- Email: [email protected]
Volatile monoterpenoid constituents of the plantlets of Mentha spicata produced by shoot tip culture
003 l-9422/90 S3.00+ 0.00 Q 1990 PergamonPressplc
Phytochemistry, Vol. 29, No. 2, pp. 493495, 1990. Printedin Great Britain.
VOLATILE MONOTERPENOID CONSTITUENTS OF THE PLANTLETS OF MENTHA SPZCATA PRODUCED BY SHOOT TIP CULTURE TOSHIFUMI HIRATA, SATORU MURAKAMI,
Department of Chemistry, Faculty of Science, Hiroshima
KAZUHITO
University,
OGIHARA
and
Higashisenda-machi,
TAKAYUKI
Naka-ku,
SUGA*
Hiroshima
730, Japan
(Received in revised form 26 May 1989)
Key Word Index-Mentha
spicata; Labiatae;
shoot tip culture;
plantlet;
essential
oil; monoterpenoids;
thiamine
hydrochloride.
Abstract-Shoot tip culture of Mentha spicata was carried out in both Murashige-Skoog and Gamborg’s B5 media supplemented with various levels of growth regulators. Cultured bodies were produced by the cultures with Gamborg’s B5 media, but no cultured body was initiated by the cultures with Murashige-Skoog media, Contents of the monoterpenoids, especially carvone and limonene, in the plantlets grown in the BS medium were higher than those in the mother plants. Production of the monoterpenoids was influenced by concentration of 1-naphthaleneacetic acid and thiamine hydrochloride in the B5 media.
INTRODUCTION
Mentha spicata has been widely used as a culinary herb for a long time. This plant is liable to cross naturally and hence many natural hybrids exist [l]. Therefore, the essential oil constituents in the Mentha species frequently fluctuate. In connection with studies on the production of beneficial substances by plant tissue cultures [2, 33, we previously tried to produce essential oils by the callus tissues of Mentha spicata, but the callus tissues did not accumulate any essential oils [43. In general the ability to produce secondary metabolites in the cultured cells is dependent on the degree of differentiation of these cells; that is to say, the highly differentiated cells have the greater ability for accumulation of secondary metabolites [S]. However, the propagation of the plantlet from M. spicata by means of shoot tip culture has not been reported [S, 63. Therefore, we have now propagated the clonal plants of M. spicata by the shoot primordia method [7] and examined the volatile constituents of the clonal plants and the influence of basal medium and growth regulators on the production of the volatile constituents. RESULTS AND DISCUSSION
The shoot tip culture of M. spicata was carried out according to the shoot primordia method [7]. Shoot tip explants of M. spicata were planted in both Gamborg’s B5 medium [S] and Murashige-Skoog’s medium [9] containing various levels of 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). The explants had been cultured for 180 days in the above media by rotating slowly under continuous fluorescent light of 9000 lux. Plantlets were produced in the B5 media supplemented with 0, 0.02, 0.2 and 2.0 ppm of BAP in addition to 0.02 and 0.2 ppm of NAA. The cultures in the
B5 media containing the higher level of NAA (2 and 4 ppm) were liable to produce callus tissues. On the other hand, no cultured body was initiated from the shoot tip explants in Murashige-Skoog media, in spite of trials under several conditions. The essential oils from the plantlets grown in the B5 media were analysed by GC and GC-MS to compare with the essential oil from the original plant. The total amounts of the essential oils from the plantlets cultured in the B5 media were 0.9-1.3 mg/g plantlet (Table 1); the total amounts were almost equal to that of the mother plants (l&1.5 mg/g plantlet). The essential oils from the plantlets consisted of constituents similar to those of the oils from the mother plants (Table 2). However, there were considerable quantitative differences. The content of a major constituent, carvone (l), in the essential oils from the plantlets was higher than that from the mother plants, while dihydrocarveol (2) and dihydrocarvone (3) were scarcely contained in the essential oils from the cultured plantlets. The content of limonene (4) in the essential oils from the plantlets was high in comparison with that from the mother plant. On the other hand, no monoterpenoids were found in the callus tissues grown in Gamborg’s B5 media containing a high level of NAA. Because no cultured body was initiated by the cultures in Murashige-Skoog media, the plantlets obtained in Gamborg’s B5 media were transferred to Murashige-
I
*Author to whom correspondence
should
be addressed.
