Documente Academic
Documente Profesional
Documente Cultură
DOI 10.1007/s00253-007-1192-5
Received: 26 April 2007 / Revised: 1 September 2007 / Accepted: 3 September 2007 / Published online: 20 September 2007
# Springer-Verlag 2007
fungi and bacteria can be used as elicitors to induce and solid, hormone-free MS medium (Murashige and Skoog
stimulate the production of secondary metabolites in plant 1962) with 8 g l−1 agar, 30 g l−1 sucrose, and 0.5 g l−1
tissue cultures (Rao and Ravishankar 2002; Zhao et al. casein hydrolysate but without ammonium nitrate, at 25°C
2005). For example, a carbohydrate fraction of yeast extract in the dark. Experiments in this study were all performed
has been used effectively to stimulate tanshinone produc- in shake-flask cultures in a liquid medium of the same
tion in S. miltiorrhiza hairy root cultures (Chen et al. 2001; composition as of the solid culture but excluding the agar
Ge and Wu 2005). To the best of our knowledge, however, as described previously (Ge and Wu 2005). Each flask
there is still no reported work on the use of live microbial (125-ml Erlenmeyer) was filled with 25 ml medium and
cells to stimulate secondary metabolite production in plant inoculated with 0.2 g fresh roots from 3-week-old shake-
tissue cultures. In routine laboratory practice of plant tissue flask cultures. The shake-flask hairy root culture was run
cultures, the presence of microbial cells in culture is re- for an overall period of 28 days, during which some flasks
garded as contamination, which would strongly inhibit or were taken out at selected days for measurement. All
completely stop the growth of plant tissue or cells. In most treatments were performed in triplicate and repeated at
previous studies on plant responses to pathogens, live least once, and the results were averaged.
microbial cells have been mainly applied to the tissue of
living plants (in vivo), and microbial cell fragments or com- Measurement of root weight and residual sucrose
pounds have been applied to plant tissue and cell cultures and total nitrogen in medium
(in vitro), to induce the responses for investigation (Ebel
and Cosio 1994; Zhao et al. 2005). Nevertheless, a few The hairy roots were picked out from the liquid medium
groups have directly inoculated live bacteria to plant cell with a pair of forceps, rinsed thoroughly with distilled
cultures, such as the zoospores of Phytophthora nicotianae water, and then blotted dry by paper towels (yielding the
(Able et al. 2001, 2003) and a mutant strain of fresh weight), and then dried at 45–50°C in an oven until
Pseudomonas syringae (Keppler et al. 1989; Atkinson et constant dry weight (dw). The liquid medium was collected
al. 1985, 1990) to tobacco cells in suspension cultures, to for the measurement of residual nutrients (sucrose and total
induce and study the pathogenic responses of plants. In nitrogen), pH, and conductivity. Sucrose content in the
these studies, these live bacteria cells were as effective as liquid medium was determined by the Anthrone method
the crude fractions or purified compounds of microbial cells using sucrose as a standard (Ebell 1969). Total nitrogen was
to induce or elicit defense responses such as hypersensitive determined by the standard Kjeldahl method (ASTM3590,
reaction, membrane cation ion fluxes (Ca2+, K+, and H+), 2002) using NH4Cl as a reference and expressed as the total
and production of reactive oxygen species (ROS). Kjeldahl nitrogen. The medium pH and conductivity were
Plant defense mechanisms can also be stimulated by measured with the respective electrodes.
beneficial and growth-promoting microorganisms, especial-
ly the rhizobacteria that colonize the roots of plants. Bacterial culture and inoculation into hairy root culture
Bacillus cereus is one of the most common rhizobacterial
species that have been shown to enhance plant resistance to The B. cereus bacterium (Gram-positive) was taken from
bacterial and fungal pathogens (Kloepper et al. 2004; Barea our departmental microbial culture storage. During this
et al. 2005). As an initiative to explore a novel and effec- study, the bacterial culture was maintained on a solid
tive means for enhancing the secondary metabolite Luria–Bertani (LB) medium with 15 g l−1 agar at 4°C. The
production in plant hairy root cultures, this study was to bacteria inocula for the root–bacteria coculture experiments
examine the growth and secondary metabolite production were prepared by growing the bacteria in liquid LB
of S. miltiorrhiza hairy roots during the coculture with a medium (pH 7.0) at 30°C in shake flasks at 250 rpm for
bacterium B. cereus, as well as the influence of hairy roots 2 h, reaching a maximum optical density (OD) value of 0.5
on bacteria growth. (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
Materials and methods root culture. Bacteria cell density in the LB medium and the
MS medium was evaluated by the OD measured at 600 nm
Hairy root culture with a spectrophotometer.
