Search

Five phenylethanoid glycosides (acteoside, isoacteoside, beta-oxoacteoside, beta-hydroxyacteoside, and salidroside) were isolated from a cell suspension culture of Olea europaea. We examined the biosynthesis of acteoside in olive cell cultures by using feeding experiments with stable isotope labeled precursors. The hydroxytyrosol moiety of acteoside is biosynthesized from tyrosine through dopamine, whereas the caffeoyl moiety of acteoside is biosynthesized from phenylalanine via a cinnamate pathway. Dopamine is incorporated into acteoside through oxidation to the corresponding aldehyde, reduction to the alcohol, and then beta-glycosylation.

We describe a 3D modeling method from multi-viewpoint images of a dynamic human body and a realtime rendering system of dynamic 3D models.This 3D modeling method reconstructs an accurate and stable 3D shape of a human body using stereo matching method supported by the regional surface feature of the visual hull.To display the generated 3D model in real-time from arbitrary viewpoints,we developed a real-time rendering system that uses aview-dependent polygon texture mapping method.Using the proposed modeling method and the rendering system,we experimentally rendered 3D models of a Noh performer and these experiments confirmed that the system was able to display 3D models in real-time from arbitrary viewpoints.Finally,comparisons with the results of other methods demonstrated the effectiveness of the proposed 3D modeling method and the rendering system.

Olive (Olea europaea) contains large quantity of triterpene acids including oleanolic acid (6) as a major one. Varieties of biological activities exhibited by triterpene acids attracted our attentions, especially from pharmaceutical viewpoints. Cell culture of olive plant was induced and its triterpene constituents were studied. From the cell suspension cultures, six ursane type triterpene acids; ursolic acid (9), pomolic acid (10), rotundic acid (11), tormentic acid (12), 2alpha-hydroxyursolic acid (13) and 19alpha-hydroxyasiatic acid (14), and two oleanane type acids; oleanolic acid and maslinic acid (7), have been isolated. Quantity of ursane type triterpene acids produced by cell cultures was larger than that of oleanane type. Further, a multifunctional oxidosqualene cyclase (OSC) named OEA was cloned by homology based PCRs from the same cultured cells. Major product of OEA is alpha-amyrin (ursane skeleton), showing good accordance to higher content of ursane-type triterpene acids in the cultured cells, and strongly suggesting OEA to be a major contributor OSC for their production.

Three lines of hairy root culture of Ambrosia maritima induced by Agrobacterium rhizogenes ATCC15834 were established. Thiarubrine A, thiarubrine A epoxide, thiarubrine A diol and their precursor pentayneene were produced by the hairy roots after elicitation with methyl jasmonate, the common signal molecule in the plant defense and development. Thiarubrine A diol was the main form detected in the medium. Maximum yield was achieved when the 13-day-old hairy root cultures were exposed to 40 μM methyl jasmonate for 72 h. Callus and cell suspension cultures were established and maintained on Murashige and Skoog medium supplied with α-naphthylacetic acid (NAA) and kinetin. When the cell suspension cultures were elicited with methyl jasmonate, pentayneene was the only polyacetylene produced. The yield of pentayneene in hairy root cultures was much higher (9.6 times) than that of cell suspension cultures.

The Eucalyptus perriniana cultured cells are widely used to biotransform a variety of compounds. The glucosyltransferase activity of a crude protein extract of E. perriniana cultured cells was maximized when cell growth was in the pre-logarithmic to logarithmic phase. We cloned a cDNA encoding glucosyltransferase (EPGT) from E. perriniana cultured cells by RT-PCR using a degenerated primer and RACE-PCR. The cDNA contained an open reading frame encoding 467 amino acids with a calculated molecular mass of 51.6 kDa. The consensus sequence of the plant glucosyltransferases was included in the deduced amino acid sequence. The amino acid sequence of EPGT showed a high identity to glucosyltransferases from tobacco and petunia. The recombinant EPGT was expressed in Escherichia coli and its substrate specificity was examined using UDP-(U- 14 C) glucose. Cinnamic acid was the best sugar acceptor in the compounds tested.

