Botanical Studies (2011) 52: 435-443.
PHYSIOLOGY
Enhanced tanshinone production in hairy roots of ‘Salvia miltiorrhiza Bunge’ under the influence of plant growth regulators in liquid culture
Sushim Kumar GUPTA1, Ren-Bang LIU2, Song-Yuan LIAW2, Hsiao-Sung CHAN1, and Hsin-Sheng
TSAY1,3,*
1Institute of Biochemical Sciences and Technology, Chaoyang University of Technology, Wufong, Taichung, Taiwan
2Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan
3Department of Agronomy, National Chung Hsing University, Taichung, Taiwan
(Received May 24, 2010; Accepted February 1, 2011)
ABSTRACT. Tanshinone constituents are the most potent diterpene diketones used to treat several diseases. We studied the influence of different plant growth regulators (PGR) on the growth and production of three tanshinone constituents using hairy root culture of Salvia miltiorrhiza Bunge. Leaves of the medicinally-im­portant plant S. miltiorrhiza were infected with Agrobacterium rhizogenes strain BCRC15010, which induced hairy roots in 78% of the explants. The best line of hairy roots was established on B5 liquid medium under dark conditions. The transformed nature of the root lines was confirmed by polymerase chain reaction using 'rol, B and C gene-specific primers. Adding different PGRs i.e., auxins, cytokinins and abscisic acid (ABA), enhanced the production of cryptotanshinone, tanshinone I and IIA. Tanshinone I and cryptotanshinone ac­cumulation was 5- and 7.5-fold higher with the addition of 1.0 mg/l ABA and TDZ, respectively, compared to root of greenhouse-grown plant. Cryptotanshinone content was 6.3-, 5.0- and 3.75-fold higher in hairy roots grown under the influence of TDZ, ABA and BA, respectively, compared to a commercial herb. The devised protocol can be used to enhance tanshinone production.
Keywords: Hairy root culture; Salvia miltiorrhiza Bunge; Tanshinone.
INTRODUCTION
and Narasu, 2000). The greatest advantage of hairy roots is that they often exhibit similar or greater biosynthetic capacity for secondary metabolite production as compared to their mother plants (Kim et al., 2002a, b). Many valu­able secondary metabolites are synthesized in roots and often the synthesis is linked to root differentiation (Flores et al., 1999). Even in cases where a particular secondary metabolite accumulates only in the aerial part of an intact plant, hairy root cultures have accumulated this same me­tabolite (Wallaart et al. , 1999). Hairy root cultures are also known to produce a spectrum of secondary metabolites that are not present in the parent plant (Veerasham, 2004). Medicinal plants have been widely explored for hairy root culture and their bioactive principles (Guillon et al., 2006; Zhou and Wu, 2006). S. miltiorrhiza Bunge (Lamiaceae) is a well-known Chinese herb and the rhizome is known as 'tanshen' or 'danshen', an ancient drug in traditional Chi­nese medicine (Duke and Avensu, 1985). It has been used widely to treat coronary heart disease, menstrual disorders, miscarriage, hypertension, and viral hepatitis (Chang and But, 1985). Quinones are biologically important com­pounds because of their cytotoxic activity and pharma­cological action. More than 50 ortho-quinone diterpenes, called tanshinone, have been isolated from danshen. S. miltiorrhiza roots contain two major classes of chemicals,
Plant cell cultures are being considered as an alterna­tive, more sustainable production method for valuable phytochemicals. Tissue culture has also become an alter­native way to obtain products when conventional methods or economic viability are challenged. Furthermore, plant metabolites are affected by climatic and seasonal varia­tions, thus their growth in controlled environments over­comes several of their production limitations. Organized cultures, especially root cultures, can make a significant contribution to secondary metabolite production. A. rhizo-genes causes hairy root disease in plants. The neoplastic (cancerous) roots produced by A. rhizogenes infection are characterized by high growth rate, genetic stability and growth in hormone-free media (Srivastava and Srivastava, 2007). These genetically transformed root cultures can produce higher levels of secondary metabolites comparable to that of intact plants. It is a valuable tool for plant bio­technology and a promising tissue culture method for mass production of useful plant secondary metabolites (Giri

