[Pharmacognostical study of Atropa belladonna].

Based on the research of plant taxonomy and botanical investigation, microscopic characteristics of the root, stem, leaf transverse section and powder of Atropa belladonna were studied for identification of the herb. The research detailed and made clear to the description identification and microscopic characteristics of officinal parts of the herbs. The work provided reference for the identification of A. belladonna herbs and pieces of work in the future, as well as a theoretical basis for the further research, development, medicinal use and the upgrading of quality standards.

Expression of functional mammalian P450 2E1 in hairy root cultures.

P450 2E1 is an important mammalian liver enzyme known to metabolize a wide range of compounds including several common environmental pollutants. The medicinal plant, Atropa belladonna, was transformed with Agrobacterium rhizogenes containing a binary vector with rabbit P450 2E1 in either the sense or antisense orientation. The resulting "hairy roots" were isolated and grown in liquid medium. Production of P450 2E1 protein was verified in the roots containing the 2E1 gene in the sense orientation. Transgenic and control root cultures were dosed with the environmental pollutant, trichloroethylene (TCE), and were analyzed for the TCE metabolites, chloral and trichloroethanol. The root cultures expressing the mammalian P450 2E1 had increased levels of the metabolites compared to the levels in the control roots. This method represents a quick way to screen transformants for expression of foreign genes before regeneration of whole plants, and also as a possible source of foreign protein for purification.

Tropane alkaloid production by hairy roots of Atropa belladonna obtained after transformation with Agrobacterium rhizogenes 15834 and Agrobacterium tumefaciens containing rol A, B, C genes only.

Atropa belladonna leaf disks were infected by a wild strain Agrobacterium rhizogenes 15834 harboring the Ri-TL-DNA and by a disarmed Agrobacterium tumefaciens strain harboring a construction with only rol ABC and npt II genes. Thirteen root lines were established and examined for their growth rate and alkaloid productivity to evaluate the possible role of rol genes in morphological differentiation and in tropane alkaloid formation. A great diversity has been observed in the growth rate of these 13 root lines. The root biomass increased up to 75 times. The total alkaloid contents were similar in the root lines obtained by infection with A. rhizogenes 15834 and A. tumefaciens rol ABC. The last ones accumulated between 4 (1.1 mg g(-1) DW) and 27 (8 mg g(-1) DW) times more alkaloids than the intact roots (0.3 mg g(-1) DW). This work has shown that the rol ABC genes were sufficient to increase tropane alkaloid production in A. belladonna hairy root cultures.

Hairy root culture in a liquid-dispersed bioreactor: characterization of spatial heterogeneity.

A liquid-dispersed reactor equipped with a vertical mesh cylinder for inoculum support was developed for culture of Atropa belladonna hairy roots. The working volume of the culture vessel was 4.4 L with an aspect ratio of 1.7. Medium was dispersed as a spray onto the top of the root bed, and the roots grew radially outward from the central mesh cylinder to the vessel wall. Significant benefits in terms of liquid drainage and reduced interstitial liquid holdup were obtained using a vertical rather than horizontal support structure for the biomass and by operating the reactor with cocurrent air and liquid flow. With root growth, a pattern of spatial heterogeneity developed in the vessel. Higher local biomass densities, lower volumes of interstitial liquid, lower sugar concentrations, and higher root atropine contents were found in the upper sections of the root bed compared with the lower sections, suggesting a greater level of metabolic activity toward the top of the reactor. Although gas-liquid oxygen transfer to the spray droplets was very rapid, there was evidence of significant oxygen limitations in the reactor. Substantial volumes of non-free-draining interstitial liquid accumulated in the root bed. Roots near the bottom of the vessel trapped up to 3-4 times their own weight in liquid, thus eliminating the advantages of improved contact with the gas phase offered by liquid-dispersed culture systems. Local nutrient and product concentrations in the non-free-draining liquid were significantly different from those in the bulk medium, indicating poor liquid mixing within the root bed. Oxygen enrichment of the gas phase improved neither growth nor atropine production, highlighting the greater importance of liquid-solid compared with gas-liquid oxygen transfer resistance. The absence of mechanical or pneumatic agitation and the tendency of the root bed to accumulate liquid and impede drainage were identified as the major limitations to reactor performance. Improved reactor operating strategies and selection or development of root lines offering minimal resistance to liquid flow and low liquid retention characteristics are possible solutions to these problems.