2 R’=H, R2=OH 3 R’,R2=0
4
T. HIRATA et al.
494 Table
1. Total amounts
of essential
Medium
BAPt 0.02
NAA$ 0
Gamborg’s B5 Murashige-Skoog TFB$
1.13+0.14 0.55 + 0.07 0.61 +0.15
oils from cultured
BAP 0.2
NAA 0
1.24 ct 0.09 0.63 + 0.05 0.70~0.10
plantlets
BAP 2.0
of M. spicata propagated
Total amounts*/mg BAP NAA 0 0.02
1.28,0.06 0.55 * 0.09 0.41 &O.lO
NAA 0.02
0.91 kO.18 0.49 + 0.04 0.52+0.10
by shoot tip culture
BAP 0.2
NAA 0.02
1.10+0.10 0.53+0.10 0.22 i_ 0.08
BAP 2.0
NAA 0.02
0.92_+0.10 0.41 & 0.07 0.14iO.06
*Weight of the essential oils per gram (fresh wt) of the plantlets t6-Benzylaminopurine (ppm). $l-Naphthaleneacetic acid (ppm). SThiamine-free 85 medium.
Skoog media. These were cultured under the same conditions as those in Gamborg’s BS media. The cultured bodies kept plantlets even in the passage culture for 60 days in Murashige-Skoog media. However, the total amounts of the essential oils from the cultured bodies were lowered (Table 1). The absolute amounts of monoterpenoids were extremely low; the contents ofcarvone{l) and limonene (4) were below one-tenth compared with the case of the plantlets grown in Gamborg’s B5 media. A difference in the ability to produce the essential oils in the plantlets grown in Gamborg’s B5 and Murashige-Skoog media seems to be attributable to a difference in concentration of thiamine hydrochloride in both basal media, because the concentration of thiamine hydrochloride is 10 ppm in the B5 medium, but only 0.1 ppm in Murashige-Skoog medium. In order to examine the contribution of thiamine hydrochloride on the formation of essential oils, the plantlets were cultured in a thiamine-free Gamborg’s B5 medium (TFB medium). The plantlets grown in ‘normal’ Gamborg’s B5 media were transferred in the TFB medium and then cultured for 60 days under the same conditions as those in Gamborg’s B5 medium. The cultured bodies kept plantlets in the TFB medium. The total amounts and constituents of the essential oils from the plantlets grown in the TFB medium were compared with those from the plantlets grown in Gamborg’s B5 and Murashige-Skoog media. The total amounts of the essential oils were low (Table 1). The constituents of the essential oils from the cultured plantlets in the TFB media were similar to those of the plantlets grown in the B5 media. However, the contents of the monoterpenoids were extremely low compared with those of the plantlets grown in the B5 media. It may be concluded that the total amounts of the essential oils and the content of monoterpenoids in the cultured plantlets produced by the shoot tip culture were influenced by the basal media and the growth regulators. The ability for monoterpene biosynthesis in the plantlets grown in both Murashige-Skoog media and TFB media was depressed; thiamine hydrochloride may be one of the essential elements for the formation of the essential oils in the cultures. It is interesting to clarify the key substances responsible for the formation of monoterpenoids in the cultured plants and the mechanism for such a regulation. EXPERIMENTAL
GC and CO-CC with authentic samples were performed on an instrument equipped with an FID and a glass capillary column
(WCOT, 0.20 mm x 50 m) coated with OV-17 (thickness: 0.25 pm) by programming the column temp. at 2”/min from 40 to 260” with N, as carrier gas. GC-MS analyses were performed on a Shimadzu QP-1000 mass spectrometer combined with a gaschromatograph instrument under conditions as follows: column, 0.20mmx50m glass capillary column coated with OV-101 (thickness: 0.25 pm); injector temp. 250’; column temp. programming at Z”/min from 40 to 230”; carrier gas He; split ratio 5O:l. Mass spectra were obtained on the above described spectrometer conducted with an ionization energy of 70 eV at 250” of the ion source temp. Shoot tip culturesfrom M. spicata. According to the reported procedure [2,7], the shoot tip cultures of M. spicata were carried out. Shoot tip explants (0.3 mm) of M. spicnta were obtained under a stereo-microscope by dissecting green segments of stems of S-week-old plants (ca 20 cm high). Each explant composed of the apical dome with 1-2 of the youngest leaf primordia was planted in a liquid medium (25 ml) in a growth vial (200 x27mm) and cultured at 22” on a gyrated drum rotated at 2 rpm under continuous fluorescent light of 9000 lux. Fifty kinds of modified media, prepared by combination of two basal media and two growth regulators, were used for the initial cultivation of the explants. Basal media used were Gamborg’s B5 [S] and Murashige-Skoog 193 media. Concentrations of the growth regulators used were 0, 0.02, 0.2. 