Appl Microbiol Biotechnol (2007) 77:543–550
DOI 10.1007/s00253-007-1192-5
BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Enhanced secondary metabolite (tanshinone) production
of Salvia miltiorrhiza hairy roots in a novel
root–bacteria coculture process
Jian-Yong Wu & Janet Ng & Ming Shi & Shu-Jing Wu
Received: 26 April 2007 / Revised: 1 September 2007 / Accepted: 3 September 2007 / Published online: 20 September 2007
# Springer-Verlag 2007
Abstract Salvia miltiorrhiza Bunge (Lamiaceae) hairy root
cultures were inoculated (at 0.02 and 0.2% v/v) and co-
cultured with Bacillus cereus bacteria. The root biomass
growth was inhibited significantly by the bacteria inocu-
lated to the root culture on the first day (day 0) but not by
the bacteria inoculated on days 14 or 21 (in a 28-day
overall period). On the other hand, the growth of the
bacteria in the hairy root culture was also strongly inhibited
by the hairy roots, partially because of the antibacterial
activity of the secondary compounds produced by the roots.
Most interestingly, the tanshinone production was promoted
by the inoculation of bacteria at any of these days but more
significantly by an earlier bacteria inoculation. With 0.2%
bacteria inoculated on day 0, for example, the total
tanshinone content of roots was increased by more than
12-fold (from 0.20 to 2.67 mg g−1 dry weight), and the
volumetric tanshinone yield increased by more than sixfold
(from 1.40 to 10.4 mg l−1). The tanshinone production was
also stimulated by bacterial water extract and bacterial
culture supernatant but less significantly than by the
inoculation of live bacteria. The results suggest that the
stimulation of tanshinone production by live bacteria in the root
cultures may be attributed to the elicitor compounds originating
from the bacteria, and the hairy root–bacteria coculture may be
an effective strategy for improving secondary metabolite
production in plant tissue cultures.
Keywords Salvia miltiorrhiza . Hairy roots .
Bacillus cereus . Coculture . Diterpenoid tanshinones .
Plant–microbe interaction
J.-Y. Wu (*) : J. Ng : M. Shi : S.-J. Wu
Department of Applied Biology and Chemical Technology,
The Hong Kong Polytechnic University,
Hung Hom,
Kowloon, Hong Kong
e-mail: bcjywu@polyu.edu.hk
Introduction
Plants synthesize a diverse array of secondary metabolites
that are important for them to survive and flourish in the
natural environment. The secondary metabolites play an
important role in plant–microbe interactions, particularly in
the plants’ defenses against pathogenic attacks. On the other
hand, secondary metabolites in plants represent a major
source of bioactive natural products as valuable pharma-
ceuticals and nutraceuticals. Plant tissue cultures (including
cell and organ cultures) are useful experimental models for
the study of plant stress physiology and secondary metab-
olism and efficient bioprocesses for the mass production of
valuable secondary metabolites, particularly from those rare
and slow-growing plant species.
Hairy roots are plant roots transformed by the infection
of wounded plant tissues with Agrobacterium rhizogenes.
Because of the rapid hormone-free growth and high genetic
stability of hairy roots, hairy root culture is a convenient
tool for plant science research and a promising production
system for medicinal plants and their active constituents
(Giri and Narasu 2000; Guillon et al. 2006). Salvia
miltiorrhiza Bunge (Lamiaceae) is a well-known herbal
plant and its root, which is known as Danshen in Chinese,
has been widely used in Chinese medicine for the treatment
of menstrual disorders and cardiovascular diseases and for
the prevention of inflammation (Tang and Eisenbrand 1992;
Wang et al. 2007). The diterpenoid pigments, which are
generally known as tanshinones, are the major bioactive
constituents of S. miltiorrhiza roots. Hairy root cultures of
S. miltiorrhiza have been established as a potential means
for the production of tanshinones and other bioactive
compounds (Hu and Alfermann 1993; Chen et al. 1999).
As the accumulation of many secondary metabolites in
plants is a defense response of the plants to microbial path-
ogens, microbial components such as cell wall fragments of
544
fungi and bacteria can be used as elicitors to induce and
stimulate the production of secondary metabolites in plant
tissue cultures (Rao and Ravishankar 2002; Zhao et al.
2005). For example, a carbohydrate fraction of yeast extract
has been used effectively to stimulate tanshinone produc-
tion in S. miltiorrhiza hairy root cultures (Chen et al. 2001;
Ge and Wu 2005). To the best of our knowledge, however,
there is still no reported work on the use of live microbial
cells to stimulate secondary metabolite production in plant
tissue cultures. In routine laboratory practice of plant tissue
cultures, the presence of microbial cells in culture is re-
garded as contamination, which would strongly inhibit or
completely stop the growth of plant tissue or cells. In most
