Plant Physiol. (1 995) 108: 1553-1 560 zyxwvutsrq Selection for Hyoscyamine and Cinnamoyl Putrescine Overproduction in Cell and Root Cultures of zy Hyoscyam zyxw us m u ticus’ Fabricio Medina-Bolivar and Hector E. Flores* Graduate Program in Plant Physiology (F.M.-B.) and Department of Plant Pathology/Biotechnology lnstitute (H.E.F.), The Pennsylvania State University, 31 5 Wartik Laboratory, University Park, Pennsylvania 16802 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 suspen- sions by addition of 1 mg/L zyxwvutsrqp 2,4-dichlorophenoxyacetic acid to Murashige-Skoog medium zyxwvutsrq (T. Murashige and F. Skoog [1962] Physiol Plant 15: 473-497) and screened for resistance to the amino acid analog pfluorophenylalanine (PFP). Cells that could grow in media containing 400 PM PFP were selected and cloned from single cells. The resistant cells accumulated high levels zyxwvutsrqp of cinnamoyl putrescines, which share the same biosynthetic precursors as hyo- scyamine. Hairy root cultures were regenerated from both PFP- sensitive and PFP-resistant cells by removing 2,4-dichlorophenoxy- acetic 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 path- way upon the regeneration of root cultures. . The use of root cultures for physiological and biochem- ical studies, in particular hairy roots obtained by transfor- mation with Agrobacterium rhizogenes (Chilton et al., 1982; Willmitzer et al., 19821, has increased steadily in recent years (Signs and Flores, 1990). Hairy roots can express root-specific pathways and have shown stable production of alkaloids, polyacetylenes, sesquiterpenes, naphthoqui- nones, and other secondary metabolites (Signs and Flores, 1990).They have been used as a model system for the study of photosynthesis and photoautotrophy in roots (Flores et al., 1993).Hairy roots also respond to funga1 elicitors by de novo expression of phytoalexins and can transform xeno- biotics into bioactive metabolites (Flores and Curtis, 1992; Flores et al., 1994).In addition to producing low-molecular- weight secondary metabolites, hairy roots have been used ’ Supported by a grant from the National Science Foundation * Corresponding author; e-mail hector-flores@agcs.psu.edu; fax (BCS-09110288). 1-814-863-1357. to study the biosynthesis of root-specific defense-related proteins (Savary and Flores, 1994). Many hairy root cultures of solanaceous species have been established to date and have been shown to produce tropane alkaloids (Flores and Filner, 1985; Knopp et al., 1988).In some cases, alkaloid levels are higher in these root cultures than in the entire plant (Mano et al., 1986; Maldo- nado-Mendoza et al., 1993). We have reported that Hyoscy- zy amus muticus hairy roots constitutively produce hyoscya- mine (Flores and Filner, 1985), an anticholinergic agent used in the treatment of Parkinson’s disease and organo- phosphate poisoning, that causes smooth muscle relaxation (Cordell, 1981). Hyoscyamine is biosynthetically derived from putrescine and Phe (Fig. 1). Putrescine is the precur- sor for the tropine ring (Leete, 1979), and Phe is used in the biosynthesis of the aromatic moiety of the alkaloid. It has been previously accepted that hyoscyamine derives from the esterification of tropine with tropic acid. However, increasing evidence suggests that phenyllactic acid, but not tropic acid, is the intermediate in the biosynthesis of hyo- scyamine (Robins et al., 1994). The production of hyoscyamine is very tightly linked to the degree of morphological organization of the culture (Flores, 1987; Lindsey and Yeoman, 1983; Robins et al., 1991a). Hyoscyamine has been shown to accumulate only in organized systems, such as hairy roots, but is present only in trace amounts or is completely absent in undiffer- entiated systems such as callus cultures (Flores and Filner, 1985). Root cultures, however, have the disadvantage of not being readily amenable to selection pressures, such as can be applied to disperse cell-suspension cultures. We have shown that Hyoscyamus hairy roots can be converted into cell suspensions by growth in 2,4-D-containing me- dium. Conversely, these root-derived cell suspensions can readily be converted back to root cultures by withdrawal of the growth regulator (Flores, 1987).The ability to intercon- vert these two morphologically (and biochemically) dis- tinct phenotypes allowed for screening at the cell leve1 of genetic mutants or somaclonal variants, which may harbor a desired trait. The selected characteristic could then be expressed in root cultures regenerated from cell suspen- Abbreviations: MS, Murashige and Skoog salts; MSD, MS me- dium supplemented with l mg/L 2,4-D; PFP, p-fluorophenylala- nine. 1553 www.plant.org on December 20, 2015 - Published by www.plantphysiol.org Downloaded from Copyright © 1995 American Society of Plant Biologists. All rights reserved.