Toxic metal pollution of soils has become a major environmental problem. Datura stramonium can become an interesting crop for the extraction of toxic metals from polluted soils because even natural populations show a tolerance. Moreover they belong to the group of accumulators and are a source of tropan alkaloids with pharmaceutical value. In this study we simultaneously study the plant tolerance against toxic metals and the by toxic metals induced changes in the synthesis of secondary metabolites in suspension cultures of Datura stramonium L., because of their relatively rapid propagation of biomass, the relatively homogenous cell types in the culture system and the ability to easily manipulate culture conditions.

The suspension cultures wee obtained from undifferentiated calli cells. Callus cultures were initiated from young leaves and stems of sterile plant Datura stramonium L., using MS media modified with addition of kinetin 2.0 mg/l, 2,4-D 0.1 mg/l, IAA 0.2 mg/l, sucrose 3%, agar 0.9% (Media G48). Suspension were shacked continually (80 ppm), at 25^oC in the dark and subcultured at 15 days before harvest. The biomass was lyophilised before extraction. They have been grown on liquid medium with increasing concentration of toxic metal.

The growth characteristics of Cd treated suspension cultures (5 for each concentration) were no similar to that of the control. The fresh weights of suspension cultures with Cd was lower than weight of the control. Inhibition of growth by 10, 20, 30, 40, and 50 mmol CdCl₂ reached 92%, 53%, 33% and 14% respectively, after 15 days. Nearly full inhibition of biomass yield was observed at 50 mmol. The growth index (GI) of all the suspension cultures shows a decreasing tendency compared to GI of the control. Further investigations were conducted to examine the maximum admissible concentration (MAC). That is the concentration at which suspension cultures continue to develop. For the suspension cultures of Datura stramonium MAC of 46 mol was established and Cd-resistant cell lines was selected by a stepwise increase in Cd concentration. They are capable of growth and division in normally lethal concentration of Cd. Cells from the resistant cultures were converted to protoplasts by enzymatic digestion and individual protoplasts were utilised to initiate new suspension cultures. Thus eliminating the chance that a suspension culture was derived from a chimera, composed partially of resistant and partially of sensitive cells. The content of total alkaloids in suspension cultures (control) was 0,83% at day 15. The alkaloid production in medium with CdCl₂ varied from 0,50 to 0,92 %(HPLC).

Treatment of suspension cultures of D. stramonium with Cd²⁺ ions in low concentration induced synthesis of tropan alkaloids. The best induction was achieved at about 20 m mol. While production was rapid over the first 9 days in medium with 44 mol Cd, little or no further increment was found thereafter. We have demonstrated that, elicitation is however effective only by the low concentration of Cd. It has been establish in Datura stramonium cultures that elicitation of tropan alkaloids includes decrease of their composition and it seems probable that the seem mechanisms are active in D. stramonium hairy roots (1,2).

References

1. Ionkova I., Demeyer K., Witte L., and Alfermann A.W. (1998): Effect of Cadmium on the Formation of Tropan Alkaloids in Hairy Root Cultures of Datura stramonium L., 46 Annual Congress of Society for Medicinal Plant Research, Vienna, 31 Aug - 4 September Austria.
2. Ionkova I., Demeyer K., Zvetanova V., Ninov S. and Alfermann A.W. (1999): Induction of Tropane alkaloid formation as a result of Heavy Metal Toxicity: Dose-Response Relation in suspension and transformed root cultures of Datura stramonium L., treated with Cadmium, Second Pharmaceutical Congress of Republic of Macedonia, Struga, Oktober, Macedonia.