Labelling experiments with [1-13C] glucose, [1-13C] sodium acetate and [1,2-13C2] sodium acetate proved that the fundamental isopentenyl diphosphate (IPP) unit, constituting GGPP in the abietane diterpenoids, were biosynthesized via the GAP/pyruvate pathway, as well as the acetate/mevalonate pathway in the cultured cells of Torreya nucifera var. radicans. In addition, the terminal IPP showed the preferential labelling from the GAP/pyruvate pathway and unfavorable labelling from the acetate/mevalonate pathway, suggesting that the biosynthetic route leading to the terminal IPP involved the GAP/pyruvate pathway more and the acetate/mevalonate pathway less, compared with the FPP part. Furthermore, it was also found that the utilization of the two pathways in the formation of the FPP part and the terminal IPP was dependent on the exogenous precursors through the simultaneous administration of [1-13C] sodium acetate and unlabelled glucose.

The new isolated compounds, namely 1-hydroxydihydropanaxacol (I) and 17-hydroxypanaxacol (II), exhibit antimicrobial activity against S.

Two new polyacetylenes, 1-hydroxydihydropanaxacol (3) and 17-hydroxypanaxacol (4), were isolated from Panax ginseng hairy root culture, along with dihydropanaxacol (1), panaxacol (2) and ginsenoyne D (5). Highly hydroxylated compounds 1-4 were isolated from the medium and compound 5, which was a biosynthetic precursor of compound 1, was isolated from the roots. Compounds 1-4 showed antimicrobial activity against Staphylococcus aureus, Bacillus subtilis, Cryptococcus neoformans and Aspergillus fumigatus. It is suggested that P. ginseng plants release antimicrobial polyacetylenes into the surrounding soil from the roots as defense compounds.

Seven new biotransformation products, (1 R ,3 Z ,5 R , 8 S, 9 S)-caryophylla-3-ene-5,14-diol 14- O -β-gentiobioside, (1 R ,5 R ,8 S , 9 S)-caryophylla-4(15)-ene-5,14-diol 14- O -β-gentiobioside, (1 R ,4 R ,5 R ,8S,9S)-4,5-epoxycaryophyllane-14-ol 14- O -β-gentiobioside, (1 R ,4 R ,5 R ,8 R , 9 S)-4,5-epoxycaryophyllane-14-ol 14- O -β- d -glucopyranoside, (1 R ,4 R ,5 R ,8 S ,9 S ,11 R )-4,5-epoxycaryophyllane-12, 14-diol 12- O -β- d -glucopyranoside, (1 R ,4 S ,5 R ,8 S ,9 S )-4,8-epoxy- caryophyllane-5,14-diol 14- O -β- d -glucopyranoside and (1 R ,4 R ,5 R ,8 S ,9 S )-4,11,11-trimethyltricyclo[6.3.1.0 1,9 ] dodecane-5,8-diol 5- O -β- d -glucopyranoside, were isolated from a cell suspension culture of Eucalyptus perriniana following administration of caryophyllene oxide.

Four new biotransformation products, (1 R , 2 R , 4 S )-1,8-epoxy- p -menthan-2-yl O -β- d -glucopyranoside, (1 S , 3 R , 4 R )- and (1 R , 3 S , 4 S )-1,8-epoxy- p -menthan-3-yl O -β- d -glucopyranosides, and (1 S , 2 S , 4 R )-1,8-epoxy- p -menthan-2-yl O -β- d -glucopyranosyl-(1→6)-β- d -glucopyranoside, together with a known (1 S , 2 S , 4 R )-1,8-epoxy- p -menthan-2-yl O -β- d -glucopyranoside were isolated from a cell suspension culture of Eucalyptus perriniana following administration of 1,8-cineole.

Activity-guided fractionations of leaf extracts from Laurus nobilis L. led to the isolation of a known sesquiterpene lactone, deacetyl laurenobiolide (1). Compound 1 showed antimicrobial activity against periopathic pathogens (Actinomyces viscosus, Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans), opportunistic Gram-positive bacteria (Staphylococcus aureus and Streptococcus pyogenes) and fungi (Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus). Furthermore, acetylation and cyclisation of deacetyl laurenobiolide (1) yielded laurenobiolide (2) and a new compound, (5S,6R,7S,8S,10R)-6,8-dihydroxyeudesma-4(15),11(13)-dien-12-oic acid 12,8-lactone (3), respectively. Compounds 2 and 3 also showed antimicrobial activities. All compounds 1-3 demonstrated growth inhibitory effects with minimum inhibitory concentrations ranging from 31 to 1000 µg mL(-1). This is the first report of compounds 1-3 showing antimicrobial activities.