*Corresponding author: E-mail: hstsay@cyut.edu.tw; Tel: 886-4-2332-3000 ext. 7580; Fax: 886-4-2330-4921.
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a class of lipid-soluble diterpene quinone pigments, gen­erally known as tanshinone, and a class of water-soluble phenolic acids (Tang and Eisenbrand, 1992). Among these tanshinones, cryptotanshinone is a typical compound hav­ing an ortho-quinone skeleton. Cryptotanshinone acts as an effective topoisomerase I inhibitor (Gantchev and Hunting, 1997; Lee and Hong, 1998) and exhibits sig­nificant cytotoxicity against a number of cultured human tumor cell lines (Ryu et al., 1997). The antitumor activities of the quinones, including naphthoquinone, were revealed more than three decades ago when the National Cancer Institute published a report in which 1500 synthetic and natural quinones were screened for their anticancer activi­ties (Driscoll et al., 1974). Many 1, 2-naphthoquinones were reported to be cytotoxic against a number of tumor cell lines. Because of their effectiveness in drug-resistant cells, these agents appear to hold promise as effective che-motherapeutic agents (Dolan et al., 1998). The hairy roots of S. miltiorrhiza have been established as an alternative method to obtain secondary metabolites without exploit­ing plants from their natural habitats (Hu and Alfermann, 1993; Chen et al., 1999). This work aims to enhance the accumulation of three tanshinones i.e. cryptotanshinone, tanshinone I and IIA in hairy roots of S. miltiorrhiza and to study the effects of different PGR on hairy root growth and tanshinone accumulation.
(LSD) test at the 5% probability level.
Agrobacterium rhizogenes-medaited transfor-mation and time course studies
Leaves were cut into 0.25 cm2 pieces and a small cut was made perpendicular to the mid rib into the center of the squared leaf. Explants were precultured on MS basal medium for one day prior to infection. A. rhizogenes strains BCRC15010 (Food Industry Development Institute, Taiwan) were grown on BEP (beef extract and peptone) medium at 28°C in the dark overnight. The cultures were inoculated into fresh BEP media and grown for 8-10 h. Cells were collected by centrifugation and resuspended in liquid MS medium at an OD580 of 0.4. Precultured explants were submerged into the bacterial suspension for 30 min by shaking. After blotting off the excess bacteria suspen­sion, discs were transferred to MS basal medium and co-cultivated for two days. They were then cultured on MS basal medium supplemented with 3% sucrose, cefotaxime (200 mg l-1) and gelled with 0.9% agar (w/v). Hairy roots, which developed from the infected areas at the end of two weeks, were individually excised after three weeks and transferred to B5 medium (Gamborg et al., 1968) containing 200 mg l-1 of cefotaxime. The induced roots were successfully cleared of bacteria by several passes through medium containing decreasing concentrations of cefotaxime. Hairy roots obtained from a single clone were transferred to 70 ml B5 liquid medium in 250 ml flasks and kept at 100 rpm. The clones were selected on the basis of rapid growth. The cultures were subcultured every three weeks and used for further analysis.
Hairy roots obtained from suspension culture were cut (1.5 cm, 5.4 mg dw) and transferred to 250 ml flask containing 70 ml of B5 liquid media. Roots were kept in growth chamber at 25±2°C at 100 rpm rotation in the dark as described by Ge and Wu (2004). The hairy roots were harvested every second week for 12 weeks and their dry weight (dw) was recorded. A growth curve was plotted between weeks of proliferation and total mass gain by the growing hairy roots (Figure 2).
MATERIALS AND METHODS
Plant materials and tissue culture
Salvia miltiorrhiza plants were collected from the mountains of China's Henan Province, Zhengzhou City, with the help of the Department of Traditional Chinese Medicine, China Medical University, Taiwan. S. milti-orrhiza leaves were washed with running tap water and excess moisture was removed with filter paper. Leaves were surface-sterilized with 70% v/v ethanol for 30 s; fol­lowed by three washes of sterile distilled water. Leaves were further sterilized with 0.5% (w/v) sodium hypochlo-rite (NaOCl) containing 0.02% Tween 20 for 10 min in an ultrasonic oscillator (15 Amp), followed by three washes of sterile distilled water (to remove traces of hypochlo-rite). The sterilized leaves were transferred aseptically on sterilized filter paper, to remove moisture from the leaf surfaces, and inoculated on MS media (Murashige and Sk-oog, 1962) supplemented with 3% of sucrose, 1.0 fiM BA (6-Benzyladenine) and 0.1 fiM NAA (naphthalene acetic acid).
Isolation of genomic DNA