2.0 and 4.0 ppm for both BAP and NAA. The plantlets were produced in the B5 media supplemented with 0, 0.02, 0.2 and 2.0 ppm of BAP in addition to 0.02 and 0.2 ppm of NAA. However, callus tissues were formed in the case of the B5 media supplemented with 0,0.02.0.2, 2.0 and 4.0 ppm of BAP in addition to 2.0 and 4.0 ppm of NAA. On the other hand, no cultured body was produced in Murashigee Skoog media supplemented with any concn of BAP (0,0.02,0.2. 2.0, and 4.0 ppm) and NAA (0, 0.02, 0.2. 2.0 and 4.0 ppm). The shoots (ca 0.5 g) of the plantlets grown in Gamborg’s B5 media were transplanted at a regular time interval for a month and cultured for ca 180 days. The plantlets were grown ca 20 times for a month and weighed 7.5-10.0 g/growth vial. Cultioation of the plantlets in Murashige-Skoog and TFB media. Shoots of the plantlets of M. spicata grown in Gamborg’s B5 media were cut off and transferred into a growth vial containing the Murashige-Skoog or thiamine-free 85 (TFB) media under sterilized conditions. The shoots were cultured for a month under the same conditions as those in the B5 media and then transplanted at regular time intervals in the same media. The cultured bodies kept plantlets and the growth rate was comparable to that in Gamborg’s B5 media. Analysis ofessential oil constiruentsfrom the shoot rip cultures. After culturing in 5 growth vials under the same conditions, the cultured masses were combined and the combined mass (ca 3CL5Og) was frozen with liquid N,, ground in a mortar and
Monoterpenoids
W-30
d
6
6
A
+I +I +I+1 hl”crr:
a +I
a +
00000
+I +I +I +I +I ‘?Y-Jz?‘lul,
ooo~o~~-
495
subjected to steam distillation. The steam distillate was extracted by a liquid-liquid continuous extractor for 5 hr with Et,O. The Et,0 extract, after drying over Na,SO,, was coned to a constant small vol. (50 ~1).The concentrate was subjected to CC and GCMS. Constituents of the concentrate were identified by co-GC with authentic samples and/or by comparison of MS spectra with those of authentic samples. The absolute amount of the constituents was determined by calibration based on the GC area of each constituent. Analyses of essential oil constituents were made on three batches of the cultured mass grown under the same conditions and the data obtained were statistically treated. The mean and deviation values of the total amounts of the essential oils and those of the relative abundances of the essential oils from the cultured plantlets are given in Tables 1 and 2 respectively.
\DbP-
c-i6
6 +I +I +I
Acknowledgements-The authors thank the Ogawa Perfumery Co. Ltd. for a gift of M. spicata and Professor Ryusou Tanaka and Dr Kenji Taniguchi (Laboratory of Plant Chromosome and Gene Stock, Faculty of Science, Hiroshima University) for permission to use a gyrated drum cultivator.
000~,2,r,p
VTY?Y
of Mentha spicata cultures
m d +I -?
REFERENCES
vi E m p. %
1. Lawrence, B. M. (1977) VII Int. Congress of Essential Oils, p. 121. 2. Sakamura, F., Ogihara, K., Suga, T., Taniguchi, K. and Tanaka, R. (1986) Phytochemistry 25, 1333. 3. Sakamura, F., Murakami, S., Hirata, T., Suga, T., Taniguchi, K. and Tanaka, R. (1987) J. Sci. Hiroshima Univ. Ser. A 50,61. 4. Suga, T., Hirata, T. and Yamamoto, Y. (1980) Agric. Biol. Chem. 44, 1817. 5. Brown, J. T. and Charlwood, B. V. (1986) in Secondary Metabolism in Plant Cell Culture, (Morris, P., Scragg, A. H., Stafford, A., Fowler, M. W., eds), p. 69. Cambridge University Press, London. 6. Bhojwani, S. S. and Razdan, M. K. (1983) in Plant Tissue Culture: Theory and Practice. Elsevier, Amsterdam. 7. Tanaka, R. and Ikeda, H. (1983) Jap. 1. Genet. 58, 65. 8. Gamborg, 0. L., Miller, R. A. and Ojima, K. (1968) Exp. Cell. Res. SO, 151. 9. Murashige, T. and Skoog, F. (1962) Physiol. Plant. 15,473.
