Applied Microbiology and Biotechnology

, Volume 87, Issue 1, pp 137–144 | Cite as

Effects of biotic and abiotic elicitors on cell growth and tanshinone accumulation in Salvia miltiorrhiza cell cultures

Biotechnological Products and Process Engineering


This study examined the effects of biotic and abiotic elicitors on the production of diterpenoid tanshinones in Salvia miltiorrhiza cell culture. Four classes of elicitors were tested, heavy metal ions (Co2+, Ag+, Cd2+), polysaccharides (yeast extract and chitosan), plant response-signaling compounds (salicylic acid and methyl jasmonate), and hyperosmotic stress (with sorbitol). Of these, Ag (silver nitrate), Cd (cadmium chloride), and polysaccharide from yeast extract (YE) were most effective to stimulate the tanshinone production, increasing the total tanshinone content of cell by more than ten-fold (2.3 mg g-1 versus 0.2 mg g-1 in control). The stimulating effect was concentration-dependent, most significant at 25 μM of Ag and Cd and 100 mg l-1 (carbohydrate content) of YE. Of the three tanshinones detected, cryptotanshinone was stimulated most dramatically by about 30-fold and tanshinones I and IIA by no more than 5-fold. Meanwhile, most of the elicitors suppressed cell growth, decreasing the biomass yield by about 50% (5.1–5.5 g l-1 versus 8.9 g l-1 in control). The elicitors also stimulated the phenylalanine ammonia lyase activity of cells and transient increases in the medium pH and conductivity. The results suggest that the elicitor-stimulated tanshinone accumulation was a stress response of the cells.


Salvia miltiorrhiza Cell culture Tanshinones Elicitors Stress response 


Salvia miltiorrhiza Bunge (Lamiaceae), called Danshen in Chinese, is a well-known and important medicinal plant because its root is an effective herb for treatment of menstrual disorders and cardiovascular diseases and for the prevention of inflammation (Tang and Eisenbrand 1992). As its Chinese name refers, Danshen root is characterized by the abundance of red pigments which are mainly ascribed to numerous diterpene quinones generally known as tanshinones, e.g., tanshinone I (T-I), tanshinone-IIA (T-IIA), and T-IIB, isotanshinone I and II, and cryptotanshinone (CT). Tanshinones constitute a major class of bioactive compounds in S. miltiorrhiza roots with proven therapeutic effects and pharmacological activities (Wang et al. 2007). Danshen in combination with a few other Chinese herbs is an effective medicine widely used for the treatment of cardiovascular diseases and used as an emergency remedy for coronary artery disease and acute ischemic stroke. According to WHO statistics, cardiovascular diseases are and will continue to be the number one cause of death in the world ( It is of significance to develop more efficient means for the production of Danshen and its active constituents.

Although field cultivation is currently the major production means for Danshen and most other plant herbs, plant tissue cultures provide more well-controlled and sustainable systems for efficient production of desired bioactive compounds of the herb. Plant tissue cultures are the most useful and convenient experimental systems for examining various factors on the biosynthesis of desired products and for exploring effective measures to enhance their production. The importance of Danshen for traditional and modern medicines has promoted the long-lasting research interest in the development of S. miltiorrhiza tissue cultures for production of bioactive compounds for more than two decades. In an early study, Nakanishi et al. (1983) induced several cell lines from plant seedlings and screened out a cell line capable of producing significant amounts of CT and another diterpene, ferruginol. In later studies, the group performed a fuller evaluation and optimization of the medium for cell growth and CT production and, eventually, derived an effective production medium with a simpler composition (ten components) than the original Murashige and Skoog (MS) medium (about 20 components), achieving a high CT yield of 110 mg l-1 (Miyasaka et al. 1987). Many recent studies have been focused on hairy root cultures of S. miltiorrhiza transformed by Agrobacterium rhizogenes (Hu and Alfermann 1993; Chen et al. 2001) and by our group (Zhang et al. 2004; Ge and Wu 2005; Shi et al. 2007).