Genomic DNA from the hairy roots and normal roots (control) of S. miltiorrhiza were isolated following the pro­cedure by Cocciolone and Cone (1993), with minor modi­fications. Fresh hairy root tissues (0.2 g) were harvested, frozen in liquid nitrogen, and ground into fine powder. The frozen powder was transferred to a 2 ml microcentrifuge tube containing 0.7 ml lysis buffer (0.30 M NaCl, 50 mM Tris-HCI pH 8.0, 20 mM EDTA, 7 M urea and 1% sarko-syl). The samples were mixed gently. One ml pheno1: chloroform: isoamyl alcohol (25:24:l) solution was added and mixed until the layers disappeared. The sample was kept at room temperature for 15 min followed by centrifu-gation at 5000 xg for 10 min. The aqueous solution was transferred to a new tube and DNA was precipitated by adding 1/10 vol of 3 M sodium acetate, pH 5.2, and 1 vol
Culture Conditions and Data Analysis
Uniform culture conditions were applied for all the ex­periments. The pH of the media was adjusted to 5.7 before autoclaving. The media was autoclaved for 15 min at a pressure of 1.05 kg cm-2 at 121°C. Cultures were incubated at 252°C in the dark. All experiments were repeated three times, each with 10 replicates. Data were analyzed statisti­cally using Fisher's protected least significant difference
GUPTA et al. ― Enhanced tanshinone production using plant growth regulators
437
of isopropanol. DNA was collected by centrifugation (5000 xg) for 10 min at 4°C. The supernatant was discarded and the pellet was washed with 500 fl of 70% ethanol fol­lowed by centrifugation for 30 s. The supernatant was dis­carded and the DNA pellet was re-suspended in 20 fl TE and stored at 4°C.
Preparation of HPLC standard and root sample.
Cryptotanshinone, tanshinone I and IIA were purchased from Jiuding Biotechnology Companies (Taiwan) for the standard. Standard solutions were prepared by dissolving 2 mg of each standard in 2 ml of MeOH. Dissolved solutions (1.0 mg ml-1) were filtered through a 0.22 fim filter (Mil-lipore, USA) and further diluted to the concentration of 0.25, 0.125, 0.0625, 0.03125, 0.015625 and 0.001 mg ml-1. Calibration curves were established based on six points covering a concentration range of 0.25-0.001 mg ml-1 for all three standards. 10 fl of standard solution was used for HPLC injections. Calibration graphs were plotted based on linear regression analyses of the peak areas in response to concentrations of standards injected. The repeatability of the migration time and peak areas of cryptotanshinone, tanshinone I and II A in the experiment were determined by repeated injection (n = 3) of each standard mixture un­der the optimum conditions. The roots were collected from the culture flask and their fresh weight was recorded. The roots were then freeze-dried for 48 h and their dry weight was determined. About 1.0 gm of dried roots was crushed into fine powder and dissolved in 30 ml of methanol. It was ultra-sonicated for 30 min and filtered through What­man No 1 filter paper. This process was repeated three times for each sample. After filtration, the combined meth-anol extracts were evaporated to dryness with the help of rotary evaporator. The residue was dissolved in 10 ml methanol and filtered through a 0.22 fim (Millipore, USA) membrane before analysis.
Isolation of plasmid DNA
Plasmid DNA from A. rhizogenes strain BCRC15010
was isolated using the alkaline lysis method (Sambrook et
al., 1989).
PCR analysis
PCR analysis was carried out in an Eppendorf Master-cycler Gradient in 25 fl reaction mixtures containing com­mercial buffer, 2.5 mM MgCl2, 200 fM dNTP, 3 U Taq
polymerase, DNA (100 ng total DNA, or 10 pg plasmids), and oligonucleotide primers in 1.0 fM final concentra­tion. To amplify internal 'rol' B and C gene fragment (780 and 540 bp respectively; Bulgakov, 2008), the following primer pairs were used:
'rol'B-1: 5'-ATGGATCCCAAATTGCTATTCCCCC
ACGA-3'
'rol'B-2: 5'-TTAGGCTTCTTTCATTCGGTTTACTG
CAGC-3'
'rol'C-1: 5'-ATGGCTGAAGACGACCTGTGTT-3'
'rol'C-2: 5'-TTAGCCGATTGCAAACTTGCAC-3'
PCR reaction profiles involved pre-denaturation at 94°C for 5 min, 35 cycles of denaturation at 94°C for 45 s, an­nealing at 57°C for 30 s, extension at 72°C for 45 s, a final extension for 10 min at 72°C, and storage at 4°C. The amplified products were analyzed on 1% agarose-ethidium bromide gels.
RESULTS
Initiation of hairy root culture and their molecu­lar analysis