In addition to live bacteria, bacterial extract and bacterial
The S. miltiorrhiza hairy root culture was derived after the culture supernatant (used medium) were also tested of their
infection of plantlets with a Ri T-DNA-bearing A. effects on the hairy root growth and tanshinone production.
rhizogenes bacterium (ATCC15834; Chen et al. 1999). For preparation of the bacterial extract, bacterial cells were
The stock culture of the hairy roots was maintained on a harvested from the B. cereus culture in the LB medium
Appl Microbiol Biotechnol (2007) 77:543–550 545
(after 2 h incubation) by filtration. The cell mass was potassium phosphate buffer (0.05 M, pH 7.9). The
resuspended in distilled water (5 g in 100 ml) and autoclaved luminescence was recorded after the last injection at an
at 121°C for 20 min. The autoclaved cell suspension was integration time of 5 s, and the luminescence intensity was
centrifuged at 6,000 rpm for 5 min, and the supernatant was calibrated to H2O2 concentration with pure H2O2 liquid
collected as the bacterial extract. The concentration of the (30 wt% in water from Junsei Chemical, Tokyo, Japan).
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 Results
100 μg ml−1 of bacterial extract was chosen based on our
previous studies using the carbohydrate fraction of yeast Effects of live bacteria on hairy root growth and nutrient
extract as an elicitor in the hairy root culture (Ge and Wu consumption
2005). The bacterial extract was fed to the root cultures on
day 0 or 21. The B. cereus bacteria inoculated into the hairy root culture
The bacterial culture supernatant for the test was on day 0 had a negative effect on the root growth,
collected from the B. cereus bacterial culture in the MS decreasing the root dw (on day 28) from 7.07 g l−1 in the
medium with or without the hairy roots in the culture at control culture to 4.25 g l−1 with 0.02% bacteria inoculum
various days (days 1, 7, 21) of the culture period. The hairy and to 3.90 g dw l−1 with 0.2% bacteria inoculum
root culture medium without the bacteria was also collected (Fig. 1a1). However, the bacteria inoculated to the root
(on day 21) and tested for comparison. The bacteria- and culture on day 14 (Fig. 1a2) or later (day 21, Table 1) did
root-free culture supernatants were obtained by filtration of not cause any significant changes in the root growth (with
the culture broth and then sterilized by filtration through a the same root weight of 7.0–7.1 g dw l−1 on day 28). The
0.2-μm membrane. In all experiments, 5 ml of the sterile bacteria inoculated to the hairy root culture on day 0 also
bacterial or root culture supernatant was mixed with 20 ml resulted in a slower consumption of the major nutrients, as
fresh MS medium on the first day (day 0) of the root culture shown by the higher residual concentrations of sucrose (as
in each culture flask. the major carbon source; Fig. 1b1) and total nitrogen
(Fig. 1c1), and a higher medium conductivity (attributed to
Analysis of tanshinones in roots metal ions and minerals) (Fig. 1d1). Similar to the effects of
bacteria on the hairy root growth, the nutrient consumption
The extraction and analysis of tanshinones from the roots rates were not significantly influenced by the inoculation of
followed the procedures as described by Ge and Wu (2005). bacteria to the root cultures on day 14 (Fig. 1b2, c2, and d2)
In brief, the dried hairy roots were ground into powder and or later (e.g., day 21, Table 1). On the other hand, the
extracted with 4:1 methanol/dichloromethane (~10 mg ml−1), inoculation of bacteria into the root cultures at any day (0, 14,
and the extract was then evaporated to dryness and or 21) caused a rapid and notable drop in the medium pH
redissolved in 9:1 methanol/dichloromethane. The tanshi- from 5.8 to about 4.0 (Fig. 1d). The addition of the bacterial
none content in the extract solution was determined by high- suspension (pH 6.5) caused a slight increase (no more than
performance liquid chromatography on a HP1100 system 0.2 units) in the initial medium pH, and the bacteria cultured
using a C18 column, 55:45 acetonitrile/water as the mobile in the MS medium without roots caused a slight pH drop (no
phase, and UV detection at 275 nm. The total tanshinone more than 0.3 units; data not shown).
