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Improvement of ginsenoside production by Jasmonic acid and some other

elicitors in hairy root culture of ginseng (Panax ginseng C. A. Meyer)

Article  in  In Vitro Cellular & Developmental Biology - Plant · September 2000

DOI: 10.1007/s11627-000-0077-4

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In Vitro Cell. Dev. Biol.ÐPlant 36:424±428, September±October 2000
q 2000 Society for In Vitro Biology
1054-5476/00 $10.0010.00

IMPROVEMENT OF GINSENOSIDE PRODUCTION BY JASMONIC ACID AND SOME OTHER ELICITORS IN

HAIRY ROOT CULTURE OF GINSENG (PANAX GINSENG C. A. MEYER)

KEE-WON YU1, WEN-YUAN GAO1, SUNG-HO SON2, and KEE-YOEUP PAEK1*

1
Research Center for the Development of Advanced Horticultural Technology, Chungbuk National University, Cheongju, 361-763, Korea
2
Division of Biotechnology, Forestry Research Institute, Forestry Administration, Omokdong, 44-3 Beongi, Kwonseon-gu, Suwon 441-350, Korea

(Received 31 January 2000; accepted 26 May 2000; editor T. A. Thorpe)

Summary
Hairy root cultures of Panax ginseng, established after the infection of root sections with Agrobacterium rhizogenes
KCTC 2703, were cultured in phytohormone-free Murashige and Skoog (MS) liquid medium containing different
concentrations of jasmonic acid and some other elicitors, in order to promote ginsenoside accumulation. Jasmonic acid in
the range 1.0±5.0 mg l21 (4.8±23.8 mM) strongly improved total ginsenoside production in ginseng hairy roots. Peptone
(300 mg l21) also showed some effect on ginsenoside improvement; however its effect was much weaker than that of
jasmonic acid. Ginsenoside content and productivity were 58.65 and 504.39 mg g21, respectively. The Rb group of
ginsenoside content was increased remarkably by jasmonic acid, while Rg group ginsenoside content changed only slightly
compared to controls. However, jasmonic acid also strongly inhibited ginseng hairy root growth.

Key words: Agrobacterium rhizogenes; catechin; ginsenoside Rb; ginsenoside Rg; peptone.

Introduction cultured cells of P. ginseng transformed with the aid of A. rhizogenes

Ginseng (Panax ginseng C. A. Meyer) belongs to the Aralilaceae
family, and is traditionally considered one of the most potent
medicinal plants. The most important active component in ginseng
root is ginsenoside. To date, more than 20 different ginsenosides
have been identified (Lee et al., 1995). The demand for ginseng
roots and ginseng extracts has increased over the years, and
currently ginseng plants are cultivated in farms throughout Korea
and China. Because the cultivation is a long (4±6 yr), laborious
process, cultivated ginseng root is an expensive commodity
(Persons, 1995). Traditional cultivation methods cannot meet the
increasing demands of the ginseng market, especially for ginseno-
sides. We recently established a bioreactor culture system for
ginseng adventitious root cultures (Seon et al., 1999; Son et al.,
1999a). However, the ginsenoside content in ginseng adventitious
roots is not very high.
Hairy root cultures resulting from the infection of plant materials
by Agrobacterium rhizogenes produce the same secondary metabo-
lites as those usually synthesized in intact parent plant roots, with
similar or higher yields (Zehra et al., 1999). This feature, together
with their genetic stability and (generally) rapid growth in simple
media lacking phytohormones, makes hairy root cultures suitable
for production of useful secondary metabolites (Pinol et al., 1999).
Yoshikawa and Furuya (1987) attempted to establish hairy root
cultures of P. ginseng; however they required phytohormones for
satisfactory growth. Accumulation of ginsenosides was found in
*Author to whom correspondence should be addressed: Email paekky@
cbucc.chungbuk.ac.kr
(Zhuravlev et al., 1990). Inomata et al. (1993) also obtained ginseng
hairy roots transformed by A. rhizogenes. These roots grew rapidly in
a phytohormone-free medium and showed biphasic growth: rapid
during the first 2 wk and slower thereafter. Yoshimatsu et al. (1996)
established hairy root cultures by infecting petiole segments with A.
rhizogenes strain 15834, and studied their cold storage at 48C and
cryopreservation by vitrification (21968C). The hairy roots
regenerated from cryopreserved root tips grew well, showing the
same ginsenoside productivity and patterns as those of control hairy
roots cultured continuously at 258C. Hwang et al. (1996) found that
growth in light conditions favored ginsenoside synthesis, while
darkness favored hairy root growth.
While hairy root cultures have thus been established in various
laboratories, ginsenoside content in these hairy root lines was
consistently low. A wide variety of elicitors have been employed to
alter cell metabolism in order to enhance the production of
secondary metabolites in plant cell culture (Dornenburg and Knorr,
1995; Chang et al., 1998). Among these elicitors, jasmonic acid has
been shown to be an effective elicitor for secondary metabolite
induction in plant cell cultures (Ketchum et al., 1999). Activation of
secondary metabolite biosynthesis by jasmonic acid was also
reported in Catharanthus roseus and Cinchona ledgeriana seedlings
(Aerts et al., 1994); in cell-suspension cultures of Rauwolfia
canescens (Gundlach et al., 1992); and in cell-suspension cultures
of Taxus canndensis (Ketchum et al., 1999). Until now, however, we
are unaware of any reports of the application of jasmonic acid to
ginseng cultures. In this experiment, jasmonic acid and some other
elicitors were tested for their ability to promote ginsenoside
accumulation in ginseng hairy root cultures.