Hairy root initiations were observed in 75-80% infected leaves after three weeks of infection with A. rhizogenes strains BCRC15010. Hairy roots were isolated individual­ly and grown on B5 solid medium supplemented with 200 mg l-1 cefotaxime. After 4-5 subcultures, the roots were able to grow without A. rhizogenes in the absence of cefo-taxime and were negatively geotropic. Transformation was confirmed by polymerase chain reaction using gene-specif­ic primers ('rol' B and C). PCR amplification with specific primers was used to show the integration of 'rol' B and C genes in to the genome of S. miltiorrhiza. When amplified with specific primers, these isolated DNA showed 'rol' B and C gene amplification of 540 and 780 bp respectively, while no amplification was observed in non-transformed roots (Figure 1).
Growth regulators and tanshinone accumula­tion
Selected lines were used to study the effect of PGR on growth and accumulation of tanshinone. B5 media supplemented with 1.0 mg l-1 concentration of different auxins (2,4 di phenoxy acetic acid {2,4-D}, indole 3 acetic acid {IAA}, NAA), cytokinins (BA, kinetin, thiadiazuron {TDZ}, zeatin) and indole butyric acid (IBA) were used to grow the hairy root. The increase in total biomass and tan-shinone content were analyzed 12 weeks after subculture.
HPLC Analysis
Reagents, materials and conditions. HPLC-grade methanol was purchased from Merck (Germany). An L-2130 pump, L-2200 auto injector and L-2459 diode ar­ray detector system were purchased from Hitachi. Symme­try Waters column C18 (5 [im, 4.6 x 250 mm) were used for HPLC analysis. Milli Q water (Millipore, Milford, MA, USA) was used for all the analysis. Acetonitrile: water (58:42) was used for the mobile phase at room tem­perature. Samples were detected at the wavelength of 245 nm with flow rate of 1 ml min-1.
Tanshinone accumulated during hairy root growth
There was a significant increase in dry weight of the hairy roots over consecutive weeks. After 12 weeks, the dry weight had increased 34 times (5.4 to 185 mg; Figure 2). HPLC analysis for the three compounds i.e. crypto-tanshinone, tanshinone I and IIA, revealed their increased
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Figure 1. PCR confirmation of transgenic hairy roots of Salvia miltiorrhiza Bunge showing the amplification of 'rol' B (780 bp) (a) and 'rol' C (540 bp) (b) genes. Lane M: marker; Lane 1: non-transgenic line; Lanes 2-7: putatively transformed hairy root lines.
accumulation over consecutive weeks (Figure 3). Crypto-tanshinone accumulation was observed four weeks after subculture and reached maximum accumulation at 10 weeks (1.301 mg g-1 dw). Accumulation of tanshinone I and IIA started six weeks after subculture and reached to maximum after 10 weeks (0.351 and 0.156 mg g-1 dw re­spectively).
Effects of different growth regulators on tanshi-none accumulation
Stable transgenic lines with high multiplication rates were selected for analysis. An 18-fold (98.67 mg from 5.4 mg) increase in dry weight (dw) was observed after 12 weeks culture in PGR-free MS medium. We investigated the effects of exogenous PGR on the growth and tanshi-none accumulation in transformed roots of S. miltiorrhiza. Hairy roots were cultured in medium containing various concentrations of PGR and the results are summarized in Table 1. There was no significant difference in hairy root multiplication with ABA, IAA, NAA and kinetin (101.67, 103.00, 116.67 and 118.33 mg) compared to the control (98.67 mg). However a significant difference in root mul­tiplication was observed with IBA, BA and TDZ (137.00, 139.33 and 185.00 mg respectively). The multiplication of hairy roots was in direct proportion to the increased rate of root branching and elongation. The total dry weight gains were 25-, 25- and 34-fold after 12 weeks with 1.0 mg l-1 IBA, BA and TDZ, respectively. Although ABA had mini­mal influence on hairy root multiplication, we observed 2-fold increase in tanshinone I accumulation and 3-fold higher tanshinone IIA and cryptotanshinone. The total tan-shinone content was thus 2.5 times higher in roots grown under the influence of ABA compared to the control. On other hand, TDZ not only influenced hairy root multiplica­tion but also tanshinone accumulation. We observed 2.0-, 1.5- and 3.5-fold higher tanshinone I, tanshinone IIA and cryptotanshinone, respectively with TDZ. Hence, the total
Figure 2. The growth course of hairy root of 'Salvia miltior-rhiza Bunge' in liquid medium* containing 1.0 mg l-1 TDZ. *B5 basal salts supplemented with 3% sucrose. Observations were recorded after 12 weeks of culture. Error bars for standard errors (SE), n=3.
2 4 6 8 ]0 12
Culture period ( weeks )
Figure 3. Effect of different culture periods on the production of tanshinone in hairy roots of Salvia miltiorrhiza Bunge. Error bars for standard errors (SE), n=3.
GUPTA et al. ― Enhanced tanshinone production using plant growth regulators
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Table 1. Effect of different PGRs on hairy root production and tanshinone accumulation in Salvia miltiorrhiza in liquid medium*.