Most of the bioactive compounds in medicinal plants belong to secondary metabolites which are usually less abundant than primary metabolites in the plants. Since the accumulation of secondary metabolites in plants is a common response of plants to biotic and abiotic stresses, their accumulation can be stimulated by biotic and abiotic elicitors. Therefore, elicitation, treatment of plant tissue cultures with elicitors, is one of the most effective strategies for improving secondary metabolite production in plant tissue cultures (Chong et al. 2005; Smetanska 2008). The most common and effective elicitors used in previous studies include the components of microbial cells especially poly- and oligosaccharides (biotic) and heavy metal ions, hyperosmotic stress, and UV radiation (abiotic), and the signaling compounds in plant defense responses such as salicylic acid (SA) and methyl jasmonate (MJ; Zhou and Wu 2006; Smetanska 2008). Some of these elicitors, yeast extract (mainly the polysaccharide fraction), silver ion Ag+, and hyperosmotic stress (by an osmoticum) have also been applied and shown effective to enhance the production of tanshinones in S. miltiorrhiza hairy root cultures (Chen et al. 2001; Zhang et al. 2004; Shi et al. 2007).

To the best of our knowledge, only a few studies have been documented on the effects of elicitors, YE, SA, and MJ, on the secondary metabolite production in Agrobacterium tumefaciens transformed S. miltiorrhiza cell cultures from one research group (Chen and Chen 1999, 2000) but not any study in normal cell cultures. The present study focuses on the effects of common biotic and abiotic elicitors including polysaccharides, heavy metal ions, SA and MJ, and osmotic stress (with sorbitol) on the growth and accumulation of three major tanshinones T-I, T-IIA, and CT in suspension culture of normal S. miltiorrhiza cells. In addition to the effects of various elicitors on the total tanshinone content of cells, the study will examine the effects on different tanshinone species and the potential relationship to plant stress response.

Material and methods

Callus induction and cell suspension culture

Young stem explants of S. miltiorrhiza Bunge were collected from the botanical garden at the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Beijing, China, in May 2005. The fresh explants were washed with tap water, surface-sterilized with 75% ethanol for 1 min, and then soaked in 0.1% mercuric chloride for 10 min and rinsed thoroughly with sterilized water. The clean and sterilized explants were cut into ∼0.5-cm segments and placed on solid MS medium (Murashige and Skoog 1962) supplemented with sucrose (30 g l-1), 2,4-D (2 mg l-1) and 6-BA (2 mg l-1) to induce callus formation. The callus culture of S. miltiorrhiza was maintained on a solid, hormone-free MS medium with 8 g l-1 agar and 30 g l-1 sucrose at 25°C in the dark and subcultured every 4 weeks. The culture was deposited in Lab Y1210 at The Hong Kong Polytechnic University with a collection number of Danshen cell-1. All experiments in this study were performed in suspension culture of S. miltiorrhiza cells in a liquid medium of the same composition as for the solid culture but excluding agar. The cell suspension culture was maintained in shake-flasks, i.e., 125-ml Erlenmeyer flasks on an orbital shaker operated at 110–120 rpm, at 25°C in the dark. Each of the flasks was filled with 25 ml medium and inoculated with 0.3 g fresh cells from 18–21-day-old shake–flask culture.