424
 GINSENOSIDE PRODUCTION IN HAIRY ROOTS OF GINSENG
Materials and Methods
Establishment of ginseng hairy root lines. Fresh ginseng (Panax ginseng
C. A. Meyer) roots (4 yr old) were collected from Pung-Kee province in
Korea. Selected roots were washed with a detergent solution (AIC Co.,
Korea) for 5±10 min, then rinsed with running tap water for 5±10 min. After
being soaked in 70% aqueous EtOH for 1 min, they were rinsed in sterile
distilled water, further sterilized with 1% (v/v) sodium hypochlorite (with a
few drops of Tween 20; Wako, Osaka, Japan) for 15±30 min, then rinsed
three times with sterile distilled water. The sterilized roots were cut into
sections of 2±10 mm, then inoculated into nutrient broth (NB; Difco,
Georgia, USA) liquid medium containing actively growing Agrobacterium
rhizogenes KCTC 2703 for 16 h at 288C. After overnight culture, explants
were transferred to MS (Murashige and Skoog, 1962) solid medium
containing 3% sucrose for 2±3 d, and finally transferred to MS solid
medium containing 300±500 mg l21 Claforan (cefotaxine sodium, Handok,
Seoul, Korea) until roots emerged. The roots which appeared on the
inoculation sites were removed individually and transferred to phytohor-
mone-free MS solid medium containing 300±500 mg l21 Claforan. Rapidly
growing roots with no bacterial contamination were used to establish lines of
hairy root cultures. Untransformed roots were excised from sterile, normal
plants. Transformed and untransformed roots were grown in 40 ml
phytohormone-free MS liquid medium supplemented with 3% sucrose, in
100 ml Erlenmeyer flasks on an orbital shaker at 100 rpm. All root cultures
were initiated from inoculation of 0.15 g FW, maintained in the dark at
25 ^ 28C; and subcultured to fresh medium every 5 wk. Transformation was
confirmed by polymerase chain reaction (PCR) experiments.
Proliferation of hairy roots. After 6 mo., hairy root line CHR 1 was
selected from seven established hairy root lines according to growth rate and
ginsenoside content. CHR 1 was subcultured every 5 wk in 400 ml
Erlenmeyer flasks with 100 ml phytohormone-free half-strength MS medium
containing 3% sucrose, on an orbital shaker (100 rpm) at 25 ^ 28C in a dark
room. An air-lift balloon-type bioreactor (Son et al., 1999b) (5 l), containing
3 l phytohormone-free MS medium with 3% sucrose, was employed to
proliferate the hairy roots, which were used as explants for further
experiments. The air-flow rate was maintained at 0.1 v/v (air volume/
medium volume) min21. The layout of the air-lift balloon-type bioreactor is
shown in Fig. 1.
Effect of jasmonic acid on ginsenoside accumulation. Ginseng hairy roots
(4 g FW) were inoculated into 400 ml Erlenmeyer flasks containing 100 ml
phytohormone-free MS liquid medium supplemented with 3% sucrose, and
different concentrations of jasmonic acid (Sigma; 0, 1, 2 and 5 mg l21; 0,
4.8, 9.5 and 23.8 mM, respectively). There were three replicate flasks for
each treatment. The hairy roots were harvested after culturing for 5 wk at
25 ^ 28C on an orbital shaker (100 rpm) in a dark room.
Effects of some other elicitors on ginsenoside accumulation. The culture
method and medium were the same as used in the jasmonic acid
experiments. The following elicitors at different concentrations were added
Fig. 1. Air-lift balloon-type bubble bioreactor layout. (a) Air compres-
sor; (b) after-cooler and air dryer; (c) air-flow meter; (d) membrane filter; (e)
glass sparger; (f) sampling port; (g) connector for medium exchange; (h)
