Hairy root cultures of Brugmansia candida (Solanaceae), a South American plant which produces scopolamine and hyoscyamine, were exposed to different elicitors (jasmonic acid (JA) and aluminum chloride (AlCl₃)) in order to increase their productivity and/or stimulate their liberation. Hairy roots of 19-day old cultures (exponential phase) were exposed to these elicitors for 24 and 48 hours. The effects on alkaloid accumulation and release into the medium were evaluated. JA was tested at 2.5 and 25 m g/ml. After 24 hours, JA promoted the release of hyoscyamine (~1200%) when the highest concentration was used. Therefore, the positive effects seen with JA could possibly be attributed in part to ethanol (EtOH), the solvent in which the acid was dissolved. At the lowest concentration tested, JA promoted an increase on scopolamine accumulation (30%) after 48 hours of exposure. When exposed to AlCl₃ for 48 hours and at concentrations of 25 and 250m M, scopolamine and hyoscyamine accumulation increased in the roots (43-83%). After 48 hours of treatment with the highest concentration of AlCl_3, release of scopolamine into the medium increased approximately 150%.

In this work, hairy roots of Brugmansia candida (Solanaceae), a South American plant which produces both alkaloids, were exposed to different elicitors (JA and AlCl₃) in order to increase their productivity and/or their release. It has been previously established that treatments with exogenous jasmonates can increase the production of several classes of alkaloids in a range of plant species (Gundlach et al, 1992) particularly tropane alkaloids in Datura stramonium (Zabetakis et al, 1999). Aluminium, a toxic soil metal, has been described as an elicitor that up-regulates genes involved in the defence of tolerant species (Hamel et al, 1998).

Establishment and maintenance of hairy root cultures. Hairy roots were obtained by infection of sterile explants of B. candida with Agrobacterium rhizogenes LBA 9402, according to the procedure described in Pitta-Alvarez and Giulietti (1995). The roots were maintained in hormone-free Gamborg (Gamborg et al, 1969) (B5) medium containing half -concentration of mineral salts and vitamins (B5_1/2) and supplemented with 15 g/l sucrose. The roots were subcultured in the medium described above every 15-20 days and incubated at 24° ± 2° C, in shakers at 100 rpm with a 16-hour photoperiod using cool white fluorescent lamps at a light intensity of approximately 1.8 wm^-2.

Assays with elicitors. Approximately 150 mg (fresh weight: FW) of hairy roots were inoculated in 20 ml of B5_1/2 medium supplemented with 15 g/l sucrose contained in 100 ml flasks. The cultures were incubated as described above.

JA was dissolved in EtOH and, after sterilising the solution by filtration, it was added in two final concentrations: 2.5 and 25 µg/ml. The AlCl₃ solution, after autoclaving at 120° C and 1atm during 20 minutes, was added in two final concentrations: 25 and 250 µM.

Hairy roots of B. candida in exponential phase (19 day-old cultures) were exposed to the elicitors for 24 and 48 hours. Growth (FW), scopolamine and hyoscyamine accumulation in the roots and release into the medium were determined.

Analytical methods. FW was determined by separating the root tissue from the medium by vacuum filtration. Alkaloid extraction from the roots and the media were carried out as described by Parr et al (1990). Hyoscyamine and scopolamine were analysed by HPLC, according to the method described by Mano et al (1986). The structures were confirmed by gas chromatography coupled with mass spectrometry, verifying also the absence of contaminants.

Statistical analysis. The treatments were carried out employing aleatory and factorial designs with three independent repetitions. Significance of treatment effects were determined using analysis of variance and comparison between the means of the determinations. Due to the heterogeneity of the variances, the variables were transformed using Taylor's method employing the SAS program (SAS Institute Inc., 1989) licensed by INTA-Argentina (National Institute of Agronomic Technology).

Chemicals. Scopolamine hydrobromide, L-hyoscyamine hemisulfate, (-) JA, AlCl₃, and all the media components were purchased from Sigma Chemical Co. (St. Louis, MO).

Effects of JA and AlCl₃ on scopolamine and hyoscyamine accumulation on hairy root cultures of B. candida.

After 24 hours of elicitor treatment, there was a reduction in scopolamine and hyoscyamine contents (Figure 1a). However, after exposing the cultures to the elicitors for 48 hours (Figure 1b), the aluminium salt, at both concentrations tested, stimulated the production of scopolamine and hyoscyamine (43-83%). JA, at the lowest concentration tested, promoted an increase in the content of scopolamine (~30%), whilst the content of both alkaloids decreased when JA was used at its highest concentration. Treatment with EtOH had a negative effect on the accumulation of scopolamine.

Effects of JA and AlCl₃ on the release of scopolamine and hyoscyamine in hairy root cultures of B. candida

Elicitation with JA and AlCl₃ showed a positive effect on release of both alkaloids (Figures 1c and 1d). The most important effect was induced by JA at the highest concentration and after 24 hours of exposure, which promoted the release of hyoscyamine (~1200%) (Figure 1c). EtOH, the solvent in which JA was dissolved, also increased the release of hyoscyamine, but in a minor proportion (Figure 1c). Furthermore, according to the level of JA tested, the alkaloid profile was inverted, with scopolamine being prevalent when the lowest concentration was employed (Figures 1c and 1d). After 48 hours, the aluminium salt increased the release of scopolamine (~150%) only at its highest concentration (Figure 1d).

Pitta-Alvarez (1998) and Pitta-Alvarez et al (2000) have reported that certain clones of hairy roots of B. candida are susceptible to elicitation, inducing the intracellular accumulation and release into the media of scopolamine and hyoscyamine. This is particularly notable during exponential phase and for periods ranging from 24 to 72 hours.

Secondary metabolism in plant cell cultures of different species could be elicited by exogenously supplied jasmonates in levels that varied from 0.1 to 100 m M (Gundlach et al, 1992, Blechert et al, 1995, Taguchi et al, 1998, Zabetakis et al, 1999). However, in our experiments, the positive effects on release with JA could be attributed in part to EtOH, although this particular interaction should be further examined.

Hamel et al (1998) found that most of the genes up-regulated by aluminium (in concentrations that ranged from 5 to 500 m M) shared homologies with pathogenesis-related ones, suggesting that aluminium may act as an elicitor. This mode of action could also be involved in the hairy root system we employed, since there was an increase in the accumulation of both alkaloids and the release of scopolamine.

Elicitors, which affect positively the release of secondary metabolites (as was observed in our research work), represent a valuable biotechnological strategy. For example, in large-scale fermentation designed to obtain molecules from plant cultures, this approach could simplify and reduce the costs of downstream processes, and at the same time allow biomass reutilization.

The authors would like to thank Marta Zanelli (PhD in Biometrics) for her help with the statistic analysis and Biochemist Carla C. Carrizo for her help with the extraction procedures and the HPLC runs.

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