(TT) content shown in the results is the sum of three
tanshinone species cryptotanshinone (CT), tanshinone-I Inhibition of bacteria growth by the hairy roots
(T1), and tanshinone-IIA (T2A), which were detected and
quantified with authentic standards obtained from the In the shake flasks at 25°C, the B. cereus bacteria cultivated
Institute for Identification of Pharmaceutical and Biological in the MS root culture medium without the roots had a
Products (Beijing). The tanshinone content in the medium specific growth rate (in the exponential phase) of 0.61 h−1
was negligible compared with that in the roots and not and a maximum cell density of 1.54 OD (on day 14), which
measured. were only slightly lower than those in the LB bacteria
medium (Fig. 2a and Table 2, all based on OD measure-
Hydrogen peroxide measurement ment). The lower bacteria growth rate in the MS medium
should be attributed mostly to its less favorable medium
Hydrogen peroxide (H2O2) in the culture medium was composition than the LB medium for bacteria growth. The
measured by chemiluminescence through ferricyanide- bacteria growth rate was further suppressed in the MS
catalyzed oxidation of luminol as described by Wang and medium cocultured with the hairy roots, with a specific
Wu (2005). Briefly, 50-μl medium was added to 750 μl growth rate of 0.57 h−1 (between 3 and 7 h) and a maximum
546 Appl Microbiol Biotechnol (2007) 77:543–550
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 became more significant as the amount of roots in the culture
the bacterial cultures with and without the roots was even increased with time.
larger over the later days (days 3–14), during which the In addition to the above quantitative results, our observa-
culture with the roots had only a small increase in the bacteria tion during the experiments indicated that the bacteria growth
cell density (OD from 0.75 to 0.90), while that in the same or survival in the hairy root culture was very dependent on
medium but without the roots increased significantly (OD casein hydrolysate, a complex organic supplement that was
from 0.69 to 1.54; Fig. 2b). The results suggest that the hairy routinely added to the medium at 0.5 g l−1. The root culture
roots had an inhibitory effect on the bacteria growth, which 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)
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
0.2%
respectively.
1.0 0.02%
120 Control for the continued growth and proliferation of the roots for
0.02% Bacteria many passages in the presence of bacteria. The more
100
0.2% Bacteria pronounced inhibition of bacteria growth in the later culture
Relative H2O2 level
1985, 1990; Keppler et al. 1989; Zhao et al. 2005). It has bacterial induction of the K+/H+ and hypersensitive responses in
tobacco. Plant Physiol 92:215–221
been suggested that the acidification or alkalinization of the
Barea JM, Pozo MJ, Azcón R, Azcón-Aguilar C (2005) Microbial co-
extracellular medium is dependant on the difference operation in the rhizosphere. J Exp Bot 56:1761–1778
between the race-specific and nonspecific microbial elic- Chen H, Chen F, Zhang YL, Song JY (1999) Production of
itors (De Wit 1995). Moreover, transient cytoplasmic lithospermic acid B and rosmarinic acid in hairy root cultures
of Salvia miltiorrhiza. J Ind Microbiol Biotechnol 22:133–138
acidification has been detected concomitantly with the
Chen H, Chen F, Chiu FCK, Lo CMY (2001) The effect of yeast elicitor
extracellular alkalinization and regarded as a necessary step on the growth and secondary metabolism of hairy root cultures of
in signaling the elicitor-induced secondary metabolite Salvia miltiorrhiza. Enzyme Microbial Technol 28:100–105
biosynthesis in plant cells (Roos et al. 1998; Sakano 2001). De Wit PJGM (1995) Fungal avirulence genes and plant resistance
genes: unravelling the molecular basis of gene for gene
In conclusion, the inoculation of a Bacillus bacterium interactions. Adv Bot Res 21:148–177
into the S. miltiorrhiza hairy root culture dramatically Ebell LF (1969) Variation in total soluble sugars of conifer tissues
enhanced the tanshinone production of roots. The enhanced with method of analysis. Phytochemistry 8:227–233
secondary metabolite accumulation in the roots may be a Ebel J, Cosio EG (1994) Elicitors of plant defense responses. Int Rev
Cytol 148:1–36
defense response of the hairy roots induced by some elicitor
Ge X, Wu J (2005) Tanshinone production and isoprenoid pathways in
compounds released from the bacteria cells living in the Salvia miltiorrhiza hairy roots induced by Ag+ and yeast elicitor.