Phytochemistry, Vol. 29, No. 2, pp. 493495, 1990. Printedin Great Britain.
VOLATILE MONOTERPENOID CONSTITUENTS OF THE PLANTLETS OF MENTHA SPZCATA PRODUCED BY SHOOT TIP CULTURE TOSHIFUMI HIRATA, SATORU MURAKAMI,
Department of Chemistry, Faculty of Science, Hiroshima
KAZUHITO
University,
OGIHARA
and
Higashisenda-machi,
TAKAYUKI
Naka-ku,
SUGA*
Hiroshima
730, Japan
(Received in revised form 26 May 1989)
Key Word Index-Mentha
spicata; Labiatae;
shoot tip culture;
plantlet;
essential
oil; monoterpenoids;
thiamine
hydrochloride.
Abstract-Shoot tip culture of Mentha spicata was carried out in both Murashige-Skoog and Gamborg’s B5 media supplemented with various levels of growth regulators. Cultured bodies were produced by the cultures with Gamborg’s B5 media, but no cultured body was initiated by the cultures with Murashige-Skoog media, Contents of the monoterpenoids, especially carvone and limonene, in the plantlets grown in the BS medium were higher than those in the mother plants. Production of the monoterpenoids was influenced by concentration of 1-naphthaleneacetic acid and thiamine hydrochloride in the B5 media.
INTRODUCTION
Mentha spicata has been widely used as a culinary herb for a long time. This plant is liable to cross naturally and hence many natural hybrids exist [l]. Therefore, the essential oil constituents in the Mentha species frequently fluctuate. In connection with studies on the production of beneficial substances by plant tissue cultures [2, 33, we previously tried to produce essential oils by the callus tissues of Mentha spicata, but the callus tissues did not accumulate any essential oils [43. In general the ability to produce secondary metabolites in the cultured cells is dependent on the degree of differentiation of these cells; that is to say, the highly differentiated cells have the greater ability for accumulation of secondary metabolites [S]. However, the propagation of the plantlet from M. spicata by means of shoot tip culture has not been reported [S, 63. Therefore, we have now propagated the clonal plants of M. spicata by the shoot primordia method [7] and examined the volatile constituents of the clonal plants and the influence of basal medium and growth regulators on the production of the volatile constituents. RESULTS AND DISCUSSION
The shoot tip culture of M. spicata was carried out according to the shoot primordia method [7]. Shoot tip explants of M. spicata were planted in both Gamborg’s B5 medium [S] and Murashige-Skoog’s medium [9] containing various levels of 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). The explants had been cultured for 180 days in the above media by rotating slowly under continuous fluorescent light of 9000 lux. Plantlets were produced in the B5 media supplemented with 0, 0.02, 0.2 and 2.0 ppm of BAP in addition to 0.02 and 0.2 ppm of NAA. The cultures in the
B5 media containing the higher level of NAA (2 and 4 ppm) were liable to produce callus tissues. On the other hand, no cultured body was initiated from the shoot tip explants in Murashige-Skoog media, in spite of trials under several conditions. The essential oils from the plantlets grown in the B5 media were analysed by GC and GC-MS to compare with the essential oil from the original plant. The total amounts of the essential oils from the plantlets cultured in the B5 media were 0.9-1.3 mg/g plantlet (Table 1); the total amounts were almost equal to that of the mother plants (l&1.5 mg/g plantlet). The essential oils from the plantlets consisted of constituents similar to those of the oils from the mother plants (Table 2). However, there were considerable quantitative differences. The content of a major constituent, carvone (l), in the essential oils from the plantlets was higher than that from the mother plants, while dihydrocarveol (2) and dihydrocarvone (3) were scarcely contained in the essential oils from the cultured plantlets. The content of limonene (4) in the essential oils from the plantlets was high in comparison with that from the mother plant. On the other hand, no monoterpenoids were found in the callus tissues grown in Gamborg’s B5 media containing a high level of NAA. Because no cultured body was initiated by the cultures in Murashige-Skoog media, the plantlets obtained in Gamborg’s B5 media were transferred to Murashige-
I
*Author to whom correspondence
should
be addressed.
