Hiroshi Miura

Glucosylation of salicyl alcohol in cultured plant cells

Abstract Salicin and isosalicin were identified as a product of glucosylation of salicyl alcohol in Lithospermum erythrorhizon suspension cultures while in Datura innoxia suspension cultures only isosalicin was isolated as a glucosylation product. In suspension cultures from seven different plant species examined, Gardenia jasminoides and L. erythrorhizon cultured cells could produce salicin from salicyl alcohol while others produced predominantly isosalicin.

Anthocyanin production of Glehnia littoralis callus cultures

A stable callus line that produces anthocyanins was established from callus derived from a petiole of a Glehnia littoralis seedling and subcultured in the dark. The major anthocyanin which made up about 60% of the total anthocyanins was determined as cyanidin 3-O-(6-O-(6-O-(E)-feruloyl-beta-D-glucopyranosyl) -2-O-beta-D-xylopyranosyl-beta-D-glucopyranoside) by chemical and spectroscopic analyses. Anthocyanin contents in the cells cultured on B5 basal medium containing NAA (1 mg l-1), kinetin (0.01 mg l-1) and 3% sucrose reached 14% (dry wt basis) and the productivity has been sustained for 5 years.

Phloem Transport of Tropane and Pyridine Alkaloids in Duboisia myoporoides

Summary The phloem sap of Duboisia myoporoides R. B R . (Solanaceae) was collected from shoots treated with EDTA solution. The usefulness of the method was confirmed by using 14 C-Iabeled atropine. Analysis of phloem sap showed existence of alkaloids (scopolamine, atropine, nicotine and anabasine) and sucrose, indicating that tropane and pyridine alkaloids are transported through not only the xylem but also the phloem in D. myoporoides plants.

Conversion of Phenylalanine and Tropic Acid into Tropane Alkaloids by Duboisia leichhardtii Root Cultures

Summary 14C-labelled phenylalanine and tropic acid, which are possible precursors of the acid moiety of the tropane alkaloids, hyoscyamine and scopolamine, were fed to Duboisia leicbhardtii root cultures. 14C-phenylalanine was efficiently incorporated into tropane alkaloids and more than 50 % of the total radioactivity extractable from the tissues and the medium was recovered in the alkaloidal fraction after 1 week in culture. On the other hand, less than 1 % of 14C-tropic acid was converted to tropane alkaloids even after 3 weeks in culture. Formation of labelled hyoscyamine, scopolamine and apoatropine was confirmed by TLC-autoradiography and that of the former two was also confirmed by enzymatic hydrolysis. A change in the ratio of radioactivity of scopolamine to hyoscyamine over 3 weeks of culture was traced, indicating that the root cultures sustained the ability to convert hyoscyamine to scopolamine for at least 21 days, although biosynthesis of hyoscyamine in the roots stopped after the first 7 days in culture. A possible reason why little 14C-tropic acid was incorporated into tropane alkaloids is discussed.

Differences of atropine esterase activity between intact roots and cultured roots of various tropane alkaloid-producing plants

Abstract The tropane alkaloid distribution and atropine esterase activity in Atropa belladonna , Datura tatula , Duboisia leichhardtii , Hyoscyamus niger and Scopolia japonica were determined at various developmental stages and compared with those in their cultured roots. High atropine esterase activity was found in the roots of every tropane alkaloid- producing plant at any stage of development, though much higher activities were detected in the roots of annual and biennial herbs at the fruiting stage. In contrast to these findings, no cultured roots contained detectable amounts of atropine esterase activity in spite of their production of hyoscyamine and scopolamine.

Isolation and culture of protoplasts from cell suspension cultures of Duboisia myoporoides with subsequent plant regeneration

Protoplasts were isolated from suspension cultures of various cell lines of Duboisia myoporoides R. Br. There were differences among cell lines with respect to optimal conditions for protoplast isolation including the amount and kind of enzymes and the osmoticum concentration. Protoplasts isolated from one cell line were successfully cultured and induced to form cell colonies in liquid modified B5 medium. Addition of conditioned medium, coconut milk and glucose as an osmoticum to protoplast culture medium as well as maintenance of high protoplast density in culture (> 105/ml) were essential to obtain protocolony formation. Reduction of osmoticum concentration and deletion of coconut milk and conditioned medium from the culture medium were necessary to allow further colony development leading to cellus formation. Intact plants regenerated from calli derived from protoplasts were successfully transferred to pots.

Phenyllactic acid in Duboisia leichhardtii root cultures B feeding of phenyl[1-14C]alanine

Abstract Phenyl[1-14C]alanine which is a precursor of the acid moiety of tropane alkaloids and one of the possible intermediates—namely phenylpyruvic, phenyllactic and tropic acids—were simultaneously added to Duboisia leichhardtii root cultures in order to trap the labelled intermediates. When phenyllactic acid was added to 0.5–5 mm, incorporation of phenyl[1-14C]alanine into the organic acid fraction was much greater in comparison with the cases using phenylpyruvic acid, tropic acid or no intermediate at all. TLC-autoradiogram of the organic acid fraction obtained from phenyllactic acid feeding experiments showed the existence of a radioactive spot corresponding to phenyllactic acid. Trapping of labelled phenyllactic acid was confirmed by recrystallization, NMR and melting point.

The Characteristics of Atropine Transport in Seedlings and Regenerated Plantlets of Duboisia myoporoides

Summary [Carbonyl- 14 C]atropine sulfate solution was fed to regenerated plantlets or seedlings of Duboisia myoporoides at various stages of development via either the root or the leaves and the subsequent distribution of radioactivity in various organs compared. Four-week-old regenerated plantlets did not transport atropine from the roots to the leaves but did from the leaves to the roots, whereas the seedlings were able to transport in both directions at all ages examined. The regenerated plantlets gradually acquired an ability to transport atropine from the roots to the leaves during development and eventually, when they were 12 weeks old, they had a transport capacity comparable with that of the seedlings. Furthermore, it was found that regenerated shoots transported labelled atropine from the stem to the leaves at any stage of growth, and that 4-week-old regenerated plantlets transported radioactive NaH 2 12 PO 4 and Na 2 35 SO 4 from the root to the leaf, in contrast to their inability to transport atropine. These results indicate that the development of the root of regenerated plantlets of D. myoporoides was incomplete in relation to the transport mechanism specific to the alkaloid, atropine, and that there must be different mechanisms operating with respect to atropine transport from root to leaf or from leaf to root.

A new radiometric determination of l-canaline

Abstract A new radiometric determination of l -canaline is described in which [2-14C]acetone is employed as a reagent. The reagent is volatile while the reaction product of the reagent with l -canaline is not. Unreacted reagent is removed in vacuo; the residue radioactivity is proportional to the l -canaline content. The major advantages of this assay are specificity and sensitivity.

Change of Atropine Esterase Activity in the Regenerated Plants of Duboisia myoporoides during Development, and its Relation to Alkaloid Accumulation

Summary Atropine esterase activity in the regenerated plants of Duboisia myoporoides was determined during development and compared with that of seedlings. High esterase activity was found in the roots of seedlings throughout growing, whereas the activity in the roots of regenerated plants was extremely low at the early stages of development, though after regeneration when they were 6-months old the activity was recovered at the levels found in seedlings. The relationship between tropane alkaloid content in the leaves and atropine esterase activity in the roots of regenerated plantlets was examined. The results showed that extremely low activity in the roots seemed to be due to no or low accumulation of alkaloids in the leaves. Alkaloid dynamics in D. myoporoides were discussed.