Elicitor preparation and administration

Eight elicitors were tested, each at three concentrations, in the initial elicitation experiments (Table 1). These are representative of the four major classes of elicitors for the induction of plant responses and the stimulation of secondary metabolite production in plant tissue cultures (Zhou and Wu 2006; Smetanska 2008). All elicitors except MJ were prepared as a concentrated stock solution in water and autoclaved at 121°C for 15 min, and stored at 4°C in a refrigerator prior to use. Yeast elicitor (YE) was the polysaccharide fraction of yeast extract (Y4250, Sigma, St. Louis, MO, USA) prepared by ethanol precipitation as described previously (Hahn and Albersheim 1978; Ge and Wu 2005). In brief, yeast extract was dissolved in distilled water (20 g/100 ml) and then mixed with 400 ml of ethanol and allowed to precipitate for 4 days at 4°C in a refrigerator. The precipitate was redissolved in 100 ml of distilled water and subjected to another round of ethanol precipitation. The final gummy precipitate was dissolved in 50 ml of distilled water and stored at 4°C before use. The concentration of YE was represented by total carbohydrate content which was determined by the Anthrone test using sucrose as a reference. Chitosan solution was prepared by dissolving 0.5 g crab shell chitosan (C3646, Sigma) in 1 ml glacial acetic acid at 55–60°C for 15 min, and then the final volume was adjusted to 50 ml with distilled water and the pH adjusted to 5.8 with NaOH (Prakash and Srivastava 2008). MJ (Cat.39, 270-7, Sigma-Aldrich) was dissolved in 95% ethanol and sterilized by filtering through a microfilter (0.2 µm). SA (10,591-0, Sigma-Aldrich), sorbitol (S3755, Sigma), and the salts of heavy metals including cobalt chloride (C8661, Sigma-Aldrich), silver nitrate (S7276, Sigma-Aldrich), and cadmium chloride (C5081, Sigma-Aldrich) were dissolved in distilled water to the desired concentrations and adjusted to pH 5.8.
Table 1

Elicitors and concentrations tested in the initial experiments







Cobalt chloride (Co)





Silver nitrate (Ag)





Cadmium chloride (Cd)





Salicylic acid (SA)





Methyl jasmonate (MJ)





Yeast elicitor (YE)

mg l-1




Chitosan (CH)

mg l-1




Sorbitol (SO)

g l-1




Elicitor treatment was administered to the shake–flask culture of S. miltiorrhiza cell on day 18, which was about 2–3 days before reaching the stationary phase. This time point is usually favorable for elicitation when the biomass concentration is high (compared with earlier days of growth), and the cell metabolism is still active (compared with that during or after stationary phase; Buitelaar et al. 1992; Cheng et al. 2006). Each of the elicitor solutions was added into the culture medium with a micropipette at the desired concentration. After the elicitor addition, the shake–flask culture of cells was maintained for another 7 days and then harvested for analysis. All treatments were performed in triplicate, and the results were averaged. After the initial experiments on the eight elicitors, the three most effective ones, Ag (25 µM), Cd (25 µM), and YE (100 mg l-1) were applied in the following experiments on the time courses of elicitor-treated cell growth and tanshinone accumulation in the S. miltiorrhiza cell culture.

Measurement of cell weight, sucrose concentration, medium pH, and conductivity

The cells were separated from the liquid medium by filtration. The cell mass on the filter paper was rinsed thoroughly with water and filtered again, and blotted dry by paper towels and then dried at 50°C in an oven to attain the dry weight. Sucrose concentration in the liquid medium was determined by the Anthrone test using sucrose as a reference (Ebell 1969), and the medium pH and conductivity were measured with the respective electrodes on an Orion 720A+ pH meter (Thermo Fisher Scientific, Inc., Beverly, MA, USA) and a CD-4303 conductivity meter (Lutron, Taiwan), respectively.

Measurement of PAL activity

Phenylalanine ammonia lyase (PAL) was extracted from fresh S. miltiorrhiza cells with borate buffer (pH 8.8). The cells were ground in the buffer (0.15 g ml-1) for 2 min with a pestle and mortar on ice, and then centrifuged at 10,000 rpm and 4°C for 20 min to obtain a solid-free extract. The PAL activity was determined based on the conversion of l-phenylalanine to cinnamic acid as described by Wu and Lin (2002).

Analysis of tanshinone contents

The cell mass from culture was dried and ground into powder and extracted with methanol/dichloromethane (4:1, v/v, 10 mg ml-1) under sonication for 60 min. After removal of the solid, the liquid extract was evaporated to dryness and redissolved in methanol/dichloromethane (9:1, v/v). Tanshinone content was determined by high performance liquid chromatography (HPLC) on a HP1100 system using C18 column, acetonitrile/water (55:45, v/v) as the mobile phase, and UV detection at 275 nm as described previously (Shi et al. 2007). Three tanshinone species CT, T-I, and T-IIA were detected and quantified with authentic standards obtained from the Institute for Identification of Pharmaceutical and Biological Products (Beijing, China). Total tanshinone content is the total content of the three tanshinones in the cells. Tanshinone content in the culture medium was negligible and not determined.


