Pkytochmrirrry.Vol. 25. No. 6. pp. 1333-1335,1986. 003I -942zjB.653.00+ 0.

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Printed ia Great Britain. 0 1986Peqamon Pras Ltd.

VOLATILE CONSTITUENTS OF ZZNGZBER OFFZCZZVALE RHIZOMES

PRODUCED BY IN JZTRO SHOOT TIP CULTURE*

FUKIKO SAKAMuRAt, KAZUHITO OGIHARA~, TAKAYUKI SUGA$$ KENJI TANIGUCHI~ and RYUSO TANAKA[[
tDcprtmcnt of Food, Oshimo Womens College, Gioncho, Asaminami-ky Hiroshima 73141, Japan; $Department of Chemistry,
Faculty of Scienoz, Hiroshima University, Higashisenda-machi,Naka-ku, Hiroshima 730, Japan; gLaboratory of Plant Chromosome
and Gene Stock of the Faculty of Science, Hiroshima University, Higashisenda-machi, Naka-ku, Hiroshima 730, Japan; IlDcpartwnt
of Botany, Faculty of Science, Hiroshima University, Higashisenda-machi, Naka-ky Hiroshima 730, Japan

(Receiwd 3 Septet&r 1985)

Key Word Index-Zingiber oRin& Zingiberaceae; 8ingcr; shoot tip culture; plantlets; rhizomes; volatile
constituents.

Abstract-Plantlets with rhizomes were produced in vitro from shoot tips of Zingiber o&We grown in both modified
Gamborgs B5 (BS) and Murashige+Skoog (MS) media supplemented with various levels of growth regulators. The
rhizomes accumulated the volatile oils similar to those formed in the original rhizome. In the oil from the rhizome
grown in the modified BS medium, the acyclic oxygenated monoterpenes predominated, while the oil from the rhizome
grown in the modified MS medium consisted mainly of sesquiterpenes.

INTRODUCTION the culture only produced precucious branches including

Ginger cultivar Oshoga, Zingiber ofiinale Rose., is
widely used as a herb, spice and additive in foods and
beverages in Japan. Previously, Sakamura et al. [ 1,2]
reported on volatile constituents from the freshly harves-
ted rhizome of the cultivar Oshoga and on changes in the
constituents during storage of the rhizome. Zingiber
ofiinule Oshoga annually propagates itself by rhizomes
stored for several months under high humidity prior to
planting. However, storage of the rhizome of this cultivar
is more difficult than that of other cultivars because the
rhizome suffers from cold injury. Therefore, an alternative
propagation technique for Z. o&i&e is needed.
Recently, Tanaka et al. [3] established the culture of shoot
primordia. The shoot primordia not only undergo rapid
propagation, but also form oil bodies. Application of the
shoot primordia method to Oshoga, however, resulted in
the formation of plantlets with rhizomes. As a continu-
ation of the previous work [l, 21, we examined the
essential oils from these rhizomes.
RESULTS AND DISCUSSION
Shoot tip explants of 2. oficinale Oshoga were planted
in both modified Gamborgs B5 (BS) medium [4] and
Murashige-Skoog (MS) medium [S] containing various
levels of 1-naphthaleneacetic acid (NAA) and 6&n-
zyladenine (BAP). According to the shoot primordia
production method [3], the explants had been cultured
for 100 days at 22 in the above media by rotating slowly
under continuous fluorescent light of !MKlO lux. However,
*Part 2 in the series Constituents of lbential Gils from
zingibero#icin&?
(To whom correspondence should be addressed.
plantlets with rhizomes. The plantlets were produced in
the modified BS media supplemented with 0.2 and
2.0ppm of BAP and with 0.2 and 2.0ppm of BAP in
addition to 0.2 ppm of NAA. The plantlets were also
formed in the modified MS media supplemented with
0.2 ppm of BAP and with 0.2 and 2.0 ppm of BAP in
addition to 0.2 ppm of NAA. The essential oils from the
rhizomes of these plantlets were analysed by GC and
GC/MS. Similarly, the essential oil from the original
rhizome was analysed for comparison.
The essential oils from these cultures contained the
same constituents as in the original rhizome (Table 1).
However, there were considerable quantitative differen-
ces. The essential oil from the rhizome grown in the
modified BS medium consisted mainly of monoterpenes.
Acyclic oxygenated compounds, such as geraniol, geranial
and geranyl acetate, together composed between 42 and
52 % of the oil. The oil pattern is similar to those in the
original rhizome and in other Japanese fresh rhizomes
[ 11. On the other hand, the essential oil from the rhizome
grown in the modified MS medium consisted mainly of
sesquiterpenes, among which zingiberene predominated.
Such an oil composition is similar to those in the ginger
rhizomes from other countries 16123. In addition, the
major oil constituents, probably diterpenes, were present
in the rhizome formed in the modified MS medium.
Obviously, the oil composition in the culture depended
on the composition of the basal culture medium, but it was
influenced by the concentration of the growth regulators
added. Thus, a change in the composition of the medium
provides a means of producing cultures with different
aromas. In the at.~ of the rhizome produced in the
modified MS medium, monoterpene biosynthesis may be
depressed. Such a depression might result from an excess
of glycine and/or from a deficiency of thiamine hydro-
chloride in comparison of the composition of MS medium
with that of BS medium.