2 R’=H, R2=OH 3 R’,R2=0
4
T. HIRATA et al.
494 Table
1. Total amounts
of essential
Medium
BAPt 0.02
NAA$ 0
Gamborg’s B5 Murashige-Skoog TFB$
1.13+0.14 0.55 + 0.07 0.61 +0.15
oils from cultured
BAP 0.2
NAA 0
1.24 ct 0.09 0.63 + 0.05 0.70~0.10
plantlets
BAP 2.0
of M. spicata propagated
Total amounts*/mg BAP NAA 0 0.02
1.28,0.06 0.55 * 0.09 0.41 &O.lO
NAA 0.02
0.91 kO.18 0.49 + 0.04 0.52+0.10
by shoot tip culture
BAP 0.2
NAA 0.02
1.10+0.10 0.53+0.10 0.22 i_ 0.08
BAP 2.0
NAA 0.02
0.92_+0.10 0.41 & 0.07 0.14iO.06
*Weight of the essential oils per gram (fresh wt) of the plantlets t6-Benzylaminopurine (ppm). $l-Naphthaleneacetic acid (ppm). SThiamine-free 85 medium.
Skoog media. These were cultured under the same conditions as those in Gamborg’s BS media. The cultured bodies kept plantlets even in the passage culture for 60 days in Murashige-Skoog media. However, the total amounts of the essential oils from the cultured bodies were lowered (Table 1). The absolute amounts of monoterpenoids were extremely low; the contents ofcarvone{l) and limonene (4) were below one-tenth compared with the case of the plantlets grown in Gamborg’s B5 media. A difference in the ability to produce the essential oils in the plantlets grown in Gamborg’s B5 and Murashige-Skoog media seems to be attributable to a difference in concentration of thiamine hydrochloride in both basal media, because the concentration of thiamine hydrochloride is 10 ppm in the B5 medium, but only 0.1 ppm in Murashige-Skoog medium. In order to examine the contribution of thiamine hydrochloride on the formation of essential oils, the plantlets were cultured in a thiamine-free Gamborg’s B5 medium (TFB medium). The plantlets grown in ‘normal’ Gamborg’s B5 media were transferred in the TFB medium and then cultured for 60 days under the same conditions as those in Gamborg’s B5 medium. The cultured bodies kept plantlets in the TFB medium. The total amounts and constituents of the essential oils from the plantlets grown in the TFB medium were compared with those from the plantlets grown in Gamborg’s B5 and Murashige-Skoog media. The total amounts of the essential oils were low (Table 1). The constituents of the essential oils from the cultured plantlets in the TFB media were similar to those of the plantlets grown in the B5 media. However, the contents of the monoterpenoids were extremely low compared with those of the plantlets grown in the B5 media. It may be concluded that the total amounts of the essential oils and the content of monoterpenoids in the cultured plantlets produced by the shoot tip culture were influenced by the basal media and the growth regulators. The ability for monoterpene biosynthesis in the plantlets grown in both Murashige-Skoog media and TFB media was depressed; thiamine hydrochloride may be one of the essential elements for the formation of the essential oils in the cultures. It is interesting to clarify the key substances responsible for the formation of monoterpenoids in the cultured plants and the mechanism for such a regulation. EXPERIMENTAL
GC and CO-CC with authentic samples were performed on an instrument equipped with an FID and a glass capillary column
(WCOT, 0.20 mm x 50 m) coated with OV-17 (thickness: 0.25 pm) by programming the column temp. at 2”/min from 40 to 260” with N, as carrier gas. GC-MS analyses were performed on a Shimadzu QP-1000 mass spectrometer combined with a gaschromatograph instrument under conditions as follows: column, 0.20mmx50m glass capillary column coated with OV-101 (thickness: 0.25 pm); injector temp. 250’; column temp. programming at Z”/min from 40 to 230”; carrier gas He; split ratio 5O:l. Mass spectra were obtained on the above described spectrometer conducted with an ionization energy of 70 eV at 250” of the ion source temp. Shoot tip culturesfrom M. spicata. According to the reported procedure [2,7], the shoot tip cultures of M. spicata were carried out. Shoot tip explants (0.3 mm) of M. spicnta were obtained under a stereo-microscope by dissecting green segments of stems of S-week-old plants (ca 20 cm high). Each explant composed of the apical dome with 1-2 of the youngest leaf primordia was planted in a liquid medium (25 ml) in a growth vial (200 x27mm) and cultured at 22” on a gyrated drum rotated at 2 rpm under continuous fluorescent light of 9000 lux. Fifty kinds of modified media, prepared by combination of two basal media and two growth regulators, were used for the initial cultivation of the explants. Basal media used were Gamborg’s B5 [S] and Murashige-Skoog 193 media. Concentrations of the growth regulators used were 0, 0.02, 0.2. 