Duboisia (Solanaceae), indigenous to Australia and New Caledonia, comprises three species; D. myoporoides R. Br., D. leichhardtii F. Muell and D. hopwoodii F. Muell. D. myoporoides is a tree with broad-lanceolate to obovate glabrous leaves 7.5 to 10 cm long and may grow 13 m tall. The flowers, are small, white, and bell-shaped with occasional mauve streakings in the throat of the corolla. The fruits is a small black globular berry about 0.5 cm in diameter. D. leichhardtii is much like D. myoporoides in general habit, though not so leafy. Its leaves are smaller and narrow, and its flowers a little larger. D. hopwoodii is a small shrub seldom exceeding 2.5 m in height with narrow lanceolate leaves which are smaller than those of D. leichhardtii ().
Duboisia contains not only tropane alkaloids but also pyridine alkaloids; and this is the first plant found to contain both types of alkaloids. The main alkaloids in the leaves of D. myoporoides and D. leichhardtii are scopolamine and hyoscyamine, both of which are commercially important anesthetic and antispasmodic drugs. On the other hand, leaves of D. hopwoodii “pituri” used by Australian aborigines contain nornicotine and nicotine as predominant alkaloids. Therefore, the former two species have been major sources of tropane alkaloids for over 40 years instead of other source plants such as Scopolia, Atropa, Hyoscyamus, or Datura. Indeed, it is a fast-growing tree and harvest can be repeated from a tree by cutting branches off.
Clonal propagation of Duboisia, especially through meristem culture, may make it possible to propagate the plants with high alkaloid content. On the other hand, somaclonal variation of secondary metabolite production in the regenerated plants, particularly obtained through callus cultures, is expected to be useful as a breeding tool. Although micropropagation of medicinal plants has recently been studied extensively, reports on the production of secondary metabolites in regenerated plants have been relatively few so far in spite of the large number of publications on the micropropagation of medicinal plants. Before using regenerated plants as plant sources, it is required to know whether or not there is a difference between original plant and regenerated plants with respect to metabolite production.
This post deals with the difference between the plants regenerated from callus cultures of D. myoporoides and the intact plant with respect to alkaloid production. In addition, the site of biosynthesis and metabolism of tropane alkaloid, which has been recently studied by us using the cultured tissues of D. myoporoides is discussed.
Original Plant and Cultured Tissues
Atropine esterase activities in callus and differentiated organs were assayed and compared with those of the original plant.
Atropine esterase activity in roots of the original plant of D. myoporoides was much higher than that in the other organs as also found in Datura (). No atropine esterase activity was found in callus or leaves of differentiated shoots or of the original plant. On the other hand, both root from callus and from the original plant exhibited atropine esterase activity, although roots of the original plant had much higher activity than adventitious roots from callus. The reason why the atropine esterase activity in adventitious roots is extremely low compared with that in the roots of the mother plant is not clear yet, but may be due to differences of growth stages, cultural conditions, and physiological states. Low activities of atropine esterase were also detected in stems of the original plant and of the differentiated shoots.
Differentiated roots from both callus and the original plant contained tropane alkaloids and exhibited atropine esterase activity. Thus the root is the most important organ for both tropane alkaloid biosynthesis and degradation. Differentiated leaves from both callus and the original plant showed no atropine esterase activity. Differentiated leaves from callus contained no tropane alkaloid unless root formation occurred, but the leaves of the original plant showed a high content of the alkaloids. These results led us to the conclusion that in the original plant of D. myoporoides atropine is synthesized in root, then transferred to and accumulated in the leaves, where atropine is not hydrolyzed. Decomposition of atropine occurs mainly in the root. However, it is not clear whether atropine synthesized in the roots is hydrolyzed directly there or whether atropine accumulated in the leaves is transported back to the roots and then hydrolyzed there.
Atropine esterase activities in leaves, stems and roots of the 1-month-old regenerated plantlets were also determined to elucidate whether or not degradation of tropane alkaloid occurred in the leaves (unpublished). There was no atropine esterase activity either in the leaves of the regenerated plants, or of the original plant, or of shoots from the callus. Metabolites of tropane alkaloids, such as scopine and tropine, were not found in leaves of the regenerated plants of D, myoporoides as expected.
From these results it is clear that tropane alkaloids are not hydrolyzed at all in the leaf. Tropane alkaloids biosynthesized in the roots cannot possibly be transported to the aerial parts of the regenerated plants at younger stages of growth. As the regenerated plantlets continued to grow well after transplanting into pots, water and nutrients must be transported from root to aerial parts, except alkaloids. Regenerated plants in which tropane alkaloids are selectively transported to leaf and pyridine alkaloids are stayed in other parts would be very useful. This type was actually found in 6-month-old regenerated plants, but not in 20-month-old ones. If more stable mutants of such a kind could be obtained, they could be used for breeding. The mechanism of alkaloid transport should be clarified, so that transport of specific alkaloids only into the leaf of plants could be controlled.
One question still remains. Does decomposition of tropane alkaloids occur only through hydrolysis? There is a possibility that the metabolism occurs through other chemical pathways. Although the metabolites of hyoscyamine and scopolamine by demethylation or by dehydration are known in D. myoporoides (), we did not find any such metabolite in leaves of the regenerated plantlets.
Duboisia spp.: Conclusion
In vitro cultures and propagation of D. myoporoides has been established as follows: Callus tissue was obtained from stem explants of a mature tree and maintained best on MS medium supplemented with kin 0.01 mg/1 and 2,4-D 0.5 mg/1 (or NAA 10 mg/1) in the dark at 26±2°C. IBA 5 mg/1 in the dark was suitable for root formation and BA 5 mg/1 in the light (or under the dark/light cycle) was for shoot-bud formation from callus. Regenerated plantlets were established routinely by induction of rooting from the shoots obtained on MS medium containing BA 5 mg/1, when the shoots were cultured on MS medium containing IBA 5 mg/1 for 2 weeks followed by transferring onto MS medium without plant hormones. The plantlets were successfully transferred into soil.
Studies on alkaloid composition and atropine esterase activity in cultured tissues of D. myoporoides gave us basic understanding with regard to the alkaloid biosynthesis and degradation that the root plays an important role in both tropane alkaloid biosynthesis and degradation whereas the leaf does not. Furthermore, specific phenomena on alkaloid distribution in the regenerated plants were found; alkaloid accumulation in leaves of the regenerated plants did not occur at the early stages of development. From the results of determination of atropine esterase activity, transport of alkaloids from root to leaf does not occur in the young regenerated plants. Therefore, the regenerated plantlets are not suitable materials for alkaloid production; regenerated plants older than 6 months should be used. Concerning cultured tissues, root cultures may be hopeful as a potential material for alkaloid biosynthesis.
Selections from the book: “Medicinal and Aromatic Plants I”, 1988.