PGR constituents
Average dry weight after 12 weeks (mg)
Weight gain
(folds)
TIa
(mg g-1 of dw)
TIIb
(mg g-1 of dw)
CTc
(mg g-1 of dw***)

0

98.677.22b

18

0.2160.06

0.1200.02

0.3740.06
NAA (5.37 fM)
116.678.95ab
21
ND
0.0690.01
0.1580.04
IAA (5.71 iM)
103.0010.07b
19
0.1340.05
0.0970.02
0.2490.05
IBA (4.90 iM)
137.0010.15a
25
ND
0.0690.02
0.1950.02
BA (4.44 iM)
139.338.76a
25
0.4300.06
0.3000.04
0.7720.09
Kinetin (4.65 fiM)
118.3311.02ab
22
0.3160.06
0.2100.02
0.5340.07
TDZ (4.54 iM)
185.008.14a
34
0.3930.03
0.1950.03
1.3100.17
ABA (3.76 iM)
101.676.69b
19
0.4510.05
0.3410.02
1.0380.11

*B5 basal salts supplemented with 3% sucrose. Observations were recorded after 12 weeks of culture.
**Value are means standard error, n = 3. Means followed by the same letter are not significantly different at 5% level by LSD (least
significant difference) test. ***Dry weight.
aTanshinone I; bTanshinone IIA; cCryptotanshinone; ND- not detected.
tanshinone content was 2.7-fold in hairy roots grown un­der the influence of TDZ.
Table 2. Comparative analysis of tanshinone contents from greenhouse-grown plant and commercial herb of Salvia milti­orrhiza.
Comparative analysis of tanshinones

Source

Tanshinone content

TIa (mg g-1) TIIb (mg g-1) CTc (mg g-1)