Cinobufacini (huachansu), an aqueous extract from the skin of the toad Bufo bufo gargarizans Cantor, is a traditional Chinese medicinal preparation widely used in clinical cancer therapy in China. Here, we screened and identified active compounds of cinobufacini and investigated their apoptosis-inducing effect on HepG2 cells. Screening was performed using bioassay-guided isolation. The effects of different fractions on the proliferation of HepG2 cells were detected by the MTT assay. The extraction and isolation of active fractions were performed by chloroform extraction, silica column chromatography, preparative thin-layer chromatography and high-performance liquid chromatography. Nuclear magnetic resonance (NMR) imaging and electron ionization-mass spectrometry (EI-MS) were used to identify the structure of the active compounds. The extent of cell apoptosis was detected by Hoechst 33258 staining and flow cytometric analysis. Western blot analysis was used to detect the expression of the apoptosis-related proteins Bax and Bcl-2. Through bioassay-guided isolation, two compounds were isolated from cinobufacini. NMR and EI-MS data revealed these compounds to be resibufogenin and cinobufagin. Cinobufagin was determined to be the more efficient of the two in inhibiting the proliferation of HepG2 cells. Hoechst 33258 staining and flow cytometric analysis indicated that cinobufagin induced marked changes in apoptotic morphology and significantly increased the proportion of apoptotic cells in HepG2 cells. Western blot analysis showed that cinobufagin up-regulated Bax expression and down-regulated Bcl-2 expression. In conclusion, we screened and identified two anti-proliferation compounds of cinibufacini, resibufogenin and cinobufagin. The most effective compound, cinobufagin, inhibited cell proliferation by inducing the apoptosis of HepG2 cells. This was potentially triggered by regulation of the Bax/ Bcl-2 ratio.

Six new biotransformation products, (-)-borneol 2-O-beta-gentiobioside, (-)-borneol 2-O-beta-sophoroside, (-)-borneol 2',6'-di-O-(beta-D-glucopyranosyl)-beta-D-glucopyranoside, 6-exo-hydroxyborneol 2-O-beta-D-glucopyranoside, 4-hydroxyborneol 2-O-beta-D-glucopyranoside and 5-exo-hydroxyborneol 2-O-beta-gentiobioside, together with 5-exo-hydroxyborneol 2-O-beta-D-glucopyranoside, were isolated from jar fermentor cultures of Eucalyptus perriniana following administration of (-)-borneol.

Publisher Summary The olive (Olea europaea L.) is a well-known evergreen tree, native to the Mediterranean coast, of which the fruit and oil are used for food and cooking. Olives contain numerous triterpene acids including oleanolic acid (1) as the major one. Recently, much attention has been paid to triterpene acids from the pharmaceutical viewpoint because of their anti-HIV, anti-inflammatory, and antitumor-promoting activities. The wax of the olive fruit contains predominantly oleanolic acid, whereas that of the leaves contains a mixture of oleanolic acid and betulinic acid in a ratio of 7:2. In addition to these triterpene acids, triterpene diols, such as erythrodiol (oleanane type) and uvaol (ursane type) are also present, though in smaller quantities than the corresponding acids. Triterpenes are concentrated mainly in the skin of the fruit. Their content in pomace olive oil, which is an olive residue, is about 10-fold higher than in other types of olive oil. This chapter summarizes the key features of triterpene acids. It describes the induction of cell cultures, production of triterpene metabolites, and cDNA cloning of oxidosqualene cyclases (OSCs) from O. europaea cell cultures.

A simple and new basket-shaped unit for agitation made of stainless steel (EGSTAR), in which immobilized coffee cells in Ca-alginate gel beads were packed, was placed in a jar fermentor (System-1). This system allowed the plant cells to grow submersed in the unit even at high agitation speed (650 rpm). Only a small amount of cells existed out of the EGSTAR. Most of the purine alkaloids produced were released into the medium. Suspended coffee cells in the jar fermentor were also possibly immobilized onto a polyurethane foam sheet fixed inside the net of the EGSTAR (System-2). The total cells in System-2 biotransformed theobromine to caffeine (77.9%). Other plant cell suspensions were also immobilized as efficiently as were the coffee cells in this system. Thus, System-2 is a simple and convenient system for immobilization of plant cells to produce secondary metabolites.

