In vitro propagation of Withania somnifera and isolation of withanolides with immunosuppressive activity.

Withania somnifera plantlets were produced in vitro from the shoot-tip of aseptically germinated seedlings. Culture conditions were optimized using different plant growth regulators which gave rise to 120 shoots from a single bud. The plantlets were then transferred to pots and maintained in greenhouse for 4 months. 90% of these in vitro propagated plantlets survived and showed normal growth. Leaves from these plants were used for isolation of the withanolides. Methanolic extract of leaves from plantlets growing in tissue culture and those transferred to the greenhouse were evaluated for immunomodulatory activity. While the extract from greenhouse samples showed potent immunosuppressive activity, those from tissue cultures samples did not show any activity. Fractionation and characterization of withanolides, using HPLC, NMR, MS methods revealed the presence of withaferin A in the greenhouse samples. Our results indicate that Withania species may require longer time and better differentiation and also natural environment for the production of withaferin A.

Use of higher plants in the biomonitoring of environmental genotoxic pollution.

Genotoxicity is recognised as being the first step in carcinogenesis. Hence the identification ambient genotoxicity represents an important step in cancer risk assessment even if non-genotoxic mechanisms also occur. Genotoxicity can be assessed after exposure of populations to chemical or physical agents, as cytogenetic alterations, mutations or production of DNA/protein adducts. Well defined higher plants represent an excellent basis for cytogenetic evaluations after exposure to genotoxic pollutants, especially that the maturation of their gametes (meiosis) follows the same patterns as in animals and humans. We present a description of the Tradescantia Micronucleus Assay (TRAD-MCN) and results of a series of field evaluations after environmental pollution (urban settings, industrial sites, landfills). A significant correlation is observed between the intensity of the pollution and the ratio of micronuclei appearing at the tetrad stage of meiosis. The method is easy, requiring no special equipment, reproducible, rather inexpensive. It allows the establishment of "genotoxicity maps" and the follow-up monitoring of the polluted sites. In environmental monitoring, we consider the TRAD-MCN assay as the first-line procedure presenting the additional advantage of not involving human populations primary evaluations, thus avoiding psychological stress.