Root from greenhouse-grown plant
Commercial herb

0.093±0.01 0.445±0.09 0.173±0.04 0.384±0.02 0.452±0.04 0.205±0.04
Three different tanshinones (cryptotanshinone, tan-shinone I and IIA) were measured from the normal roots grown in greenhouse and from the commercial herb avail­able in the market. The contents were compared with the contents of hairy roots grown under the influence of differ­ent PGRs. Tanshinone I content was approximately 4-fold (0.384 mg g-1) higher in the commercial herb, while, it was 5.0- (0.451 mg g-1), 4.6- (0.430 mg g-1), 4.2-fold (0.393 mg g-1) higher in the hairy root grown under the influence of ABA, BA and TDZ respectively, compared to the root of greenhouse-grown plant (0.093 mg g-1). Tanshinone IIA content in the commercial herb (0.452 mg g-1) was similar to the root of the greenhouse-grown plant; and no significant influence of ABA, BA and TDZ on tanshinone IIA accumulation was observed. However, these regulators had profound influence on cryptotanshinone accumulation in hairy roots, cryptotanshinone content increased 7.6-fold (1.310 mg g-1), 6-fold (1.038 mg g-1) and 4.5-fold (0.772 mg g-1) under the influence of TDZ, ABA and BA respec­tively, compared to roots from the greenhouse-grown plants. There were no significant difference in tanshinone I and IIA content in PGR-influenced hairy roots compared with commercial herbs, however a significant increase in cryptotanshinone content was observed in PGR-influenced hairy roots compared to commercial herbs. Cryptotanshi-none content was 6.3- (1.310 mg g-1), 5.0- (1.038 mg g-1) and 3.75- (0.772 mg g-1) fold higher in hairy roots grown with TDZ, ABA and BA respectively (Tables 1 and 2).

