M

Figure 5 Photographs of Duboisia tissue culture plants obtained (a) from nodal stem sections or (b) via callus formation and subsequent regeneration from leaf explants.

plant transport purposes, particularly where local quarantine regulations restrict the import of conventional plants. However, vegetative propagation of Duboisia clones using conventional cuttings is relatively straightforward and is done routinely to provide the plants needed for the ongoing planting program on Boehringer Ingelheim's farms. Hence, mass propagation of Duboisia clones in vitro is not a cost-effective alternative.

In collaboration with the University of Barcelona in Spain, we also examined the potential of Duboisia hairy root cultures for biotechnological production of scopolamine. Hairy roots were obtained after leaf disc infection with the Agrobacterium rhizogenes strain A4. Selected hairy root lines were able to produce up to 1.8 mg/L/day in a 4-L airlift bioreactor (42). This productivity of the bioreactor process using hairy roots is far too low to compete with the conventional farming approach. In addition, upscaling of the process to an 80-L bioreactor was found to be difficult. As a consequence, we did not pursue this approach further.

C. Agronomical Performance of Hairy Root-Derived Plants

As with other hairy root systems, Duboisia hairy root cultures may regenerate shoots spontaneously (see also earlier). Regenerated plants were transferred to soil and grown under greenhouse conditions. The plants displayed the typical symptoms of the hairy root syndrome to varying extents. We wanted to know whether these plants could outperform conventional Duboisia plants and conducted a field trial to analyze growth and scopolamine content of these plants (Fig. 6). Only the plants displaying the strongest hairy root syndrome symptoms had a significantly higher scopolamine content than control plants of the same clone. However, because of their stunted growth, overall productivity was strongly reduced compared with the control plants: the control plants produced almost twice as much scopolamine as the best hairy root plants (Table 2) (43). Thus, our data suggest that hairy root-derived plants do not represent a viable alternative to the cultivation of conventional plants.

D. Genetic Transformation

Genetic transformation of Duboisia so far has been reported only for Agrobacterium rhizogenes-mediated tranformation methodologies (44). As mentioned before, the disadvantage of this system is that regenerated plants typically display the symptoms of the hairy root syndrome. To supplement our conventional breeding program, we therefore started to develop genetic transformation protocols for Duboisia clones based on Agrobacterium tu-mefaciens—mediated transformation of leaf discs. The transformation effi-

Figure 6 Field performance of Duboisia plants regenerated from hairy roots, (a) Overview of field trial site. Hairy root-derived plants with minor hairy root syndrome symptoms can be seen in the foreground; plants with strong symptoms are located in the background, (b) Photograph of a hairy root-derived Duboisia plant with strong hairy root syndrome symptoms (43).

Figure 6 Field performance of Duboisia plants regenerated from hairy roots, (a) Overview of field trial site. Hairy root-derived plants with minor hairy root syndrome symptoms can be seen in the foreground; plants with strong symptoms are located in the background, (b) Photograph of a hairy root-derived Duboisia plant with strong hairy root syndrome symptoms (43).

ciency was evaluated using different A. tumefaciens strains in combination with a binary vector system harboring an intron-containing /3-glucuronidase (GUS) gene (45) and the neomycinphosphotransferase II (NPT1I) gene as a selectable marker. Using our shoot regeneration protocol for leaf discs following Agrobacterium transformation, we were able to obtain transgenic Duboisia plants constitutively expressing the GUS gene (Fig. 7). In contrast to the plants regenerated from hairy roots, no morphological difference could be detected between control plants and the plants obtained after A. tumefa-ciens-mediated transformation, as expected.

Of particular interest to the commercial application of genetic modification technology to Duboisia is the gene encoding hyoscyamine-6/3-hy-droxylase (H6H). Based on the published DNA sequence data (46), we cloned the gene from Hyoscyamus niger root culture RNA. Transgenic plants expressing the gene under the control of the cauliflower mosaic virus (CaMV) 35S promotor will be tested with respect to its effect on the levels of hyoscyamine, 6/3-hydroxy-hyoscyamine, and scopolamine. As Duboisia contains high amounts of scopolamine naturally, it will be interesting to see whether overexpression of the h6h gene will lead to a further improvement of the scopolamine content, similar to what has been observed in Atropa belladonna plants (36) and Hyoscyamus muticus hairy roots (85).

Table 2 Size and Scopolamine Content of Field-Tested Plants Regenerated from A. rhizogenes-Transformed Hairy Roots of Duboisia (43)

Average

Number of plants Average size Plant line3 tested (cm)

scopolamine Total content scopolamine (% DW) yield (g)

Control

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