Cell growth and tanshinone accumulation in S. miltiorrhiza cell culture

The time course of S. miltiorrhiza cell growth exhibited a lag phase or slow growth period in the first 3–6 days, a rapid, linear growth period between day 9–18, and a stationary or declining phase in the later days, reaching the maximum biomass concentration (8.1 g l-1) around day 21. The total tanshinone content of cells remained at a very low level from days 1–12 and then increased steadily from days 12–27 to a maximum of 0.16 mg g-1. A significant portion (65%) of the tanshinone accumulation or content increase occurred during the stationary phase from days 21–27 (Fig. 1a), which is characteristic of secondary metabolite production in a batch culture process. The time course of sugar (sucrose) concentration (Fig. 1b) was nearly symmetrical to that of cell growth, indicating a direct correlation of the cell growth (or biomass production) to sugar consumption. As the major carbon source, sugar was required for the S. miltiorrhiza cell growth, and when it was depleted (around day 21), the cell growth stopped, and the biomass concentration began to drop. As seen from Fig. 1b, the medium pH showed a notable drop in the first 3 days (due to consumption of NH4+ and release of protons) and a gradual increase after day 6 (due to consumption of nitrate NO3-) (Morard et al. 1998).
Fig. 1

Time courses of biomass and total tanshinone content (a), residue sugar (sucrose) and medium pH (b) in S. miltiorrhiza cell cultures (error bars for standard deviations, n = 3)

Effects of various elicitors on cell growth and tanshinone production

Figure 2 shows the effects of elicitor treatments on the cell growth and tanshinone accumulation in S. miltiorrhiza cell cultures, which were dependent both on the elicitor species and elicitor dose. As seen from Fig. 2a, most of the elicitor treatments except Co2+ and sorbitol at lower concentrations suppressed the cell growth to a lower biomass concentration than that of the untreated control culture, and the growth suppression was more severe at a high elicitor dose. On the other hand, most of the elicitor treatments except Co2+, sorbitol, SA, and MJ at lower concentrations increased the total tanshinone content of cell to a higher level than in the control (Fig. 2b). Overall the results indicated that the enhancement of tanshinone accumulation by an elicitor treatment concurred with a notable suppression of cell growth or biomass production. Nevertheless, some of the elicitors had a much stronger stimulating effect on the tanshinone accumulation than the suppressing effect on the cell growth. In particular, Ag and Cd both at 25 μM, and YE at 100 mg l-1 increased the total tanshinone content to 2.30 mg g-1, about 11.5-fold versus that of the control (0.20 mg g-1), but decreased the biomass production by no more than 50% (5.1–5.5 g l-1 versus 8.9 g l-1). Another three elicitors, SA, MJ (both at 50 μM), and sorbitol (50 g l-1) increased the total tanshinone content by 2–3-fold but decreased the biomass by 30–45% compared with the control. The stimulating effect of chitosan on tanshinone accumulation (about 6-fold) was stronger than SA, MJ, and sorbitol but much weaker than Ag, Cd, and YE, while its suppressing effect on the cell growth was as severe as Ag, Cd, and YE. In summary, the results indicate that Ag, Cd, YE are the most favorable elicitors for the tanshinone production in S. miltiorrhiza cell culture and were used in the following experiments.
Fig. 2

Effects of various elicitors on biomass growth (a) and tanshinone production (b) in S. miltiorrhiza cell cultures (elicitors added to cultures on day 18 at three concentrations C1, C2, and C3 as shown in Table 1, and cultures harvested on day 25; error bars for standard deviations, n = 3)