previous studies on plant responses to pathogens, live
microbial cells have been mainly applied to the tissue of
living plants (in vivo), and microbial cell fragments or com-
pounds have been applied to plant tissue and cell cultures
(in vitro), to induce the responses for investigation (Ebel
and Cosio 1994; Zhao et al. 2005). Nevertheless, a few
groups have directly inoculated live bacteria to plant cell
cultures, such as the zoospores of Phytophthora nicotianae
(Able et al. 2001, 2003) and a mutant strain of
Pseudomonas syringae (Keppler et al. 1989; Atkinson et
al. 1985, 1990) to tobacco cells in suspension cultures, to
induce and study the pathogenic responses of plants. In
these studies, these live bacteria cells were as effective as
the crude fractions or purified compounds of microbial cells
to induce or elicit defense responses such as hypersensitive
reaction, membrane cation ion fluxes (Ca2+, K+, and H+),
and production of reactive oxygen species (ROS).
Plant defense mechanisms can also be stimulated by
beneficial and growth-promoting microorganisms, especial-
ly the rhizobacteria that colonize the roots of plants.
Bacillus cereus is one of the most common rhizobacterial
species that have been shown to enhance plant resistance to
bacterial and fungal pathogens (Kloepper et al. 2004; Barea
et al. 2005). As an initiative to explore a novel and effec-
tive means for enhancing the secondary metabolite
production in plant hairy root cultures, this study was to
examine the growth and secondary metabolite production
of S. miltiorrhiza hairy roots during the coculture with a
bacterium B. cereus, as well as the influence of hairy roots
on bacteria growth.
Materials and methods
Hairy root culture
The S. miltiorrhiza hairy root culture was derived after the
infection of plantlets with a Ri T-DNA-bearing A.
rhizogenes bacterium (ATCC15834; Chen et al. 1999).
The stock culture of the hairy roots was maintained on a
Appl Microbiol Biotechnol (2007) 77:543–550
solid, hormone-free MS medium (Murashige and Skoog
1962) with 8 g l−1 agar, 30 g l−1 sucrose, and 0.5 g l−1
casein hydrolysate but without ammonium nitrate, at 25°C
in the dark. Experiments in this study were all performed
in shake-flask cultures in a liquid medium of the same
composition as of the solid culture but excluding the agar
as described previously (Ge and Wu 2005). Each flask
(125-ml Erlenmeyer) was filled with 25 ml medium and
inoculated with 0.2 g fresh roots from 3-week-old shake-
flask cultures. The shake-flask hairy root culture was run
for an overall period of 28 days, during which some flasks
were taken out at selected days for measurement. All
treatments were performed in triplicate and repeated at
least once, and the results were averaged.
Measurement of root weight and residual sucrose
and total nitrogen in medium
The hairy roots were picked out from the liquid medium
with a pair of forceps, rinsed thoroughly with distilled
water, and then blotted dry by paper towels (yielding the
fresh weight), and then dried at 45–50°C in an oven until
constant dry weight (dw). The liquid medium was collected
for the measurement of residual nutrients (sucrose and total
nitrogen), pH, and conductivity. Sucrose content in the
liquid medium was determined by the Anthrone method
using sucrose as a standard (Ebell 1969). Total nitrogen was
determined by the standard Kjeldahl method (ASTM3590,
2002) using NH4Cl as a reference and expressed as the total
Kjeldahl nitrogen. The medium pH and conductivity were
measured with the respective electrodes.
Bacterial culture and inoculation into hairy root culture
The B. cereus bacterium (Gram-positive) was taken from
our departmental microbial culture storage. During this
study, the bacterial culture was maintained on a solid
Luria–Bertani (LB) medium with 15 g l−1 agar at 4°C. The
bacteria inocula for the root–bacteria coculture experiments
were prepared by growing the bacteria in liquid LB
medium (pH 7.0) at 30°C in shake flasks at 250 rpm for
2 h, reaching a maximum optical density (OD) value of 0.5
(pH 6.5). The culture broth was inoculated into the hairy
root culture in shake flasks at 0.02 and 0.2% (v/v; 5–50 μl
broth in 25 ml medium) on days 0, 14, or 21 of the hairy
root culture. Bacteria cell density in the LB medium and the
MS medium was evaluated by the OD measured at 600 nm
with a spectrophotometer.
In addition to live bacteria, bacterial extract and bacterial
culture supernatant (used medium) were also tested of their
effects on the hairy root growth and tanshinone production.
For preparation of the bacterial extract, bacterial cells were
harvested from the B. cereus culture in the LB medium
Appl Microbiol Biotechnol (2007) 77:543–550
(after 2 h incubation) by filtration. The cell mass was
resuspended in distilled water (5 g in 100 ml) and autoclaved
at 121°C for 20 min. The autoclaved cell suspension was
centrifuged at 6,000 rpm for 5 min, and the supernatant was
collected as the bacterial extract. The concentration of the
