India figures among developing countries that have the potential biotechnological competence to add value to the natural
resources, though its current share (1.6%) of the projected world market for medicinal plants and derivative products (US$ 5 trillion by the year 2010) is remarkably low. In an effort to develop appropriate technologies for bioproduction of the antineoplastic compound, plumbagin, hairy roots of Plumbago rosea L. were raised by infecting shoot cultures with Agrobacterium rhizogenes A4 strain and the bacteria-free roots (500mg. f.wt) established in 250ml agitated flask cultures, each flask containing 50ml Murashige-Skoog liquid medium devoid of growth regulators. The 12-16 fold increase in root biomass production and parallel synthesis of plumbagin with mean concentration of the compound (1.1% d.wt) through 25-day culture cycles indicated the stability of product synthesis and negligible differences between different hairy root clones. Cultivation of 6 gm fresh roots in 2.0 lit working volume in Infors (Labfors) airlift bioreactor through the same culture period consistently yielded 35-fold increase in root biomass without affecting plumbagin synthesis. An ointment made out of hairy root extracts in IP grade paraffin otherwise free from toxic principles was tested successfully against allergy induced skin eruptions (eczema, warts etc.). Additional tests are to be completed before a patentable product is developed. In another patented process using in vitro cultures, auxin mediated production of hypericin, a high value compound having antidepressant, anticancer and antiretroviral activities was achieved. Metabolic inhibitors (mevinolin, fosmidomycin and glyphosphate) at varying concentrations when fed to the shoot cultures did not inhibit synthesis of hypericin, thereby implicating mediation of polyketide synthase in the process. Attempts were made to isolate full length PKS gene(s) using RT-PCR followed by RACE analysis. 5' and 3'RLM-RACE analysis using gene specific primers designed to two RT-PCR products yielded 5'products of size range 650, 580 and 850 bp and 3' products of 750, 620 bp and 600bp. Sequence analysis of these RACE products yielded a full length cDNA having an open reading frame of 1173 bp encoding a 42 kDa protein having 390 amino acids. The deduced amino acid sequence of the of the PKS from Hypericum sp showed 85-99% identity with CHS-related enzymes of other plant species with highest similarity of 98-99% with Hypericum perforatum and H. androsaemum. Further, the catalytic triad (Cys 164, His 303 & Asp 336), CHS active site residues (Met 137, Gly 211, 216, Phe 215, 265 & Pro 375) and the conserved CHS residues that form the floor of the active site cavity (Thr 197, Gly 256, Ser 338 & Ile 254) are well conserved thereby indicating that the isolated gene corresponds to CHS. Isolation and characterization of other novel forms of PKS is in progress. The study reveals PKS mediated synthesis of hypericin in Hypericum sp. and the leads obtained hopefully result in eventual isolation and characterization of PKS gene(s) involved in the biosynthesis of hypericin.

Key words:plumbagin, hypericin.