Figure 3 shows the time courses of cell growth and tanshinone production after treatment with the three most effective elicitors Ag (25 μM), Cd (25 μM), and YE (100 mg l-1) and the control culture. All three elicitor treatments caused a steady decline of biomass concentration from initially 8.5 g l-1 to 5.3 g l-1 on day 6 while biomass in the control culture was increased during this period (Fig. 3a). In the meantime, the tanshinone content of cells in the three elicitor-treated cultures increased sharply and most rapidly by Ag (from 0.14 to 1.98 mg g-1), while that of control increased slightly (from 0.14 to 0.21 mg g-1; Fig. 3b). The volumetric total tanshinone yields (the products of total tanshinone content and cell dry weight) were 1.9 mg l-1 in the control, and 9.2 mg l-1, 10.7 mg l-1 and 11.7 mg l-1 in cultures treated with 100 mg l-1 YE, 25 μM Cd, and Ag, respectively (on day 6).
Fig. 3

Time courses of biomass (a) and total tanshinone content (b) in S. miltiorrhiza cell cultures after treatment with Ag (25 µM), Cd (25 µM), and YE (100 mg l-1; error bars for standard deviations, n = 3)

Another test was performed on the effects of two and three elicitors in combinations in the S. miltiorrhiza cell culture. As shown in Fig. 4, the tanshinone content was increased about 20% with either two elicitors and about 40% with all three elicitors in combination compared with that with a single elicitor. The results suggest an additive or synergistic effect of these elicitors on the tanshinone accumulation in the cells. However, the combined use of two or three elicitors also suppressed the cell growth (biomass) more severely than with a single elicitor.
Fig. 4

Effects of single and combined elicitors on S. miltiorrhiza cell growth and tanshinone accumulation (elicitors added to cell cultures on day 18 at the same concentrations as in Fig. 3, and cultures harvested on day 25; error bars for standard deviations, n = 3)

Effects of elicitor treatments on different tanshinone species

Of the three tanshinone species detected, CT was stimulated most significantly by all elicitors without exception; T-IIA was stimulated by most elicitors, and T-I was stimulated significantly only by chitosan but slightly stimulated or suppressed by other elicitors (Table 2). The highest CT content was about 2 mg g-1 (1,854–2,011 μg g-1) in cell cultures treated with 25 μM Ag and Cd, and 100 mg l-1 YE, about 31–34 fold of the control level (60 μg g-1), the highest T-I content 0.27 mg g-1 with 100 mg l-1 chitosan (3.4-fold of the control 80 μg g-1) and the highest T-IIA content 0.37 mg g-1 with 25 μM Cd (6-fold of the control 60 μg g-1). As seen from the HPLC chromatograms (Fig. 5), the cultures treated with the three different elicitors exhibited a similar profile with virtually identical major peaks. The experimental results do not suggest any specificity of particular tanshinone species to the type of elicitors, YE and chitosan as biotic polysaccharides, Cd and Ag as abiotic heavy metals, or SA and MJ as plant stress signaling compounds. Compared with that of control, the HPLC profiles of elicitor-treated cultures also had three new unknown peaks appearing before the CT peak, between 10.0–11.5 min and a high peak at 11.1 min, which may be ascribed to tanshinone relatives of higher polarity than CT induced by the elicitors.
Table 2

Effects of various elicitors on the accumulation of three tanshinones in S. miltiorrhiza cells


Content, μg/g (fold of content control)





59.9 (1)

81.6 (1)

57.6 (1)


263.7 (4.4)

67.5 (0.83)

55.5 (0.96)


1,817.5 (30)

71.0 (0.87)

225.8 (3.9)


1,854.0 (31)

80.3 (0.98)

369.0 (6.4)


390.0 (6.5)

78.5 (0.96)

72.8 (1.3)


299.8 (5.0)

109.5 (1.3)

82.6 (1.4)


2,011.4 (34)

90.3 (1.1)

190.3 (3.3)


597.2 (10)

276.0 (3.4)

98.8 (1.7)


584.6 (9.8)

56.9 (0.70)

83.0 (1.4)

CT cryptotanshinone, T-I tanshinone I, T-IIA tanshinone-IIA

aNumber after each elicitor symbol represents the elicitor concentration as shown in Table 1

Fig. 5

HPLC profiles of tanshinones in S. miltiorrhiza cells from a control culture and culture treated with b Ag (25 µM), c Cd (25 µM), and d YE (100 mg l-1; peak 1 for CT, 2 for T-I, and three for T-IIA)

