Selection for Hyoscyamine and Cinnamoyl Putrescine Overproduction in Cell and Root Cultures of Hyoscyamus muticus.
Hairy root cultures of Hyoscyamus muticus have been shown to produce stable levels of tropane alkaloids comparable to those found in whole plants. In contrast, cell cultures of this and other solanaceous species produce only trace amounts of alkaloids but can be used for selection of metabolic variants. We have taken advantage of both systems and the ability to convert between them in vitro in an effort to select for increased production of the tropane alkaloid hyoscyamine. Hairy roots were converted into cell suspensions by addition of 1 mg/L 2,4-dichlorophenoxyacetic acid to Murashige-Skoog medium (T. Murashige and F. Skoog [1962] Physiol Plant 15: 473-497) and screened for resistance to the amino acid analog p-fluorophenylalanine (PFP). Cells that could grow in media containing 400 [mu]M PFP were selected and cloned from single cells. The resistant cells accumulated high levels of cinnamoyl putrescines, which share the same biosynthetic precursors as hyoscyamine. Hairy root cultures were regenerated from both PFP-sensitive and PFP-resistant cells by removing 2,4-dichlorophenoxyacetic acid from the medium. Resistance to PFP continued to be expressed in regenerated roots. Higher levels of hyoscyamine were found in hairy roots regenerated from PFP-resistant cells than were found in controls. We suggest that the precursors overproduced by the PFP-resistant cells can be diverted into the hyoscyamine pathway upon the regeneration of root cultures.