Selection for Hyoscyamine and Cinnamoyl Putrescine Overproduction in Cell and Root Cultures of Hyoscyamus muticus

Abstract

Hairy root cultures of Hyoscyamus muticus have been shown to produce stable levels of tropane alkaloids comparable to those found i n whole plants. I n 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 t o 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 t o Murashige-Skoog medium (T. Murashige and F. Skoog [1962] Physiol Plant 15: 473-497) and screened for resistance t o 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 of cinnamoyl putrescines, which share the same biosynthetic precursors as hyoscyamine. Hairy root cultures were regenerated from both PFPsensitive and PFP-resistant cells by removing 2,4-dichlorophenoxyacetic acid from the medium. Resistance t o PFP continued to be expressed in regenerated roots. Higher levels of hyoscyamine were found i n 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..

In vivo nuclear-magnetic-resonance analysis of polyamine and alkaloid metabolism in transformed root cultures of Datura stramonium L.: evidence for the involvement of putrescine in phytohormone-induced de-differentiation

“…Parr et al (1990) also found that the conjugated-putrescine content of D. stramonium increased rapidly in the ®rst few days after subculture and in their experiments the subsequent decline in the conjugated-polyamine pool was correlated with a steady increase in the hyoscyamine pool. Similarly, Medina-Bolivar and Flores (1995), working with Hyoscyamus muticus, found an inverse relationship between the formation of conjugated putrescine and the accumulation of hyoscyamine. While the NMR experiment shows no delay in the increase in hyoscyamine (Fig.…”

“…3) while the labelling of the hyoscyamine pool continued to increase provides further evidence that putrescine can be stored as putrescine conjugates prior to its utilisation in other pathways Medina-Bolivar and Flores 1995).…”

“…Under normal subculturing conditions, putrescine is apparently directed towards the synthesis of conjugates, such as caeoyl-and feruloyl-putrescine, and is then liberated and used as a substrate for alkaloid synthesis (Medina-Bolivar and Flores 1995;Parr et al 1990). In de-dierentiating roots, it is unlikely that putrescine released from the conjugates would be used for hyoscyamine biosynthesis because of the loss of the methylating capacity (Robins et al 1991c), and this again suggests that the plant growth regulator treatments could increase the availability of putrescine for polyamine synthesis.…”

Root-specific metabolism: The biology and biochemistry of underground organs

“…The ability to convert root cultures into disorganized cell suspension and to readily regenerate the organized phenotype has allowed for selection of root clones with desirable characteristics. Roots regenerated from Hyoscyamus muticus suspension cultures and selected for resistance to p-fluorophenylalanine (PFP), showed significantly higher and stable levels of hyoscyamine than the parental hairy roots, apparently resulting from the turnover of increased cinnamoyl putrescine pools (Medina-Bolivar and Flores, 1995). This suggests that it may be possible to overproduce root-specific metabolites by diversion of (2) fungal cell wall elicitor from P. cinnamoni; (3) chitosan (0.12% w/v) treatment; (4) jasmonic acid (100 mM ) treatment; (5) salicylic acid (800 mM ) treatment; (6) non-elicited control.…”

Root herbivory in vitro: Interactions between roots and aphids grown in aseptic coculture

“…A fungal elicitor from P. drechsleri mycelial cell walls increased the production of polyacetylenes in safflower and Bidens sulphureus hairy roots (our laboratory, unpublished data and Flores et al, 1988). Recent work in our laboratory showed that henbane (Hyoscyamus muticus L.) hairy roots accumulated more hyoscyamine under abiotic and biotic stresses as compared to controls (Medina-Bolivar and Flores, 1995;Halperin and Flores, 1997). On the basis of our previous work, we explored the possibility that hairy root culture may help in the study of root morphological, physical, and biochemical responses to insect herbivory.…”