A new biotransformation product, steviol 19-β-gentiobiosyl ester, together with steviol 19-β-glucopyranosyl ester and steviol-13- O -β-glucopyranoside 19-β-glucopyranosyl ester (rubusoside), was isolated from Eucalyptus perriniana jar fermentor culture following the administration of steviol. Only rubusoside was isolated as a biotransforination product of steviol from Coffea arabica cell suspension culture.

Two new biotransformation products, 28-hydroxy-18β-glycyrrhetinic acid 30-β- glucopyranosyl ester and 23,28-dihydroxy-18β-glycyrrhetinic acid 30-β-glucopyranosyl ester, together with 18β-glycyrrhetinic acid 30-β-glucopyranosyl ester were isolated from Eucalyptus perriniana cell suspension culture following administration of 18β- glycyrrhetinic acid. Only 18β-glycyrrhetinic acid 30-β-glucopyranosyl ester was isolated as a biotransformation product of 18β-glycyrrhetinic acid from Coffea arabica cell suspension culture.

Camellia sinensis cultured cells can produce purine alkaloids, caffeine and theobromine, but the productivity is lower than that of cultured coffee cells. Total amino acid content in C. sinensis cultured cells was higher than that in commercial green tea. The main amino acid in cultured cells was glutamine (40–70% total) and the presence of theanine was also confirmed. α-Spinasterol, α-spinasterol β- d -glucopyranoside and 22-dihydro-α-spinasterol β- d -glucopyranoside were also isolated from an ethyl acetate extract of C. sinensis cultured cells. A 1 -Barrigenol was isolated from the acid hydrolysate of the saponin fraction.

Ganciclovir, foscarnet, vidarabine and ribavirin, which are used to treat viral infections in humans, inhibited the proliferation of a baculovirus (Bombyx mori nucleopolyhedrovirus) in BmN4 cells, a cultured silkworm cell line. These antiviral agents inhibited the proliferation of baculovirus in silkworm body fluid and had therapeutic effects. Using the silkworm infection model, the antiviral activity of Kampo medicines was screened and it was found that cinnamon bark, a component of the traditional Japanese medicine Mao-to, had a therapeutic effect. Based on the therapeutic activity, the antiviral substance was purified. Nuclear magnetic resonance analysis of the purified fraction revealed that the antiviral activity was due to cinnzeylanine, which has previously been isolated from Cinnamomum zeylanicum. Cinnzeylanine inhibits the proliferation of herpes simplex virus type 1 in Vero cells. These results suggest that the silkworm–baculovirus infection model is useful for screening antiviral agents that are effective for treating humans infected with DNA viruses.

Thiophene A and thiophene A diol are the major polyacetylenes isolated from the hairy roots of AMBROSIA MARITIMA (Asteraceae) cultured under continuous light irradiation. The biosynthesis of thiophene A was studied using [1 - 13 C]-, [2 - 13 C]-, [1,2 - 13 C]-acetates. The biosynthesis of thiarubrine A, produced by hairy roots cultured in the dark, was studied using [1,2 - 13 C]-acetates and [18 - 13 C]-linoleic acid. Our results suggest a catabolic pathway for polyacetylenes biosynthesis from linoleic acid in hairy roots of AMBROSIA MARITIMA. DEPT:distortionless enhancement of polarization transfer HMBC:heteronuclear multiple correlation MeJ:methyl jasmonate MS:Murashige and Skoog medium

In this paper, we present a high-resolution dynamic 3D object generating method from multi-viewpoint images. This dynamic 3D object can display fine images of the moving human body form arbitrary viewpoints, and consists of subject's 3D model generated for each video frame. To create a high-resolution dynamic 3D object, we propose a 3D-model-generation method from multi-viewpoint images. The method uses stereo matching to refine an approximate 3D model obtained by the volume intersection method. Furthermore, to reproduce high-resolution textures, we have developed a new technique which obtains the visibility of vertices and polygons of 3D models. A modeling experiment performed with 19 fire-wire cameras confirmed that the proposed method effectively generates high-resolution dynamic 3D objects.

Three abietane diterpenoids were isolated from the suspension cultured cells of Torreya nucifera var. radicans along with four known abietane diterpenoids. Based on spectroscopic evidence, the structures of the three were elucidated as (3S,5R,10S)-7-oxo-12-methoxyabieta-8,11,13-triene-3,11-diol, (3S,5R,10S)-7-oxo-12-methoxyabieta-8,11,13-triene-3,11,14-triol and (5R,10S)-3-oxo-7R,12-dimethoxyabieta-8,11,13-trien-11-ol, respectively.