bacterial extract was represented by the total carbohydrate
content, which was determined by the Anthrone test using
glucose as a standard. In this study, a fixed concentration
100 μg ml−1 of bacterial extract was chosen based on our
previous studies using the carbohydrate fraction of yeast
extract as an elicitor in the hairy root culture (Ge and Wu
2005). The bacterial extract was fed to the root cultures on
day 0 or 21.
The bacterial culture supernatant for the test was
collected from the B. cereus bacterial culture in the MS
medium with or without the hairy roots in the culture at
various days (days 1, 7, 21) of the culture period. The hairy
root culture medium without the bacteria was also collected
(on day 21) and tested for comparison. The bacteria- and
root-free culture supernatants were obtained by filtration of
the culture broth and then sterilized by filtration through a
0.2-μm membrane. In all experiments, 5 ml of the sterile
bacterial or root culture supernatant was mixed with 20 ml
fresh MS medium on the first day (day 0) of the root culture
in each culture flask.
Analysis of tanshinones in roots
The extraction and analysis of tanshinones from the roots
followed the procedures as described by Ge and Wu (2005).
In brief, the dried hairy roots were ground into powder and
extracted with 4:1 methanol/dichloromethane (~10 mg ml−1),
and the extract was then evaporated to dryness and
redissolved in 9:1 methanol/dichloromethane. The tanshi-
none content in the extract solution was determined by high-
performance liquid chromatography on a HP1100 system
using a C18 column, 55:45 acetonitrile/water as the mobile
phase, and UV detection at 275 nm. The total tanshinone
(TT) content shown in the results is the sum of three
tanshinone species cryptotanshinone (CT), tanshinone-I
(T1), and tanshinone-IIA (T2A), which were detected and
quantified with authentic standards obtained from the
Institute for Identification of Pharmaceutical and Biological
Products (Beijing). The tanshinone content in the medium
was negligible compared with that in the roots and not
measured.
Hydrogen peroxide measurement
Hydrogen peroxide (H2O2) in the culture medium was
measured by chemiluminescence through ferricyanide-
catalyzed oxidation of luminol as described by Wang and
Wu (2005). Briefly, 50-μl medium was added to 750 μl
545
potassium phosphate buffer (0.05 M, pH 7.9). The
luminescence was recorded after the last injection at an
integration time of 5 s, and the luminescence intensity was
calibrated to H2O2 concentration with pure H2O2 liquid
(30 wt% in water from Junsei Chemical, Tokyo, Japan).
Results
Effects of live bacteria on hairy root growth and nutrient
consumption
The B. cereus bacteria inoculated into the hairy root culture
on day 0 had a negative effect on the root growth,
decreasing the root dw (on day 28) from 7.07 g l−1 in the
control culture to 4.25 g l−1 with 0.02% bacteria inoculum
and to 3.90 g dw l−1 with 0.2% bacteria inoculum
(Fig. 1a1). However, the bacteria inoculated to the root
culture on day 14 (Fig. 1a2) or later (day 21, Table 1) did
not cause any significant changes in the root growth (with
the same root weight of 7.0–7.1 g dw l−1 on day 28). The
bacteria inoculated to the hairy root culture on day 0 also
resulted in a slower consumption of the major nutrients, as
shown by the higher residual concentrations of sucrose (as
the major carbon source; Fig. 1b1) and total nitrogen
(Fig. 1c1), and a higher medium conductivity (attributed to
metal ions and minerals) (Fig. 1d1). Similar to the effects of
bacteria on the hairy root growth, the nutrient consumption
rates were not significantly influenced by the inoculation of
bacteria to the root cultures on day 14 (Fig. 1b2, c2, and d2)
or later (e.g., day 21, Table 1). On the other hand, the
inoculation of bacteria into the root cultures at any day (0, 14,
or 21) caused a rapid and notable drop in the medium pH
from 5.8 to about 4.0 (Fig. 1d). The addition of the bacterial
suspension (pH 6.5) caused a slight increase (no more than
0.2 units) in the initial medium pH, and the bacteria cultured
in the MS medium without roots caused a slight pH drop (no
more than 0.3 units; data not shown).
Inhibition of bacteria growth by the hairy roots
In the shake flasks at 25°C, the B. cereus bacteria cultivated
in the MS root culture medium without the roots had a
specific growth rate (in the exponential phase) of 0.61 h−1
and a maximum cell density of 1.54 OD (on day 14), which
were only slightly lower than those in the LB bacteria
medium (Fig. 2a and Table 2, all based on OD measure-
ment). The lower bacteria growth rate in the MS medium
should be attributed mostly to its less favorable medium
composition than the LB medium for bacteria growth. The
bacteria growth rate was further suppressed in the MS
medium cocultured with the hairy roots, with a specific
growth rate of 0.57 h−1 (between 3 and 7 h) and a maximum
546 Appl Microbiol Biotechnol (2007) 77:543–550