2.0 and 4.0 ppm for both BAP and NAA. The plantlets were produced in the B5 media supplemented with 0, 0.02, 0.2 and 2.0 ppm of BAP in addition to 0.02 and 0.2 ppm of NAA. However, callus tissues were formed in the case of the B5 media supplemented with 0,0.02.0.2, 2.0 and 4.0 ppm of BAP in addition to 2.0 and 4.0 ppm of NAA. On the other hand, no cultured body was produced in Murashigee Skoog media supplemented with any concn of BAP (0,0.02,0.2. 2.0, and 4.0 ppm) and NAA (0, 0.02, 0.2. 2.0 and 4.0 ppm). The shoots (ca 0.5 g) of the plantlets grown in Gamborg’s B5 media were transplanted at a regular time interval for a month and cultured for ca 180 days. The plantlets were grown ca 20 times for a month and weighed 7.5-10.0 g/growth vial. Cultioation of the plantlets in Murashige-Skoog and TFB media. Shoots of the plantlets of M. spicata grown in Gamborg’s B5 media were cut off and transferred into a growth vial containing the Murashige-Skoog or thiamine-free 85 (TFB) media under sterilized conditions. The shoots were cultured for a month under the same conditions as those in the B5 media and then transplanted at regular time intervals in the same media. The cultured bodies kept plantlets and the growth rate was comparable to that in Gamborg’s B5 media. Analysis ofessential oil constiruentsfrom the shoot rip cultures. After culturing in 5 growth vials under the same conditions, the cultured masses were combined and the combined mass (ca 3CL5Og) was frozen with liquid N,, ground in a mortar and
Monoterpenoids
W-30
d
6
6
A
+I +I +I+1 hl”crr:
a +I
a +
00000
+I +I +I +I +I ‘?Y-Jz?‘lul,
ooo~o~~-
495
subjected to steam distillation. The steam distillate was extracted by a liquid-liquid continuous extractor for 5 hr with Et,O. The Et,0 extract, after drying over Na,SO,, was coned to a constant small vol. (50 ~1).The concentrate was subjected to CC and GCMS. Constituents of the concentrate were identified by co-GC with authentic samples and/or by comparison of MS spectra with those of authentic samples. The absolute amount of the constituents was determined by calibration based on the GC area of each constituent. Analyses of essential oil constituents were made on three batches of the cultured mass grown under the same conditions and the data obtained were statistically treated. The mean and deviation values of the total amounts of the essential oils and those of the relative abundances of the essential oils from the cultured plantlets are given in Tables 1 and 2 respectively.
\DbP-
c-i6
6 +I +I +I
Acknowledgements-The authors thank the Ogawa Perfumery Co. Ltd. for a gift of M. spicata and Professor Ryusou Tanaka and Dr Kenji Taniguchi (Laboratory of Plant Chromosome and Gene Stock, Faculty of Science, Hiroshima University) for permission to use a gyrated drum cultivator.
000~,2,r,p
VTY?Y
of Mentha spicata cultures
m d +I -?
REFERENCES
vi E m p. %
1. Lawrence, B. M. (1977) VII Int. Congress of Essential Oils, p. 121. 2. Sakamura, F., Ogihara, K., Suga, T., Taniguchi, K. and Tanaka, R. (1986) Phytochemistry 25, 1333. 3. Sakamura, F., Murakami, S., Hirata, T., Suga, T., Taniguchi, K. and Tanaka, R. (1987) J. Sci. Hiroshima Univ. Ser. A 50,61. 4. Suga, T., Hirata, T. and Yamamoto, Y. (1980) Agric. Biol. Chem. 44, 1817. 5. Brown, J. T. and Charlwood, B. V. (1986) in Secondary Metabolism in Plant Cell Culture, (Morris, P., Scragg, A. H., Stafford, A., Fowler, M. W., eds), p. 69. Cambridge University Press, London. 6. Bhojwani, S. S. and Razdan, M. K. (1983) in Plant Tissue Culture: Theory and Practice. Elsevier, Amsterdam. 7. Tanaka, R. and Ikeda, H. (1983) Jap. 1. Genet. 58, 65. 8. Gamborg, 0. L., Miller, R. A. and Ojima, K. (1968) Exp. Cell. Res. SO, 151. 9. Murashige, T. and Skoog, F. (1962) Physiol. Plant. 15,473.