condenser.
425
to the medium according to their effects on other plants (Dornenburg and
Knorr, 1995): 300 mg l21 phenylalanine; 300 mg l21 caffeic acid; 100 and
300 mg l21 catechin; 1000 mg l21 chitin; 1000 mg l21 gum karaya;
200 mg l21 fucoidan; and 300 mg l21 peptone (all Sigma).
Determination of root weight and growth yield. Root FW and DW were
determined as follows. Roots were separated from the medium by passing
through a 1 mm stainless steel sieve. Root FW was measured after rinsing
once with tap water and blotting away surface water, and root DW was
recorded after roots were dried to a constant weight at 708C for several days.
The hairy root growth yield was calculated as:
Growth yield ˆ harvested DW …g†=inoculated DW …g†:
Determination of ginsenoside content and productivity. Analysis of
ginseng ginsenosides was modified according to Son et al. (1999a). Dried,
pulverized roots (1 g) were extracted with 60% methanol (40 ml) at 1008C
for 3 h and vacuum-filtered through filter paper (Advantec, Tokyo). The
extract was evaporated to dryness and dissolved in 10 ml HPLC-grade water.
The water-soluble extract was passed slowly through a SEP-PAK C18
cartridge (Waters, Massachusetts, USA), which was then eluted with 10 ml
methanol. The resulting ginsenoside fraction was analyzed using a Waters
2690 HPLC system with a 996 photodiode array detector and Millenium
2010 data manager under Altec Platinum C18 column (diameter 1.5 mm,
33  7 mm†; with water and acetonitrile. The ratios of water to acetonitrile
for the first 10 and last 25 min were 75:25 and 63:37, respectively. The
mobile phase flow rate was 1.0 ml min21, and monitoring of ginsenosides
was conducted at 203 nm. Authentic ginsenosides were purchased from
Wako. The total ginsenoside content was calculated as the sum of individual
ginsenoside fractions.
The ginsenoside content of ginseng hairy roots was calculated as:
Ginsenoside content …mg g21 †
ˆ sample ginsenoside concentration …from HPLC† …mg l21 †
 sample volume …l†=hairy root DW …g†:
The ginsenoside productivity of ginseng hairy roots was calculated as:
Ginsenoside productivity …mg l21 † ˆ total ginsenoside content …mg g21 †
 harvested hairy root DW …g†=volume of culture medium …l†:
Results
Effects of jasmonic acid on ginseng hairy root growth and
ginsenoside production. Jasmonic acid showed obvious effects on
both ginseng hairy root growth and ginsenoside production (Table 1).
Jasmonic acid strongly inhibited ginseng hairy root growth. The
FW, DW and growth yield all decreased with jasmonic acid
concentration in the range 1.0±5.0 mg l21. On the other hand,
jasmonic acid strongly improved ginsenoside production. After
growth in 5.0 mg l21 jasmonic acid for 5 wk, the greatest total
ginsenoside contents attained were 58 mg g21 (four times the
control) and 504 mg l21 (twice the control), respectively. However,
this was not a universal effect, as Rb group ginsenoside content was
more obviously improved than Rg group ginsenoside content. The
Rb group content increased steadily with the jasmonic acid
concentration in the range 1.0±5.0 mg l21, while the Rg group
was only slightly improved. The Rg group of ginsenoside showed no
significant change in the presence of 1.0±5.0 mg l21 jasmonic
acid.
Table 2 presents detailed content changes of the different
ginsenosides. The ginsenosides Rb1, Rb2, Rc and Rd belong to the
Rb group (protopanaxadiols), while Re, Rg1 and Rf belong to the Rg
group (protopanaxatriols) (Kushiro et al., 1997). Rb1 and Rb2
426 YU ET AL.