Fig. 1 Time courses of S.

Bacteria inoculation on day 0 Inoculation on day 14
miltiorrhiza hairy root cultures
inoculated with 0.02 and 0.2% 10
B. cereus on days 0 (block 1) or Control a1 a2
14 (block 2): a Root biomass 8 0.02% Bacteria

Root dry wt (g l-1)

growth or dry weight; b sucrose
consumption (residual sucrose 0.2% Bacteria
6
in culture medium); c nitrogen
consumption (residual total
4
Kjeldahl nitrogen in culture me-
dium); d Medium pH and con-
ductivity (error bars for 2
standard deviation, n=3) 0
35
Sucrose inmedium (g l-1) b1 b2
30
25
20
15
10
5
250
TKN in medium (mg l-1)

225 c1 c2
200
175
150
125
100

d1 d2
6.0
Conductivity (mS).

5.0
Medium pH

4.0
3.0
2.0 pH
1.0 Conductivity
0.0
0 4 8 12 16 20 24 28 12 16 20 24 28
Time (day)

cell density (OD) of 0.89 (on day 14). The difference between
the bacterial cultures with and without the roots was even
larger over the later days (days 3–14), during which the
culture with the roots had only a small increase in the bacteria
cell density (OD from 0.75 to 0.90), while that in the same
medium but without the roots increased significantly (OD
from 0.69 to 1.54; Fig. 2b). The results suggest that the hairy
roots had an inhibitory effect on the bacteria growth, which
became more significant as the amount of roots in the culture
increased with time.
In addition to the above quantitative results, our observa-
tion during the experiments indicated that the bacteria growth
or survival in the hairy root culture was very dependent on
casein hydrolysate, a complex organic supplement that was
routinely added to the medium at 0.5 g l−1. The root culture
medium was fairly clear and transparent (low OD) when
Appl Microbiol Biotechnol (2007) 77:543–550 547

Table 1 Root growth and tanshinone production of S. miltiorrhiza hairy root cultures with various bacteria treatments (28-day overall culture
period)

Treatment Root dry weight (g l−1) Tanshinone content (mg g−1 dw) Tanshinone yield (mg l−1)

Live bacteria (bacteria inoculation vol.% and time)