PAL activity, pH, and conductivity changes induced by elicitors

Figure 6 shows the changes of intracellular PAL activity and medium pH and conductivity in the S. miltiorrhiza cell culture after the treatment by Ag (25 μM), Cd (25 μM), and YE (100 mg l-1). The PAL activity of cells was stimulated by all three elicitors to the similar level, from 1.4- to 1.9-fold of the control level over 6 days (Fig. 6a). PAL is a key enzyme at the entrance step in the phenylpropanoid pathway in plants, and its activity increase stimulated by the elicitors is suggestive of an enhanced secondary metabolism in the plant cells (Taiz and Zeiger 2006). The pH and conductivity of culture medium were also increased (to higher levels than those of the control) by all three elicitors but more significantly by YE (Fig. 6b, c). Most significant increases (differences from the control level) in the medium pH and conductivity were shown in the very early period from day 0–1. The increase in medium conductivity in the early period was most probably attributed to the release of potassium K+ ion from the cells or K+ efflux across the cell membrane (Zhang et al. 2004). Transient medium pH increase (alkalinization) and K+ efflux across the cell membrane are early and important events in the elicitation of plant responses and phytoalexin production (Ebel and Mithöer 1994; Roos et al. 1998). The conductivity decline in the later period after day 1 of Ag+ and Cd2+-treated cultures and the control cultures can be attributed to the consumption of inorganic and mineral nutrients in the culture medium (Kinooka et al. 1991). Overall, the results here provide further evidence for the elicitor activities of Ag, Cd, and YE in stimulating the stress responses and secondary metabolism of the S. miltiorrhiza cells.
Fig. 6

Time courses of PAL activity (a), medium pH (b), and conductivity (c) of S. miltiorrhiza cell cultures after elicitor treatments in comparison with the control (error bars for standard deviation, n = 3)


The effects of various elicitors on tanshinone accumulation found here in the normal S. miltiorrhiza cell cultures are in general agreement with those found in transformed cell and hairy root cultures of S. miltiorrhiza. In transformed cell cultures (Chen and Chen 1999), the CT accumulation was also stimulated significantly by YE but not by SA or MJ alone, and YE also inhibited the cell growth. The tanshinone (mainly CT) production in hairy root cultures was also enhanced significantly (3–4 fold) by Ag (Zhang et al. 2004) and YE (Shi et al. 2007). In all these culture systems, CT was the major tanshinone species stimulated by various elicitor treatments. CT has been identified as a phytoalexin in S. miltiorrhiza plant which plays a defense role against pathogen invasion of the plant (Chen and Chen 2000). In this connection, the stimulated CT accumulation by the elicitors may be a defense or stress response of the cells. CT was also the major diterpenoid produced by a normal S. miltiorrhiza cell line which was initially grown in the MS medium and then transferred to a production medium containing only about half of the nutrient components of the MS medium (Miyasaka et al. 1987). It is very possible that the improvement of CT yield in this production medium was also attributed, at least partially, to the stress imposed by the nutrient deficiency which suppressed growth but stimulated secondary metabolite accumulation.

MJ or its relative jasmonic acid has been shown effective for stimulating a variety of secondary metabolites in plant tissue cultures such as hypericin in Hypericum perforatum L. (St. John’s Wort) cell cultures (Walker et al. 2002), paclitaxol (diterpenoid) and related taxanes in various Taxus spp. and ginsenosides in Panax spp. (Zhong and Yue 2005), and bilobalide and ginkgolides in Ginkgo biloba cell cultures (Kang et al. 2006). However, MJ showed only a moderate or insignificant stimulating effect on tanshinone accumulation in normal and transformed S. miltiorrhiza cell cultures. The discrepancy suggests that the effects of various elicitors on secondary metabolite production in plant tissue cultures are dependent on the specific secondary metabolites. This argument is also supported by the much stronger stimulation of CT than T-I and T-IIA by most elicitors found in our S. miltiorrhiza cell cultures. In addition, the hairy roots appeared more tolerant to the elicitor stress, and the growth was less inhibited by the elicitors or even enhanced in some cases, e.g., by YE (Chen et al. 2001) and sorbitol (Shi et al. 2007). Moreover, sorbitol as an osmotic agent significantly stimulated the tanshinone accumulation (3–4 folds) in S. miltiorrhiza hairy root cultures, but not so significantly in the cell cultures. This shows that the elicitor activities for the same metabolites can vary with the tissue culture systems.