TABLE 1

EFFECTS OF JASMONIC ACID ON GROWTH AND GINSENOSIDE PRODUCTION OF GINSENG HAIRY ROOTS AFTER CULTURE FOR 5 WK

Biomass Ginsenoside (mg g21 DW) Rb

Ginsenoside
Jasmonic acid (mg l21) FW (g) DW (g) Growth yield Rb Rg Total Rg productivity (mg l21)
0.0 30.2 a 1.52 a 7.12 10.31 d 5.51 a 15.85 d 1.92 d 240.92 d
1.0 24.5 b 1.31 b 6.12 30.08 c 5.87 a 35.98 c 5.14 c 471.34 c
2.0 20.0 c 1.08 c 5.04 41.59 b 6.05 a 47.69 b 7.24 b 515.05 b
5.0 14.1 d 0.86 d 4.04 52.98 a 5.60 a 58.65 a 9.28 b 504.39 a

Means followed by different letters within a column are significant different at P , 0:05 by Duncan's multiple range test. Each treatment was repeated three
times.

TABLE 2 Effects of other elicitors on ginseng hairy root growth and

ginsenoside production. Phenylalanine, caffeic acid, catechin,
EFFECTS OF JASMONIC ACID ON GROWTH AND GINSENOSIDE Rg1, chitin, gum karaya, fucoidan, and peptone were tested as stimulants
Re, Rf, Rb1, Rc, Rb2 AND Rd PRODUCTION OF GINSENG HAIRY
ROOTS AFTER CULTURE FOR 5 WK
in ginseng hairy root cultures. After 5 wk culture, none showed a
promotive effect on ginseng hairy root growth except 100 mg l21
Ginsenoside (mg m21 DW) catechin (Table 3). The greatest ginseng hairy root growth yield
Jasmonic Rg group Rb group achieved was eight (1.04 times the control) after culture in
acid 100 mg l21 catechin for 5 wk; caffeic acid and fucoidan strongly
(mg l21) Rg1 Re Rf Rb1 Rc Rb2 Rc Total
inhibited ginseng hairy root growth. Addition of 300 mg l21
0 1.63 ab 3.02 a 0.87 a 7.23 d 1.20 b 1.13 d 0.1 15.85 d peptone resulted in a total ginsenoside content and productivity
1 1.85 a 3.49 a 0.52 a 18.99 c 5.56 a 3.44 c 2.3 35.98 c of 19 mg g21 (1.18 times the control) and 244 mg l21 (1.18 times
2 1.73 ab 3.69 a 0.63 a 24.09 b 7.26 a 4.62 b 4.6 47.69 b
5 1.34 b 3.74 a 0.61 a 33.70 a 6.19 a 8.80 a 4.3 58.65 a
the control), respectively, while showing no inhibition of ginseng
hairy root growth. These results suggest that peptone can also be
Means followed by different letters within a column are significant used to improve ginsenoside accumulation, while catechin could be
different at P , 0:05 by Duncan's multiple range test. Each treatment was employed to increase growth.
repeated three times.

ginsenosides increased 4.6 and 7.7 times, respectively, more than
controls lacking jasmonic acid. Although the Rc and Rd ginseno-
sides were not produced in as great overall quantities as the Rb1
and Rb2 ginsenosides, they also increased dramatically in response
to jasmonic acid applications of 5 mg l21. Jasmonic acid at
5.0 mg l21 appeared to inhibit production of Rc and Rd ginseno-
sides. In contrast, production of Rg ginsenoside was only slightly
affected by jasmonic acid (Table 2), but Rf decreased after
treatment with jasmonic acid (both non-significantly).
Discussion
As far as we know, this is the first report of the effects of jasmonic
acid and some other supplements on ginseng hairy root growth and
ginsenoside accumulation. Jasmonic acid in the range of 1.0±
5.0 mg l21 strongly promoted total ginsenoside content and
ginsenoside productivity, with particular stimulation of Rb group
ginsenoside content. At the same time, however, jasmonic acid
strongly inhibited ginseng hairy root growth. These results suggest
that it may be desirable to use a two-stage culture system for