Control (no bacteria) 7.07±0.72 0.20±0.02 1.40
0.02%, day 0 4.25±0.13 1.98±0.12 8.41
0.2%, day 0 3.90±0.27 2.67±0.16 10.40
0.2%, day 14 6.82±0.45 1.25±0.05 8.54
0.2%, day 21 7.10±0.21 0.79±0.07 5.59
Bacterial extract (100 mg l−1)
Control 6.78±0.32 0.17±0.02 1.15
Fed on day 0 9.90±0.64 0.54±0.10 5.35
Fed on day 21 8.20±0.35 0.41±0.07 3.36
Bacterial culture medium (25% v/v, fed to hairy root culture on day 0)
Control 5.31±0.14 0.16±0.01 0.85
24-h bacterial culture without roots 5.14±0.18 0.75±0.06 3.84
21-day bacterial–root coculture medium 5.01±0.26 0.79±0.09 3.95

casein hydrolysate was excluded from the medium, suggest- the volumetric tanshinone yield was increased by about
ing that the bacteria did not grow well in the MS medium 3.5-fold.
without casein hydrolysate. The bacterial culture supernatant or used bacterial
culture medium (in the absence of hairy roots) had a
negligible effect on the root growth if the culture
Effects of live bacteria on tanshinone production
of hairy roots
2.0
The hairy root culture receiving 0.02 and 0.2% bacteria Without roots a
inoculum on day 0, 14, or 21 all achieved a higher TT 1.5 With roots
OD at 600 nm

content in the roots than the bacteria-free control culture;

the culture receiving a larger bacteria inoculum at an earlier 1.0
day during the culture usually had a higher tanshinone
content, i.e., day 0>day 14>day 21 (Fig. 3a, Table 1). The
root culture inoculated with bacteria also attained a higher 0.5
volumetric tanshinone yield (the product of root dw and TT
content), although the final root dw was significantly lower 0.0
than that of the control (3.90 vs 7.07 g dw l−1; Fig. 3b, 0 5 10 15
Table 1). In this set of results, the culture inoculated with
Time (day)
0.2% bacteria on day 0 attained the highest tanshinone
content of 2.67 mg g−1 dw and the highest volumetric yield
of 10.4 mg l−1 (on day 28), which were 13.5- and 7.6-fold 2% inoculum b
1.5
of those in the control, 0.20 mg g−1 dw and 1.40 mg l−1,
OD at 600 nm

0.2%
respectively.
1.0 0.02%

Effects of bacterial extract and culture supernatant

on root growth and tanshinone production 0.5

The bacterial extract enhanced both the hairy root growth

and tanshinone biosynthesis, more significantly when it was 0.0
fed to the hairy root culture on day 0 than on day 21 0 2 4 6 8
(Table 1). With the bacterial extract fed on day 0, the root
Time (h)
weight increased by nearly 50% (9.9 vs 6.78 g dw l−1 in
Fig. 2 Bacteria growth curves a in the MS root culture medium with
the control), and the TT content of roots increased by about and without the hairy roots and b in the LB bacteria medium (all with
twofold, 0.54 vs 0.17 mg g−1 dw in control. Accordingly, 0.2% bacteria inoculum)
548 Appl Microbiol Biotechnol (2007) 77:543–550

Table 2 Bacteria growth data at various conditions (inoculated at 0.2%)

Culture Maximum OD Growth rate Log-growth

conditions (μmax, h−1) period (h)