Cultured cells of Eucalyptus perriniana biotransformed (+)-menthol to its gentiobioside and triglucoside [2,6-di-O-(beta-D-glucopyranosyl)-beta-D-glucopyranoside]. The structures of these compounds were determined by means of NMR techniques.

Seven new biotransformation products were isolated from a jar fermentor culture of Eucalyptus perriniana following administration of (+)-camphor. The main product was (1S,4R,6S)-6-hydroxybornan-2-one 6-O-beta-D-glucopyranoside. The minor components were (1S,4R,6R)-6-hydroxybornan-2-one 6-O-beta-D-glucopyranoside, (1R,4S,6S)-6-hydroxybornan-2-one 6-O-beta-D-glucopyranoside, (1R,4R,5R)-5-hydroxybornan-2-one 5-O-beta-D-glucopyranoside, (1R,3R,4S)-3-hydroxybornan-2-one 3-O-beta-D-glucopyranoside, (1R,4R,7R)-8-hydroxybornan-2-one 8-O-beta-D-glucopyranoside and 2-(4-oxo-2,2,3-trimethylcyclopentyl)-ethyl-beta-D-glucopyranoside. All products were mono-glucosides and the oxygen function was introduced before glucosylation. Reduction of the ketone group of camphor was not observed.

Plant cells may be capable of stably supplying their secondary metabolites for commercial use when they are cultured on a large scale. Immobilization enables the re-use of plant cells which grow very slowly and which are sensitive to shear forces. In particular, adhesion of plant cells on reticulate polyurethane foam is one of the inexpensive and efficient methods for immobilization [1]. This method does not involve reagents and complicated operations which cause damage to cells and contamination with microbes. Moreover, foam particles could immobilize more cells than gel matrices.

The pore size and shape of porous matrices were evaluated as to their effect on the immobilization efficiency in cultured coffee (Coffea arabica L.)/cells. A hydrophilic porous matrix (13–20 pores/25 mm) and reticulate polyurethane foam (30 pores/25 mm) indicated more efficient immobilization than the others, in small cubes (1 cm3 × 9) and a strip (1 × 1 × 9 cm3) at the end of the fourth subculture. Among the large cubes (9 cm3), the reticulate one with the largest pore size (13 pores/25 mm) was the most advantageous for immobilization. In the strip-shaped matrices (1 × 1 × 9 cm3), immobilization was the most efficient in spite of its lower surface area as compared to the small cubes, except for those with the largest pore size. The strip-shaped foams, which were fixed on the inside of the flask against shaking, were effective for immobilization. Finally, strips (30 pores/25 mm) with slits to increase the surface area of the foam immobilized the largest amount of cells at the end of the fourth subculture. Caffeine production was not changed by diffenences in pore size.

Biotransformations using isolated enzymes and miro-organisms have been applied to the field of organic synthesis [1]. Biotransformations using plant cultured cells, in particular glycosylation reactions, have been the subject of increasing attention [2–4]. We have already reported that the cultured cells of Eucalyptus perriniana can biotransform monoterpenes, (-)- menthol [5], (+)-menthol [6] and (-)-borneol [7], diterpene, steviol [8], and triterpene, 18β-glycyrrhetinic acid [9], to their hydroxylation, oxidation, reduction and glucosylation products. For example, from the experiments of (-)-menthol and (-)-borneol various biotransformation products (10 and 8, respectively) were obtained. On the point of view of the industrial application, the biotransformation that resulted a useful product (not complicated mixture) would be preferred. In this paper, the effects of bioregulators, glucose and substrate feeding methods on the biotransformation of (-)-borneol by cultured cells of E. perriniana are reported.

Coffee (Coffea arabica L.) cells could be immobilized in polyurethane foam and subcultured repeatedly for a long time. Four phases were observed for cell growth and caffeine production, I; immobilization, II; growth, III; caffeine production, IV; regrowth. Their periods were influenced by the number of foam particles. Especially in the phase III, the immobilized cells produced a relatively large amount of caffeine in the subculture numbers 5-8 (34 cubes) when the fresh weight of the immobilized cells decreased despite culture in growth medium (DK medium). Caffeine production appeared to have a negative correlation with the growth of the immobilized cells throughout the subcultures.

