Production of Tropane Alkaloids by Cell Cultures

For the isolation of atropine, Atropa belladonna. Datura species (e.g., D. stramonium and D. innoxia), and Hyoscyamus species are presently being used, which have atropine as the major compound. Hyoscyamus muticus is of interest for industrial-scale isolation, having an average alkaloid content of about 0.035-2.15% (365) in the leaves, with only small amounts of other alkaloids. Duboisia species, small trees whose leaves contain scopolamine as the major alkaloid during a certain period of seasonal development, are the main source for the industrial isolation of scopolamine. The leaves of Hyoscyamus niger and Datura stramonium were widely used in pharmacy, they contain, besides atropine as the main alkaloid, considerable amounts of scopolamine, the ratio of atropine to scopolamine being, respectively, about 2 1 and 1.2 1. They contain, respectively, 0.1-0.16% (365) and 0.02-0.08% total alkaloid. In Atropa belladonna, atropine is the m or alkaloid, with only small amounts of scopolamine the alkaloid content of the roots is about 0.3-0.7%, that of the leaves 0.2-0.6%. 

Differentiation into root-type tissues is apparently necessary for alkaloid production. Consequently the transformation of solanaceous plants with Agrobacterium rhizogenes, yielding so-called hairy roots which can easily be grown in vitro, was a logical step in efforts to produce tropane alkaloids in vitro 135,268,269,293,296,314-316,319,328,329, 338,347,348). In Table XIX results obtained with Agrobacterium rhi-zogenej-transformed hairy root cultures are summarized. 

Generally the Solanaceae cell and tissue cultures follow the alkaloid production pattern as found in the plant. For the production of atropine, Atropa belladonna. Datura stramonium, and Datura innoxia (see Tables XIV and XV, e.g., 291,292,296) cultures are particularly suited. For the production of scopolamine, Hyoscyamus species (see Table XVII, e.g., 291,292,296,339,365), Duboisia species (see Table XVI, e.g., 291,292, 296), Scopolia species (e.g., 52,296,348), Anisodus acutangulus (349), and some Datura species (see Table XV, e.g., 291,292,296,316) are of interest. 

The production in Hyoscyamus hairy roots was reported to be stable during at least 10 subcultures (337). Hairy root cultures of Scopolia japonic a were selected for high alkaloid production. Media were optimized for growth and alkaloid production. One cell line, SI, produced scopolamine as the major alkaloid (0.5% of DW) the productivity was approximately 17 mg/liter in 4 weeks. Cell line S22 had atropine as the major alkaloid (1.3% of DW), from which a productivity of about 117 mg/liter in 4 weeks can be calculated (347,348). A similar productivity for atropine was found in Datura stramonium hairy roots (50-100 mg/liter in 4 weeks) (314,317). The Datura roots excreted about 5% of the atropine into the medium in the stationary phase this increases to about 30% with a concomitant decrease in total alkaloids (317). Datura wrightii was found to produce about 180 mg/liter of atropine, a production which was reached after only 15 days, which is considerably faster than the 45 days after which maximum alkaloid production is achieved in D. stramonium (317). 

Alkaloid Content of Agrobacterium rhizogenes-TnANSFORMED Hairy Roots Cultures 

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