title: Coculture of genetically transformed roots and shoots for synthesis, translocation, and biotransformation of secondary metabolites.
abstract: Genetically transformed shooty teratomas of Atropa belladonna and a Duboisia leichhardtii x D. myoporoides hybrid were studied for biotransformation of tropane alkaloids in shake flasks and bioreactors. Although de novo synthesis of hyoscyamine and scopolamine was limited, shoots of both species were able to translocate and accumulate significant quantities of exogenous alkaloid. The maximum yield of scopolamine from hyoscyamine fed to the Duboisia hybrid shoots was 35% w/w; the yield of the scopolamine precursor, 6beta-hydroxyhyoscyamine, was 37% w/w. Biotransformation activity was poor in A. belladonna shooty teratomas provided with exogenous hyoscyamine; however, scopolamine levels comparable with those in leaves of the whole plant accumulated in shoots fed with hairy root extract. Coculture of A. belladonna shooty teratomas and hairy roots in the same hormone-free medium was investigated as a means of providing a continuous source of hyoscyamine for conversion to scopolamine. Of the biotransformation systems tested with A. belladonna, coculture produced the highest levels of scopolamine and the highest scopolamine: hyoscyamine ratios. Cocultured shoots accumulated up to 0.84 mg g(-1) dry weight scopolamine, or 3-11 times the average concentrations found in leaves of the whole plant. The scopolamine: hyoscyamine ratio in coculture ranged from 0.07 to 1.9, a significant improvement over levels of 0-0.03 normally found in A. belladonna hairy roots. Addition of Pluronic F-68 or copper sulfate to the medium and variation in initial medium pH did not improve hyoscyamine release from hairy roots. Scopolamine levels were increased using 1 microM copper sulfate or initial medium pH between 6.0 and 8.0; however, results from elicitation of hairy roots could not match the beneficial effect on scopolamine synthesis of root-shoot coculture. Addition of 0.001-1.0% (w/v) Pluronic F-68 to the roots reduced hyoscyamine release but postponed necrosis in the root tissue for up to 60 d.
authors.affiliation: Department of Biotechnology, University of New South Wales, Sydney, NSW 2052, Australia.
authors.forename: M A
authors.initials: MA
authors.lastname: Subroto
authors.forename: K H
authors.initials: KH
authors.lastname: Kwok
authors.forename: J D
authors.initials: JD
authors.lastname: Hamill
authors.forename: P M
authors.initials: PM
authors.lastname: Doran