TABLE 3

EFFECTS OF SOME OTHER SUPPLEMENTS ON GROWTH AND GINSENOSIDE PRODUCTION OF GINSENG HAIRY ROOTS AFTER CULTURE FOR
5 WK

Biomass
21
Treatment (mg l ) FW (g) DW (g) Growth yield Ginsenoside (mg g21 DW) Ginsenoside productivity (mg l21)
Control 0 22.5 cd 1.23 b 7.68 16.71 b 205.5 b
Phenylalanine 300 24.2 ab 1.11 c 6.94 16.11 b 178.8 c
Caffeic acid 300 17.5 e 0.79 d 4.94 8.93 f 70.5 f
Catechin 100 24.2 a 1.29 a 8.06 15.77 bc 203.4 b
Catechin 300 23.2 bc 1.22 b 7.63 14.86 cd 181.3 c
Chitin 1000 22.2 d 1.16 c 7.63 13.27 e 153.9 d
Gum karaya 1000 21.7 d 1.11 c 6.94 14.44 d 160.3 d
Fucoidan 200 17.3 e 0.81 d 5.06 12.58 e 101.9 e
Peptone 300 21.7 d 1.23 b 7.69 19.84 a 244.0 a

Means followed by different letters within a column are significant different at P , 0:05 by Duncan's multiple range test. Each treatment was repeated three
times.
 GINSENOSIDE PRODUCTION IN HAIRY ROOTS OF GINSENG
ginseng hairy root culture. In the first stage a medium without
elicitor could be used for growth, while in the second stage the
accumulated hairy root tissue would be transferred to fresh medium
containing jasmonic acid, in order to increase ginsenoside content.
The role of jasmonate as an elicitor, as well as its role in plant
signal transduction, has been reviewed by Sembdner and Parthier
(1993). In Lithospermum cell cultures, jasmonic acid and methyl
jasmonate were shown to induce biosynthesis of shikonin and
dihydroechinofuran (Yazaki et al., 1997), while Taxus cell
suspensions also respond to jasmonic acid and methyl jasmonate
elicitation with enhanced production of paclitaxel (Ketchum et al.,
1999). In C. roseus suspension cells cultured in a 2,4-D-starved
medium, exogenous jasmonic acid greatly increased alkaloid
production (Gantet et al., 1998). However, there are few reports
of the effects of jasmonates on hairy root cultures. Rijhwani and
Shanks (1998) reported that jasmonic acid induced variable
increases in the content of different indole alkaloids in hairy root
cultures of C. roseus, with no inhibitory effects on root growth. In our
experiments jasmonic acid also yielded a differential increase in
ginsenosides, but growth was inhibited. It may be that jasmonic acid
has a similar inductive effect on various parts of secondary
metabolisms, but may affect primary metabolism differently in
different species of hairy root.
Jasmonic acid (or its derivatives) is thought to be involved in
signal transduction controlling particular enzymes that catalyze
biosynthetic reactions to form low molecular-weight defense
compounds in plants (Mizukami et al., 1993; Urbanek et al.,
1996). In Lithospermum cell cultures, jasmonates caused a rapid
increase in the activities of enzymes involved in the biosynthesis of
shikonins such as p-hydroxybenzoate geranyltransferase (Yazaki et
al., 1997). Ginsenosides possess a triterpenoid dammarene skeleton
in their aglycone moiety. The aglycones of two major ginsenosides,
ginsenoside Rb and ginsenoside Rg, are protopanaxadiol and
protopanaxatriol, respectively, which are derived from dammar-
enediol by specific hydroxylations (Kushiro et al., 1997). It is not
known which enzymes are involved in the synthesis of protopanax-
adiol and protopanaxatriol ginsenosides. However, jasmonic acid
improved the accumulation of protopanaxadiol ginsenosides (Rb)
much more than the accumulation of protopanaxatriol ginsenosides
(Rg), which suggests that jasmonic acid stimulates the pathway
leading to protopanaxadiol ginsenoside synthesis more than
protopanaxatriol ginsenoside synthesis. Jasmonate-treated cultures
may provide a useful experimental system in which to learn more
about these key enzymes in ginsenoside biosynthesis.
Acknowledgments
This work was supported in part by a grant from the Research Center for
the Development of Advanced Horticultural Technology (HortTech),
Cheongju, Korea, and by a grant from the Ministry of Science and
Technology, Korea.
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