LB medium 1.75 (at 7 h) 0.755 1.5–7

(30°C)
LB medium 1.64 (at 7 h) 0.677 3–7
(25°C)
MS with roots 0.90 (on day14) 0.571 3–7
(25°C)
MS without roots 1.54 (on day 14) 0.614 3–7 Fig. 4 Effects of bacterial culture broth (used MS medium, collected at
(25°C) various days, days 1, 7, and 21) on hairy root growth and total tanshinone
(TT) content (25 vol.% used medium added to the root cultures on
day 0; 28-day overall culture period; error bars for SD, n=3)
supernatant was collected from the bacterial culture on
day 7 or earlier but had a positive effect when it was coculture supernatant collected on day 21, increasing the
collected on day 21 (Fig. 4; Table 1). The stimulating effect tanshinone content of roots by nearly fourfold from 0.16 to
of the older bacteria medium (on day 21 but not on day 7) 0.79 mg g−1 dw.
was perhaps due to the release of root growth-stimulating Figure 5 shows the contents of three individual tanshi-
compounds from the bacteria, which was consistent with none species CT, T1, and T2A in the roots, which were all
the stimulating effect of the bacterial extract on the root stimulated by the three bacterial treatments, live bacteria,
growth. The root culture supernatant collected on day 21 bacterial extract, and bacterial culture medium. There was a
caused a negative effect on the root growth probably because notable difference in the extents of stimulation of the
of the presence of waste metabolites. The bacterial culture different tanshinone species by these treatments.
and the root–bacteria coculture supernatants collected at any
of the 3 days stimulated the tanshinone accumulation in the H2O2 production induced by live bacteria in hairy root
hairy roots, most significantly with the root–bacteria cultures

As shown in Fig. 6, the inoculation of 0.2% bacteria into

the hairy root cultures induced a sharp and rapid increase in
the hydrogen peroxide (H2O2) level, reaching a peak of
about 100 times over the initial and control level within
1.5 h after inoculation. The inoculation of bacteria at a
lower concentration of 0.02% (v/v) also induced a rapid but