In conclusion, the polysaccharide fraction of yeast extract and two heavy metal ions Ag+ and Cd2+ were potent elicitors for stimulating the tanshinone production in S. miltiorrhiza cell culture. The stimulated tanshinone production by most elicitors was associated with notable growth suppression. CT was more responsive to the elicitors and enhanced more dramatically than another two tanshinones, T-I and IIA. The results from this study in the S. miltiorrhiza cell cultures and from previous studies in hairy root cultures suggest that the cell and hairy root cultures may be effective systems for CT production, provided with the elicitors. As most of the elicitor chemicals are commercially available or can be readily prepared in the laboratory and easily administered to the cell and root cultures, they are suitable for practical applications in the laboratory or large-scale production.



This work was supported by grants from The Hong Kong Polytechnic University (G-U502 and 1-BB80) and the China Hi-Tech Research and Development Program (2006AA10A209).


  1. Buitelaar RM, Cesário MT, Tramper J (1992) Elicitation of thiophene production by hairy roots of Tagetes patula. Enzyme Microb Technol 14:2–7CrossRefGoogle Scholar
  2. Chen H, Chen F (1999) Effects of methyl jasmonate and salicylic acid on cell growth and cryptotanshinone formation in Ti transformed Salvia miltiorrhiza cell suspension cultures. Biotechnol Lett 21:803–807CrossRefGoogle Scholar
  3. Chen H, Chen F (2000) Effect of yeast elicitor on the secondary metabolism of Ti-transformed Salvia miltiorrhiza cell suspension cultures. Plant Cell Rep 19:710–717CrossRefGoogle Scholar
  4. Chen H, Chen F, Chiu FCK, Lo CMY (2001) The effect of yeast elicitor on the growth and secondary metabolism of hairy root cultures of Salvia miltiorrhiza. Enzyme Microb Technol 28:100–105CrossRefGoogle Scholar
  5. Cheng XY, Zhou HY, Cui X, Ni W, Liu CZ (2006) Improvement of phenylethanoid glycosides biosynthesis in Cistanche deserticola cell suspension cultures by chitosan elicitor. J Biotechnol 121:253–260CrossRefGoogle Scholar
  6. Chong TM, Abdullah MA, Lai QM, Nor’Aini FM, Lajis NH (2005) Effective elicitation factors in Morinda elliptica cell suspension culture. Process Biochem 40:3397–3405CrossRefGoogle Scholar
  7. Ebel J, Mithöer A (1994) Early events in the elicitation of plant defence. Planta 206:335–348CrossRefGoogle Scholar
  8. Ebell LF (1969) Variation in total soluble sugars of conifer tissues with method of analysis. Phytochemistry 8:227–233CrossRefGoogle Scholar
  9. Ge XC, Wu JY (2005) Tanshinone production and isoprenoid pathways in Salvia miltiorrhiza hairy roots induced by Ag+ and yeast elicitor. Plant Sci 168:487–491CrossRefGoogle Scholar
  10. Hahn MG, Albersheim P (1978) Host-pathogen interactions XIV. Isolation and partial characterization of elicitor from yeast extract. Plant Physiol 62:107–111CrossRefGoogle Scholar
  11. Hu ZB, Alfermann AW (1993) Diterpenoid production in hairy root cultures of Salvia miltiorrhiza. Phytochemistry 32:699–703CrossRefGoogle Scholar
  12. Kang SM, Min JY, Kim YD, Kang YM, Park DJ, Jung HN, Kim SW, Choi MS (2006) Effects of methyl jasmonate and salicylic acid on the production of bilobalide and ginkgolides in cell cultures of Ginkgo biloba. In Vitro Cell Dev Biol Plant 42:44–49CrossRefGoogle Scholar
  13. Kinooka M, Taya M, Tone S (1991) Conductometric estimation of main inorganic nutrients in plant cell culture. J Chem Eng Jpn 24:381–384CrossRefGoogle Scholar