1333
 1334 F. SAKAMURA
et al.

Table 1. Percentage relative abundances of essential oil constituents from Zingiber O@I& rhizomes produced by shoot tip
culture

Culture

Gamborgs BS medium Murashige-Skoog medium

NAA BAPt NM BAP NAA BAP NM BAP NAA BAP NAA BAP
chiginal
Constituents 0 0.2 0.2 0.2 0.2 2.0 0 0.2 0.2 0.2 0.2 2.0 rhizome$

Monoterpenes8 65.2 60.0 63.3 31.3 23.4 44.1 72.1

g+inene 2.2 1.1 2.1 0.9 1.4 1.6 tr
Myrcene 0.5 0.2 0.7 0.2 0.2 0.3 tr
IJt-Cineole 14.2 5.6 17.6 5.8 5.5 9.4 1.8
Linalool 2.3 2.0 23 1.2 0.8 1.3 1.0
lsobomeol 0.5 0.3 0.3 tr tr 0.3 0.1
a-Terpineol 0.8 0.6 0.7 0.8 0.7 0.7 0.9
Citronellol 1.0 0.6 1.0 0.5 0.7 0.7 21
Nero1 23 1.6 1.0 1.1 0.7 1.1 0.9
Neral 1.5 4.4 4.4 3.8 24 5.2 8.1
Geraniol 10.5 8.9 9.1 28 1.7 5.0 20.8
Geranial 16.7 12.1 I20 8.5 8.0 13.7 25.2
Geranyl acetate 6.7 22.6 120 5.7 1.3 5.1 11.2
SesquiterpenesQ 29.0 320 29.8 50.4 61.1 40.4 12.6
a-Copaene 0.1 0.4 tr 0.4 0.2 0.2 0.1
/?-Bisabolene 2.7 1.7 1.4 29 2.8 1.7 0.3
Zingiberene 8.5 9.0 8.5 16.4 26.6 15.4 3.2
a-Curcumene 4.0 4.8 4.4 5.2 6.2 5.3 1.6
/3-Sesquiphellandrene 2.6 1.6 24 4.3 4.4 2.4 0.7
Nerolidol 0.8 1.2 1.2 1.8 0.9 0.9 1.0
/?-Sesquiphellandrol 0.4 0.6 0.6 0.9
Others)1 5.8 8.0 6.9 18.3 15.5 14.9 -

l 1-Naphthaleneacetic acid @pm).

t6Benxyladenine @pm).
*The rhizome of &wee.k-oki plants.
QGrder of elution from an OV-101 GC column.
[IOther higher boiling compounds. tr: trace (< 0.1%)

EXPERIMENTAL extracted by a liquid-liquid continuous extractor for 6 hr with

EtsO. The EtsO ~01%dried over anhydrous NasSO,, was coned
Shoot tipculruresfiomginger. Shoot tipexplants (0.3 mm)of Z.
to a small vol. under red. pres. The concentrate was assayed by a
o&inale Rose. Oshoga were obtained under stereo-microscope
combination of GCand GC/MS under theconditionsas follows.
by dissecting pale yellow segments of stems of 5-wee.k-old plants
GC a 50 m x 0.25 mm i.d. WCOT glass capilhuy column coated
(10-25 cm high). Each explant composed of the apical dome with
with 2% OV-101, column temp. lC0-250 at Z/min, injector
l-2 of the youngest leaf primordia was planted in a liquid me-
temp. 250, detector temp. 250; GC/MS: a 50 m x 0.25 mm i.d.
dium (25 ml) in a growth vial (2oOmm x 27 mm), and cultured at
WCOT glass capillary column coated with 2% OV-101, con-
22 for 100 days on a gyrated drum rotated at 2rpm under
nected to a Shimaxu QP-loo0 mass spectrometer, column temp.
continuous fluorescent light of 9000 lx [3]. Culture media used
100-250 at Z/min, ion source temp. 250. ionization voltage
were modified B5 [4] and MS [5] media containing 0.0.02,0.2,
70 eV.
2.0 and 4.0 ppm of NAA and 0.2 and 2.0 ppm of BAP, as shown in
Table 1. Plantlets were produced in the modified B5 media
supplemented with 0.2 and 2.0 ppm of BAP and with 0.2 and
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