The earliest examples of engineering an alkaloid pathway in a medicinal plant/ plant tissue culture come from the tropane alkaloid field. The commercial source of scopolamine is Duboisia, originally cultivated in Australia. A. belladonna accumulates hyoscyamine rather than scopolamine as the major alkaloid. The cDNA encoding h6h from H. niger was introduced into A. belladonna using either A. tumefaciens- or A. rhizogenes-mediated transformation. Elevated levels of scopolamine accumulated in the transgenic plants (up to 1.2% dry weight compared to trace levels in control plants) and 0.3% in hairy roots compared to 0.03% in control roots (Yun et al., 1992). Since these original experiments, the tobacco pmt has been expressed under transcriptional control of the dual CaMV 35S promoter in A. belladonna (Sato et al., 2001). Regardless of the transgene transcript level, the plants were phenotypically normal and had levels of hyoscyamine comparable to control plants. The alkaloid profiles of transgenic hairy root lines were also quantitatively and qualitatively similar to wild type. In contrast, N. sylvestris overexpressing the tobacco pmt showed increases in leaf nicotine of up to 40% compared to wild type (Sato et al., 2001). Cosuppressed plants accumulated only 2% of wild-type nicotine levels and contained elevated levels of putrescine and spermidine. Cosuppressed plants also showed phenotypic abnormalities.
The tobacco pmt under transcritional control of the CaMV 35S promoter has also been introduced into a scopolamine-rich Duboisia hybrid (Moyano et al., 2002). The N-methylputrescine levels of the resultant hairy roots increased twoto fourfold compared to wild type, but levels of tropane alkaloids were not increased. When the tobacco pmt gene was introduced into Datura metel and Hyoscyamus muticus, the resultant hairy roots showed improved hyoscyamine and scopolamine production in D. metel, but only hyoscyamine content increased in H. muticus (Moyano et al., 2003). A similar pmt-construct has been introduced into A. belladonna (Rothe et al., 2003). Transgenic plants and derived root cultures showed alkaloid profiles similar to those of controls. In summary, the expression level of pmt is apparently not rate limiting in all tropane alkaloid-producing species.
Efforts have also been made to establish particle bombardment-mediated transformation of H. muticus to facilitate future metabolic engineering of this species (Zeef et al., 2000). Importantly in these experiments, fertile, mature plants were recovered that could be self-pollinated.
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