Coffee (Coffea arabica L.) cells are capable of biotransforming theobromine to caffeine. In suspension culture of B2K medium, which is the “production medium” for caffeine, biotransformation was also more efficient than in DK medium. More caffeine was finally produced than calculated based on theobromine added to the medium. On the other hand, the efficiency of the biotransformation using immobilized cells in reticulate polyurethane foam cubes as a matrix varied with the phases. The biotransformation tended to be efficient under conditions which allowed the coffee cells to vigorously produce caffeine de novo.

Thiarubrine A, thiarubrine A epoxide, thiarubrine A diol, and their precursor pentayneene were produced by hairy roots of Ambrosia maritima. The molluscicidal activity of the lipophilic extract of the hairy roots and isolated polyacetylenes were investigated. The activity was studied against Schistosome intermediate host Biomphalaria glabrata. Thiarubrine A diol, the main form detected in the medium, showed the highest activity with LC50 = 0.37 ppm. This activity is 23 times higher than that of ambrosin, a sesquiterpene lactone that has long been regarded as the main molluscicidal agent of the plant. Thiarubrine A diol can be an environmentally accepted molluscicidal agent in areas endemic with schistosomiasis.

コーヒーノキ (Coffea arabica L.) 培養細胞は, プリンアルカロイドであるカフェインおよびテオブロミンを生産し, その70～90%を培地中に放出した. カフェイン生産量はオーキシンの種類によって影響を受け, 3-indole butyric acid; 2mg/l kinetin; 0.1mg/lを含むMS基本培地 (B2K培地) において最大であり, 全体で43.1mg/flask, 培地中の濃度は131mg/lであった. アルギン酸カルシウムゲルで包括固定化した細胞は4代目まで培養可能であり, この場合は成長培地として用いた2,4-D; 1mg/l, kinetin; 0.1mg/lを含むMS基本培地 (DK培地) で4代目に最も高い生産量を示した.

Ten biotransformation products have been isolated from Eucalyptus perriniana cultured cells administered (–)-menthol. The main product is (–)-menthol 3-O-β-D-gentiobioside (1) and the minor products are (–)-menthol 3-O-β-D-cellobioside (2), (+)-neomenthol 3-O-β-D-gentiobioside (3), (1R,3R,4R)-p-menthane-3,8-diol 3-O-β-D-glucoside (4) and 8-O-β-D-glucoside (5), (1R,3R,4R)-p-menthane-3,8-diol 3-O-β-D-gentiobioside (6) and 8-O-β-D-gentobioside (7), (1S,3R,4S)-p-menthane-1,3-diol 3-O-β-D-glucoside (8), (1R,2S,3S,4S)-p-menthane-2,3-diol 3-O-β-D-gentiobioside (9), and 2-O-β-D-gentiobioside (10).

Suspension cultures of Coffea arabica are able to convert phenylacetic acid and 2-( R , S )-phenylpropionic acid and its ethyl ester into their sucrose esters, β- d -fructofuranosyl 6- O -phenylacetyl-α- d -glucopyranoside and β- d -fructofuranosyl 6- O -[2-( R , S )-phenylpropionyl]-α- d -glucopyranoside, which are accumulated in the cells. The diastereomeric mixture of the sucrose ester of 2-( R , S )-phenylpropionic acid (R: S::1:1) was resolved by HPLC.

Eight triterpenes were isolated from the methanol extracts of Eucalyptus perriniana cultured cells as their methyl esters. The structures were elucidated by spectral data to be oleanolic acid, ursolic acid, maslinic acid, 2α-hydroxyursolic acid, hederagenin, 23-hydroxyursolic acid, arjunolic acid and asiatic acid.

Six types of cultured tissues at various stages of differentiation were induced from rhizome segments of Wasabia japonica Matsum. (Japanese name: wa-sa-bi); NC callus, rootlets, embryoids, differentiated tissues I and II, and plantlets. Myrosinase activity was detected at all stages of differentiation, but sinigrin could not be detected from NC callus, rootlets and embryoids, so that pungency could be felt from differentiated tissues I, II and plantlets. It is supposed that the process of differentiation stages (the increase of the rhizome weight) is parallel to the sinigrin content in the tissues.