Production of paclitaxel and baccatin III in a 20-L airlift bioreactor by a cell suspension of Taxus wallichiana.

A cell suspension culture of Taxus wallichiana (Himalayan Yew) was grown in shake flasks and a 20-L airlift bioreactor running for 28 days in a batch mode, and its capacity to accumulate paclitaxel and baccatin III was measured. When both culture types were in the highest productive state (from day 24 to day 28), there was a greater accumulation of paclitaxel and baccatin III in the bioreactor culture than in the shake flask culture (factor of 2.0 and 1.2, respectively). These increases in paclitaxel and baccatin III production cannot be related to the difference observed between the growth rates of both cultures, because when the bioreactor culture was at maximum productivity, its cell biomass, expressed in g L(-1) of dry weight, was similar to that obtained in the shake flask culture. It seems that these improvements were mainly due to adequate aeration and mixing of the culture in the bioreactor. The maximum yield observed for paclitaxel (20.84 mg x L(-1) day 24) and baccatin III (25.67 mg x L(-1) day 28) represents a productivity of 0.90 mg x L(-1) d(-1) and 0.93 mg x L(-1) x d(-1) respectively.

Ginsenoside production in different phenotypes of Panax ginseng transformed roots.

Transformed roots were obtained after the inoculation of sterile root discs of Panax ginseng C.A. Meyer with Agrobacterium rhizogenes A4. The established hairy root lines displayed three morphological phenotypes when cultured on hormone-free liquid Schenk and Hildebrandt medium. Most of the cultures showed the characteristic traits of hairy roots (HR-M), while others were either callus-like (C-M) or thin (T-M) without branching. The growth rate of the transformed root lines was always higher than that of untransformed roots, showing that the genetic changes caused by the A. rhizogenes transformation conditioned a higher biomass formation. When considering the different transformed root phenotypes, we can observe that the highest ginsenoside production was achieved by HR-M root lines, closely followed by C-M ones, whereas the lowest yield was reached by T-M root phenotype. The study of the integration of the TL-DNA and TR-DNA fragments of the pRiA4 in the root genome showed that the aux1 gene was always detected in HR-M and C-M root phenotypes which presented the highest biomass and ginsenoside productions. This fact suggests a significant role of aux genes in the morphology of Panax ginseng transformed roots. The ginsenoside pattern of transformed roots varied according to their morphology, although the ginsenoside contents of the Rg group was always higher than that of the Rb group. From our results, we can infer the potential of some root phenotypes of Panax ginseng hairy root cultures for an improved ginsenoside production.

Decreased scopolamine yield in field-grown Duboisia plants regenerated from hairy roots.

Hairy root cultures were obtained from hybrid clones of Duboisia myoporoides x D. leichhardtii following transformation by Agrobacterium rhizogenes strain A4. Shoots spontaneously regenerating from the hairy root cultures were rooted and transferred to soil. The plants displayed typical morphological alterations known as hairy root syndrome to varying degrees. PCR analysis confirmed that all transformed plants contained the rolA, rolB and rolC genes, irrespective of the degree of morphological alterations. A field test of the transformed regenerated plants revealed that those plants displaying the strongest hairy root syndrome symptoms had the highest content of the tropane alkaloid scopolamine. However, the overall scopolamine and hyoscyamine yield of all transformed plants was clearly reduced compared to untransformed control plants. These results demonstrate that the A. rhizogenes-transformed plants tested in this study do not provide a viable alternative to agricultural farming of hybrid clones of D. myoporoides x D. leichhardtii obtained by conventional breeding.

Datura metel: in vitro production of tropane alkaloids.

Hairy root lines of Datura metel were established following infection of aseptic stem segments with Agrobacterium rhizogenes strain A4 and cultured in hormone-free B5 solid medium. The growth and production of hyoscyamine and scopolamine (mg/g dry wt.) of these root cultures was encouraged by using B5 liquid medium with half-strength salts. In these culture conditions, the capacity of the highest productive root line 25 to excrete scopolamine into the culture medium rose from 8.7% to 70% when the permeabilizing agent Tween 20 was added for 24 h to the medium, after 2 and 4 weeks of culture. Using an airlift bioreactor (41) with modifications in order to increase root anchorage, the Tween 20 treatment encouraged both growth and alkaloid productivity of cultured root line 25. After 4 weeks their biomass yield was 2.3 mg/l/day and 0.84 mg/l/day of scopolamine was produced (70% extracellular). The scopolamine released into the culture medium was separated with an Amberlite XAD-2 column located in the media exit.

Expression of the rolC gene and nicotine production in transgenic roots and their regenerated plants.

Transformation of Nicotiana tabacum cv. Xanthi leaf sections with the pPCV002-ABC (rol genes A, B and C together under the control of their own promoter) or pPCV002-CaMVC (rol gene C alone under the control of the CaMV 35S promoter) construction present in trans-acting Agrobacterium tumefaciens vectors yielded several transgenic root lines. The two types (rolABC and rolC) of transgenic root lines were examined for their nicotine productivity in relation to growth rate and the amount of rolC gene product measured with specific antibodies. In all cases, the changes in the amount of this polypeptide were positively correlated with the capacity of the transgenic roots to grow and produce nicotine. Both capacities were greatly increased when the rolA, rolB and rolC genes were present together, which demonstrates that the activity of the three rol-gene-encoded functions is synergistic. Consistent observations were also made in the corresponding regenerated plants.

Activation of plant plasma membrane H(+)-ATPase by the non-ionic detergent Brij 58.

Plasma membrane vesicles were purified from tobacco callii and the modulation of H(+)-ATPase by detergents was investigated. The nonionic detergent Brij 58 not only activated ATP hydrolysis (2-fold) but also proton pumping (more than 4-fold). Triton X-100, within a more limited concentration range, produced a similar effect. The simultaneous activation of ATP hydrolysis and proton pumping is not compatible with current interpretations of effects of nonionic detergents on the H(+)-ATPase based on latency of the enzyme and opening of vesicles.