  14. Miyasaka H, Nasu M, Yamamoto T, Shiomi Y, Ohno H, Endo Y, Yoneda K (1987) Effect of nutritional factors on cryptotanshinone and ferruginol production by cell suspension cultures of Salvia miltiorrhiza. Phytochemistry 26:1421–142CrossRefGoogle Scholar
  15. Morard P, Fulcheri C, Henry M (1998) Kinetics of mineral nutrient uptake by Saponaria officinalis L. suspension cell cultures in different media. Plant Cell Rep 18:260–265CrossRefGoogle Scholar
  16. Murashige T, Skoog FA (1962) A revised medium for rapid growth and bioassays with tobacco cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  17. Nakanishi T, Miyasaka H, Nasu M, Hashimoto H, Yoneda K (1983) Production of cryptotanshinone and ferruginol in cultured cells of Salvia miltiorrhiza. Phytochemistry 22:721–722CrossRefGoogle Scholar
  18. Prakash G, Srivastava AK (2008) Statistical elicitor optimization studies for the enhancement of azadirachtin production in bioreactor Azadirachta indica cell cultivation. Biochem Eng J 40:218–226CrossRefGoogle Scholar
  19. Roos W, Evers S, Hieke M, Tschöe M, Schumann B (1998) Shifts of intracellular pH distribution as a part of the signal mechanism leading to the elicitation of benzophenanthridine alkaloids phytoalexin biosynthesis in cultured cells of Eschscholtzia californica. Plant Physiol 118:349–364CrossRefGoogle Scholar
  20. Shi M, Kwork KW, Wu JY (2007) Enhancement of tanshinone production in Salvia miltiorrhiza Bunge (red or Chinese sage) hairy-root culture by hyperosmotic stress and yeast elicitor. Biotechnol Appl Biochem 46:191–196CrossRefGoogle Scholar
  21. Smetanska I (2008) Production of secondary metabolites using plant cell cultures. Adv Biochem Eng Biotechnol 111:187–228Google Scholar
  22. Taiz L, Zeiger E (2006) Plant physiology, 4th edn. Sinauer Assoc., SunderlandGoogle Scholar
  23. Tang W, Eisenbrand G (1992) Chinese drugs of plant origin: chemistry, pharmacology and use in traditional and modern medicine. Springer, Berlin, pp 891–902Google Scholar
  24. Walker TS, Bais HP, Vivanco JM (2002) Jasmonic acid-induced hypericin production in cell suspension cultures of Hypericum perforatum L. (St. John’s wort). Phytochemistry 20:289–293CrossRefGoogle Scholar
  25. Wang XB, Morris-Natschke SL, Lee KH (2007) New developments in the chemistry and biology of the bioactive constituents of Tanshen. Med Res Rev 27:133–148CrossRefGoogle Scholar
  26. Wu JY, Lin LD (2002) Ultrasound-induced stress responses of Panax ginseng cells: enzymatic browning and phenolics production. Biotechnol Prog 18:862–866CrossRefGoogle Scholar
  27. Zhang C, Yan Q, Cheuk WK, Wu JY (2004) Enhancement of tanshinone production in Salvia miltiorrhiza hairy root culture by Ag+ elicitation and nutrient feeding. Planta Med 70:147–151CrossRefGoogle Scholar
  28. Zhong JJ, Yue CJ (2005) Plant cells: secondary metabolite heterogeneity and its manipulation. Adv Biochem Eng Biotechnol 100:53–88Google Scholar
  29. Zhou LG, Wu JY (2006) Development and application of medicinal plant tissue cultures for production of drugs and herbal medicinals in China. Nat Prod Rep 23:789–810CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  1. 1.Department of Plant PathologyChina Agricultural UniversityBeijingChina
  2. 2.Department of Applied Biology and Chemical TechnologyThe Hong Kong Polytechnic UniversityKowloonHong Kong

Personalised recommendations