American Institute of Chemical Engineers)

This study assessed the effect of two precursors (l-phenylalanine and p-amino benzoic acid) used alone or in combination with methyl jasmonate, on the growth and accumulation of paclitaxel, baccatin III and 10-deacetylbaccatin III in hairy root cultures of Taxus x media var. Hicksii. The greatest increase in dry biomass was observed after 4 weeks of culturing hairy roots in medium supplemented with 1microM of l-phenylalanine (6.2gL(-1)). Addition of 1microM of l-phenylalanine to the medium also resulted in the greatest 10-deacetylbaccatin III accumulation (422.7microg L(-1)), which was not detected in the untreated control culture. Supplementation with 100microM of l-phenylalanine together with 100microM of methyl jasmonate resulted in the enhancement of paclitaxel production from 40.3microg L(-1) (control untreated culture) to 568.2microg L(-1), the highest paclitaxel content detected in the study. The effect of p-amino benzoic acid on taxane production was less pronounced, and the highest yield of paclitaxel (221.8microg L(-1)) was observed when the medium was supplemented with 100microM of the precursor in combination with methyl jasmonate. Baccatin III was not detected under the conditions used in this experiment and the investigated taxanes were not excreted into the medium.

In vitro cultivated plant cells and tissues were found to synthesize polyisoprenoids. Taxus baccata suspension cell cultures accumulated polyisoprenoids of the same pattern as the parental tissue; methyl jasmonate or chitosan treatment almost doubled their content. All the root cultures studied accumulated dolichols as predominant polyisoprenoids. Roots of Ocimum sanctum grown in vitro accumulated approx. 2.5-fold higher amount of dolichols than the roots of soil-grown plants. Dolichols dominated over polyprenols in all Triticum sp. tissues studied.

Paclitaxel and 10-deacetylbaccatin III (10-DAB III) were produced in suspension cultures of Taxus x media var. Hicksii grown in shake-flasks and in a 7-l bioreactor reaching, in the bioreactor, 4.4 mg l(-1) (on day 14) and 37.5 mg l(-1) (on day 11). In shake-flasks the highest total content of paclitaxel and 10-DAB III was 7.3 mg l(-1) (on day 4) and 8.8 mg l(-1) (on day 18). Phenylalanine, at 0.05 mM, increased paclitaxel accumulation in cells cultivated in bioreactor and flasks 30-fold and 9-fold (from 0.02 mg l(-1) to 0.6 mg l(-1) and to 0.2 mg l(-1), respectively). The 10-DAB III content in cells from flasks was increased from 0.4 mg l(-1) to 1.6 mg l(-1).