Fig. 5 Contents of three major tanshinones, cryptotanshinone (CT),

Fig. 3 Tanshinone production of S. miltiorrhiza hairy root cultures
inoculated with 0.2% bacteria on days 0 and 14 (28-day overall
culture period;): a total tanshinone (TT) content of roots; b volumetric
yield of total tanshinone (error bars for SD, n=3)
tanshinone-I (T1), and tanshinone-IIA (T2A) in the hairy roots treated
with various bacterial culture components: 0.2% B. 0.2% live bacteria,
B.E. 100 mg l−1 bacteria cell extract, B.med. bacterial culture
supernatant collected at 24 h, B.-R. med. root–bacteria coculture
supernatant collected on day 21 (all fed to the hairy root cultures on
day 0; error bars for SD, n=3)
Appl Microbiol Biotechnol (2007) 77:543–550
120 Control
0.02% Bacteria
100
0.2% Bacteria
Relative H2O2 level
8 period (Fig. 2a) may be due partially to the accumulation of
80
6 antibacterial compounds in the culture medium. Moreover,
60 4 the significantly lower medium pH in the root–bacteria
2 coculture than the optimal pH 6.5–7.0 (in the LB medium)
40
0
0 2
20
0
0 2 4 6 8 10
Time after inoculation (h)
Fig. 6 Hydrogen peroxide (H2O2) level (relative to the initial level in
the control culture) in hairy root cultures after bacteria inoculation
(error bars for SD, n=3)
smaller increase in the H2O2 level, to a peak of about 6.8
times higher than the initial and control level in 1 h. At both
bacteria concentrations, the bacteria-induced H2O2 increase
in the hairy root cultures could be detected within 10 min of
inoculation. The results show that the bacteria inoculation
induced a rapid and transient production of H2O2 in the
hairy root cultures, and the production level was bacteria
concentration dependent. Likewise, the increase in H2O2
production also occurred in the root cultures treated with
the bacterial extract and culture media (data not shown).
Discussion
The above experimental results clearly show that the
coculture of S. miltiorrhiza hairy roots with the Bacillus
bacteria can enhance the accumulation of diterpenoid
tanshinones in the hairy roots. Although the presence of
live bacteria in the hairy root culture also caused an
inhibition of the hairy root growth, the stimulation of
secondary metabolite accumulation was much more sub-
stantial, thus leading to a significant net increase in the
volumetric yield of tanshinones. On the other hand, the
bacteria growth in the hairy root culture was also inhibited
by the hairy roots, perhaps because of the antimicrobial
effects of compounds produced by the roots. It has been
reported that some of the tanshinones in Danshen, such as
CT and dihydrotanshinone-I, have a strong antibacterial
activity on a broad range of Gram-positive bacteria including
two strains of Bacillus subtilis, although they are less active
against Gram-negative bacteria (Lee et al. 1999). The
antibacterial compounds released to the medium (although
at very low concentrations) may contribute to the preven-
tion of proliferation of B. cereus (also a Gram-positive
species) in the S. miltiorrhiza hairy root culture, allowing
549
for the continued growth and proliferation of the roots for
many passages in the presence of bacteria. The more
pronounced inhibition of bacteria growth in the later culture
for the bacteria growth could also contribute to the bacteria
4 6 growth inhibition.
The most possible cause for the enhanced tanshinone
production in the hairy root–bacteria coculture is the action
of some elicitor compounds generated and released by the
12
B. cereus bacteria. The rapid and transient production of
ROS H2O2 in the hairy root cultures induced by the bacteria
(Fig. 6) is a characteristic response of plants to elicitors
(Ebel and Cosio 1994; Zhao et al. 2005). This deduction is
further supported by the stimulating effects of the bacteria
cell extract and the used bacterial culture medium as shown
in our experiments. Because the bacterial culture and the
root–bacteria coculture supernatants had a similar stimulat-
ing effect on the tanshinone production, these elicitor
compounds should mainly be the constituents or the
extracellular products of bacteria cells instead of the
products of root–bacteria interaction. The much stronger
stimulation by the live bacteria than the bacterial extract
and the bacterial culture supernatant may be due to that the
live bacteria’s provision of a source of fresh and active
elicitors or stimulants for the secondary metabolite biosyn-
thesis. Another possible cause for the enhanced tanshinone
production in the root–bacteria coculture is the stress
conditions imposed by the bacteria to the roots, such as
the medium pH drop, which may stimulate the secondary
metabolite biosynthesis of roots. However, the stress be-
cause of the competition for nutrients or nutrient deficiency
can be ruled out as a significant factor because the con-
sumption rates of nutrients including sugar, nitrogen, and
metal and minerals did not show any increase after the
bacteria inoculation. In this connection, the nutrients should
be utilized mainly by the roots but not by the bacteria. The
very low maximum cell density as well as the low growth
rate of bacteria in the hairy root culture (Fig. 1 and Table 2)
also imply that the bacteria cells did not consume much of
the major growth nutrients available in the culture medium.
The pH drop after bacteria inoculation (Fig. 2d) was
most probably caused by the extrusion of protons H+ from
plant cells (by the action of plasma membrane H+-ATPase)
and/or the production of acidic metabolites by the bacteria
cells. Similar to our results, a pH drop was also observed in
bacteria-challenged tobacco cell culture within a few hours
of postbacteria inoculation (Able et al. 2001). This seems to
be in contrast to the medium alkalinization resulting from the
H+ influx/K+ efflux exchange, which commonly occurs in
plant tissue cultures after elicitor treatment (Atkinson et al.
550
1985, 1990; Keppler et al. 1989; Zhao et al. 2005). It has
been suggested that the acidification or alkalinization of the
extracellular medium is dependant on the difference
between the race-specific and nonspecific microbial elic-
itors (De Wit 1995). Moreover, transient cytoplasmic
acidification has been detected concomitantly with the
extracellular alkalinization and regarded as a necessary step
in signaling the elicitor-induced secondary metabolite
biosynthesis in plant cells (Roos et al. 1998; Sakano 2001).
In conclusion, the inoculation of a Bacillus bacterium
into the S. miltiorrhiza hairy root culture dramatically
enhanced the tanshinone production of roots. The enhanced
secondary metabolite accumulation in the roots may be a
defense response of the hairy roots induced by some elicitor
compounds released from the bacteria cells living in the
root cultures. Compared with the chemical elicitors,
however, the live bacteria may have different and more
complex interactions with the roots such as cell–cell, gene–
gene, and protein–protein interactions, and their involve-
ment in the root–bacteria coculture process remains to be
investigated. To the best of our knowledge, this is the first
report on plant tissue growth and secondary metabolite
production in plant tissue–microbe coculture system and on
the stimulation of secondary metabolite biosynthesis in
plant tissue cultures by live microbial cells. The hairy root–
bacteria coculture used in this study may present a novel
and effective strategy for improving the production of
valuable secondary metabolites in plant tissue cultures as
well as a convenient experimental system for the study of
plant–microbe interactions.
Acknowledgments This work was supported by grants (G-U150
and A-PE94) from The Hong Kong Polytechnic University.
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