root cultures. Compared with the chemical elicitors, Plant Sci 168:487–491
however, the live bacteria may have different and more Giri A, Narasu L (2000) Transformed hairy roots: recent trends and
applications. Biotechnol Adv 18:1–22
complex interactions with the roots such as cell–cell, gene–
Guillon S, Trémouillaux-Guiller J, Pati PK, Rideau M, Gantet P
gene, and protein–protein interactions, and their involve- (2006) Hairy root research: recent scenario and exciting
ment in the root–bacteria coculture process remains to be prospects. Curr Op Plant Biol 9:341–346
investigated. To the best of our knowledge, this is the first Hu ZB, Alfermann AW (1993) Diterpenoid production in hairy root
cultures of Salvia miltiorrhiza. Phytochemistry 32:699–703
report on plant tissue growth and secondary metabolite Keppler LD, Baker CJ, Atkinson MM (1989) Active oxygen
production in plant tissue–microbe coculture system and on production during a bacteria-induced hypersensitive reaction in
the stimulation of secondary metabolite biosynthesis in tobacco suspension cells. Phytopathology 79:974–978
plant tissue cultures by live microbial cells. The hairy root– Kloepper JW, Ryu CM, Zhang SA (2004) Induced systemic resistance
and promotion of plant growth by Bacillus spp. Phytopathology
bacteria coculture used in this study may present a novel
94:1259–1266
and effective strategy for improving the production of Lee DS, Lee SH, Noh JG, Hong SD (1999) Antibacterial activities of
valuable secondary metabolites in plant tissue cultures as cryptotanshinone and dihydrotanshinone I from a medicinal herb,
well as a convenient experimental system for the study of Salvia miltiorrhiza Bunge. Biosci Biotechnol Biochem 63:2236–
2239
plant–microbe interactions.
Murashige T, Skoog FA (1962) A revised medium for rapid growth
and biosynthesis with tobacco tissue cultures. Physiol Plant
Acknowledgments This work was supported by grants (G-U150 15:473–497
and A-PE94) from The Hong Kong Polytechnic University. Rao SR, Ravishankar GA (2002) Plant cell cultures: Chemical
factories of secondary metabolites. Biotechnol Adv 20:101–153
Roos W, Evers S, Hieke M, Tschöpe M, Schumann B (1998) Shifts of
intracellular pH distribution as a part of the signal mechanism
leading to the elicitation of benzophenan thridine alkaloids. Plant
References
Physiol 118:349–364
Sakano K (2001) Metabolic regulation of pH in plant cells: role of
Able AJ, Guest DI, Sutherland MW (2001) Relationship between cytoplasmic pH in defense reaction and secondary metabolism.
transmembrane ion movements, production of reactive oxygen Int Rev Cytol 206:1–44
species and the hypersensitive response during the challenge of Tang W, Eisenbrand G (1992) Chinese drugs of plant origin:
tobacco suspension cells by zoospores of Phytophthora nicotia- chemistry, pharmacology, and use in traditional and modern
nae. Physiol Mol Plant Pathol 58:189–198 medicine. Springer, Berlin, pp 891–902
Able AJ, Sutherland MW, Guest DI (2003) Production of reactive Wang JW, Wu JY (2005) Nitric oxide is involved in methyl
oxygen species during non-specific elicitation, non-host resis- jasmonate-induced defense responses and secondary metabolism
tance and field resistance expression in cultured tobacco cells. activities of Taxus cells. Plant Cell Physiol 46:923–930
Funct Plant Biol 30:91–99 Wang X, Morris-Natschke SL, Lee KH (2007) New developments in
Atkinson MM, Huang JS, Knopp JA (1985) The hypersensitive the chemistry and biology of the bioactive constituents of
reaction of tobacco to . Pseudomonas syringae pv. pisi. Plant Tanshen. Med Res Rev 27:133–148
Physiol 79:843–847 Zhao J, Davis LC, Verpoorte R (2005) Elicitor signal transduction
Atkinson MM, Keppler LD, Orlandi EW, Baker CJ, Mischke CF leading to production of plant secondary metabolites. Biotechnol
(1990) Involvement of plasma membrane calcium influx in Adv 23:283–333
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