Polyacetylenes in hairy root cultures of Trachelium caeruleum L

• • • W• • ALOF. Short Communication Pl.uPII,. ...., © 1998 by Gustav Fischer Verlag, lena Polyacetylenes in Hairy Root Cultures of Trachelium caeruleum L. 2 l 3 YOSHIE MURAKAMI , KOICHIRO SHIMOMURA , KUNITOSHI YOSHIHIRA , l KANJI ISHIMARU and 1 Department of Applied Biological Sciences, Faculty of Agriculture, Saga University, 1 Honjo, Saga 840, Japan 2 Tsukuba Medicinal Plant Research Station, National Institute of Health Sciences, 1 Hachimandai, Tsukuba, Ibaraki 305, Japan 3 Division of Applied Sciences, Graduate School of Integrated Science and Art, University of East Asia, 2-1 IchinomiyaGakuen, Shimonoseki 751, Japan Received April 17, 1997· Accepted June 30,1997 Summary Trachelium caerukum hairy roots were induced by the infection with Agrobacterium rhizogenes ATCC 15834. The hairy roots grew well in various hormone-free liquid media such as Murashige-Skoog (MS), ~ MS, Woody Plant (WP) and Gamborg B5, and produced a high amount of the polyacetylene mono-glucoside lobetyolin [maximum: 4.72 % as dry weight (dw), in WP medium]. The maximum level oflobetyolin content in the hairy root cultures was over 16 times higher than that of the intact plant (0.28 % as dw, in root portion). This study firsdy showed the identification of the secondary metabolites (polyacetylenes) of the genus Trachelium. Key words: Trachelium caerukum L., Campanulaceae, hairy root, polyacetylme, lobetyol, lobetyolin, lobetyo-linin. Abbreviations: MS = Murashige-Skoog; HPLC weight; dw =dry weight. Introduction The genus Trachelium, consisting of six to seven species, ranges mainly in the districts along the shore of the Mediterranean. Only one species, T. caerukum, is cultivated for horticultural purpose (decorative plant for cut flowers, sometimes for pot, veranda and garden). However, the secondary metabolites of the species have not been investigated. In this study, some tissue cultures (shoot, hairy root transformed by the infection with Agrobacterium rhizogenes ATCC 15834, etc.) of this plant were established and the secondary metabolism [production of polyacetylenes, i.e. lobetyol, lobetyolin and lobetyolinin (Fig. 1) (Ishimaru et al., 1991 and 1992)] in the tissues was determined. J Plant Physiol liIl 152. pp. 574-576 (1998) = high performance liquid chromatography; fw = fresh Material and Method Shoot and hairy root cullUm ofTrachelium caeruleum L. Seeds of Trachelium caeruleum L. were purchased in a market. The seeds were surface sterilized (in 2 % NaOCI with Tween 20, for 8 min) and germinated aseptically on lh Murashige-Skoog (MS) (Murashige and Skoog, 1962) solid medium containing 0.25 % gelrite under illumination (16 hiday light condition, 60 limol m -2 s-1, cool white fluorescent lamp: HITACHI FLR 40SW/M-G). The in vitro axenic plantlets, maintained for over a half year by subculturing at two-month intervals, were used for the explants for infection with Agrobacttrium. A. rhizogenes ATCC 15834 strain subcultured on YEB agar medium (Vervliet et al., 1975) was inoculated onto the cut ends of the stems (ca. 1cm length) prepared &om the axenic shoot cultures. About 20 days mer infection, several hairy roots Polyacetylenes in Trachelium caeruleum H H I I Me--C=C-C=C-C=C-CH-CH-C=C-CHzCHzCHzOH I I I I H H ~ ~ lobetyol : R=H lobetyolin : R=Glc lobetyolinin : R=GIe'·GIe Fig. 1: Structures of polyacetylenes. appeared at the inoculated sites in the light. The hairy roots were cut off and placed on hormone-free ~ MS solid medium containing the antibiotic Claforan (0.5 giL) and incubated for 1 week at 25 ·C in the dark to eliminate the bacteria. The axenic hairy roots obtained, which yield no bacterial colony when they were placc;d on YEB medium, were maintained in hormone-free MS liquid medium in the dark (100 rpm). The clone that showed best growth was selected and used for the experiment. The transformation of the hairy roots was proved by the detection of opine using paper electrophoresis (Petit et al., 1983). Hairy root cultures ofT. caerukum L. in four basal liquid media Hairy roots [ca. 200 mg, fresh weight (fw)] were inoculated into four hormone-free liquid media {MS, ~ MS, Woody Plant (WP) (Lloyd and McCown, 1980) and Gamborg B5 (B5) (Gamborg et al., 1968), 50 mUlOO mL Erlenmeyer flask} and cultured (100 rpm) in the dark. The hairy roots were harvested at 5 weeks of culture and both the growth [fresh weight (fw) and dry weight (dw)] and the polyacetylene contents were determined. HPLC analysis for polyacetyknes HPLC analysis for polyacetylenes was performed as described in Ishimaru et al. (1993). Time course experiment ofT. caerukum hairy root cultures in WP medium study, the induction of the transformed tissues (hairy roots) of T. caeruleum by the infection with A. rhizogenes ATCC 15834 and the determination of the polyacetylene metabolism were succesful for the first time. T. caeruleum hairy roots proliferated well in various hormone-free liquid media such as MS, ~ MS, WP and B 5 (Fig. 2). The maximum growth (fw: 5.50 glflask and dw: 292 mgl flask) of the cultures was observed in WP medium. The hairy root cultures also produced three polyacetylenes, i.e. lobetyol, lobetyolin and lobetyolinin, particularly, the predominant amount of the mono-glucoside constituent lobetyolin in all culture media tested. The maximum content (4.72 % as dw) of lobetyolin observed in the tissues cultured in WP medium was almost comparable to those in Lobelia species and was much higher than those in Campanula and Platycodon. The predominant production of lobetyolin was similar to the case in Campanula plantS. The much higher productivity of lobetyolin in T. caeruleum hairy roots compared with those of the in vitro shoot cultures (leaf: 0.35 %, root: 1.12 % as dw, on ~ MS solid medium) and in vivo plantlets (leaf: 0.03 %, root: 0.28 % as dw) also coincided with that observed in the other campanulaceous plants. Time course experiments on the growth and polyacetylene production of T. caeruleum hairy roots cultured in hormonefree WP liquid medium were performed. The root biomass, both fw and dw, increased almost linearly at all culture periods, showing the maximum level at week 8 (fw: 1.8 g/flask and dw: 94 mglflask). The hairy roots produced a high content of lobetyolin (1.65-3.63 %, as dw) and very low levels of both lobetyol and lobetyolinin (below 0.1 % as dw) at all periods of culture (Fig. 3). Although lobetyolin content in the hairy root cultures was fairly variable, the maximum level observed at week 5 was comparable to those of the hairy root cultures of Lobelia species. The maximum yield (2.2 mglflask) of lobetyolin in the cultures was observed at week 8 (data are not shown as a figure). In the near future, transgenic T. caeruleum having both a well-modified secondary metabolism and horticulturally val- Fresh hairy roots (ca. 100mg) were inoculated into hormone-free WP liquid medium (50 mUflask) and cultured in the dark (100 rpm). The hairy roots were harvested periodically (once a week, 1-8 weeks) and afrer measurement of the weights (fw and dw), the polyacetylene production was determined. Results and Discussion Agrobacterium rhizogenes-mediated transformation, which results in the induction of hairy roots from the infected plant tissues, has been available for various species of Campanulaceae. In recent research, several hairy root cultures of the genera Lobelia (Ishimaru et al., 1991, 1992, 1993, 1994, 1995; Tada et al., 1995 b; Yamanaka et al., 1996), Campanula (Tada et al., 1996; Tanaka et al., 1996 b), and Platycodon (Tada et al., 1995 a; Ahn et al., 1996) have been established, which have also been clarified to be peculiarly rich in polyacetylene glycosides, lobetyolin (mono-glucoside type) and sometimes lobetyolinin (di-glucoside type). These polyacetylenes are expected to be important marker products for the chemotaxonomic research in campanulaceous plants. In the present 575 (2.99 ± 0.20) [190±13.3] (5.50±0.01) [292±6.64] ( ): fw (gtOask) [ 1: dw (mgt ftask) 5.0 .~ ... ..c:: (4.62 ± 0.87) [272±30.0] 4.0 (3.34±0.04) [170± 1.70] ~ ~ tOO ...110 3.0 III Iobetyol • lobetyolin • lobetyolinin 2.0 ~ 1.0 0.0 MS 1/2MS WP BS medium Fig. 2: Growth (fw and dw) and polyacetylene content of Trachelium caerukum hairy roots cultured in various hormone-free liquid media (bars represent standard errors). 576 YOSHIE MURAKAMI, KOICHIRO SHIMOMURA, KUNITOSHI YOSHIHIRA, and KANJI ISHIMARU References ~ 4.0 i•• 3.0 •• GI ~ ~ 2.0 ." fI.I ell ~ 1.0 o.-l~"'I o 1 2 3 4 5 weeks 6 7 8 Fig. 3: Polyacerylene content in Trachtlium caeruleum hairy roots cultured in hormone-free WP liquid medium (bars represent standard errors). uable morphology might be obtained using the Agrobacterium-mediated gene transformation method. 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