**Value are means standard error, n = 3. Means followed by the same letter are not significantly different at 5 % level by LSD (least significant difference) test.
aTanshinone I; bTanshinone IIA; cCryptotanshinone.
tically-useful metabolites including diterpenes, quinones and phenolics. Since these are primarily located in the roots, the hairy root culturing method provides a promising means for large scale metabolite production. Several inves­tigations have been carried out on in vitro regeneration of roots using leaves as the starting material (Zhi and Alfer-mann, 1993; Kintzios et al., 2004; Tao and Li, 2005; Yan et al., 2005; Lee et al., 2007b). The success of hairy root induction depends on the type of explants and Agrobac-terium strains. Different strains of A. rhizogenes have dif-ferentially influenced hairy root induction in 'golden flax Linum flavum, leaves, (Lin et al., 2003). Sudha et al. (2003) have reported that leaf tissue produced more hairy roots than other plant tissue upon induction in Rauwolfia mi-crantha. We have found that amongst two different strains of A. rhizogenes (BCRC15010 and BCRC15722), the former responded better to hairy root induction (data not shown). Approximately 78% of leaves responded to hairy root induction. The response of hairy root proliferation on hormone-free B5 solid media was similar to that reported by Liao and Jhuang (2007) for Paulownia (Paulownia
DISCUSSION
Salvia miltiorrhiza contains a large number of therapeu-
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Botanical Studies, Vol. 52, 2011
fortunei). The proliferating roots were subcultured every three weeks to select the best line. During the subculture most of the hairy root growth was arrested, they turned brown and some died. A few roots grew rapidly with mul­tiple branching and negative geotropism. The thick and fast-growing hairy roots without buds were maintained on B5 liquid medium and used for further experiments. Hairy root growth response varied with different transgenic lines and even in the branches of the same lines; a similar phe­nomenon has been reported by Kang et al. (2006) in Aralia (Aralia elata) hairy roots, where the growth and multipli­cation of hairy roots depended on the expression of the 'rol' gene. The multiplication rate of Datura hairy roots varied between different lines and doubling time took 24 to 90 h (Payne et al., 1987). The doubling time of S. milti-orrhiza hairy root was 18 days. Many studies have pointed out that hairy root growth and metabolite production de­pend on 'rol' gene expression (Palazon et al., 1998; Shkryl et al., 2008; Bulgakov, 2008). A growth curve was plotted to monitor the growth of hairy root and metabolite accu­mulation (Figure 2). Only cryptotanshinone accumulated four weeks after subculture (0.109 mg g-1 of dw), while, tanshinone I and IIA have started accumulating six weeks after subculture (0.218 and 0.122 mg g-1 of dw respec­tively). The maximum accumulations of cryptotanshinone, tanshinone I and IIA was observed 10 weeks after culture (Figure 3).
in S. miltiorrhiza hairy roots. The studies by Yoshimatsu et al. (2004) on Australian eggplant (Duboisia myoporo-ides) showed a negative correlation between auxin (IAA) concentration and secondary metabolite accumulation. The highest number of roots, and thus metabolites accumula­tion, was obtained when the leaf of Plumbago zeylanicas was cultured on MS medium with 1.0 mg l-1 IBA and 0.5 mg l-1 NAA (Sivanesan and Jeong, 2009). S. miltiorrhiza hairy roots showed similar responses. ABA can inhibit seed germination, leaf aging, stomatal closure, and trig­ger defensive gene activation (Zhao et al., 2005), but it has no significant effect on hairy root growth in henbane (Hyoscyamus muticus) or, as we found, in S. miltiorrhiza. ABA does trigger metabolite accumulation and all three tanshinones together increased 2.6-fold with 1.0 mg 1-l ABA. There were no significant differences in tanshinone I and IIA accumulation in PGR-influenced hairy roots and the commercial herb, however differences were found in their cryptotanshinone content. Cryptotanshinone content in hairy roots grown under the influence of TDZ, ABA and BA, respectively, was 6.3- (1.310 mg g-1), 5.0- (1.038 mg g-1) and 3.75-fold (0.772 mg g-1) that of the commercial herb. Zhang et al. (2004) have reported the total tanshi-none content 0.54, and 2.50 mg g-1, when hairy roots of S. miltiorrhiza were supplemented with sucrose alone or in combination with Ag+. However, we reported total tanshinone content in hairy root of S. miltiorrhiza, which was 1.898 and 1.830 mg g-1 with TDZ and ABA, respectively. The total tanshinone content have increased 4.5- and 10­fold, when hairy root of S. miltiorrhiza, supplemented with either sorbitol or sorbitol and yeast elicitor together (Shi et al., 2007). Hence, the synergistic effects of biotic and abiotic elicitors are more effective for tanshinone accumu­lation. We have not yet tried or studied elicitors for their effects on tanshinone accumulation, but since they may increase total tanshinone content, they may be the subject of further research. Plant secondary metabolite analysis is an important way to measure their responses to biotic and abiotic stresses. Plants use multiple signal pathways to mediate their response to different stresses, and these signal pathways can converge at certain points, leading to common cellular processes (Zhao et al., 2005). The results also showed that different PGRs induced higher cryptotan-shinone accumulation.
In vitro plant cell culture usually requires the pres­ence of PGR, mainly auxins and cytokinins. As for hairy roots, one characteristic of their phenotype is the rapid hormone- independent growth. As a result, the media used to culture hairy roots generally lacks PGR. Even more, as demonstrated in transformed roots of 'Datura stramo­nium', cultures treated with 2.0 and 0.2 mg l-1 NAA and kinetin respectively have induced root tissue de-differ­entiation (Ford et al., 1996). In several experiments, this de-differentiation have influenced a significant decrease or even the cessation of alkaloid production (Robbins et al., 1996). However, a more recent systematic test on the effects of different types of phyto-hormones upon root growth and secondary metabolite production showed that some of them have enhanced either growth or metabolite production. The growth of hairy roots of Artemisia an-nua (Weathers et al., 2005) has been evaluated under the influence of five different hormones: auxins, cytokinins, ethylene, gibberellins (GA) and abscissic acid (ABA). The highest biomass was obtained with 1-5 mg l-1 ABA, while 0.5-1.0 mg 1-l 2-isopentenyladenine inhibited root growth but enhanced artemisinin accumulation. Different auxins have different effects on hairy root morphology and root formation. Washida et al. (2004) have reported that in­creased ginsenosides (ginsenoside) content was observed in ginseng (Panax ginseng) hairy root culture medium supplemented with individual or combined auxins. How­ever, Lee et al. (2007a) have reported the inhibitory effect of different auxins on Ludin (rutin) levels in buckwheat (Fagopyrum esculentum M.). Similarly, auxins have an inhibitory effect on different tanshinone accumulations
CONCLUSION
Tanshinones are the major compounds of danshen and are used to treat Alzheimer's disease, insomnia, vascular dementia, memory loss, attention disorders, heart disease, cancer, etc. The present study was designed to enhance the production of tanshinones in hairy roots of Salvia in liquid culture using PGRs. A. rhizogenes (BCRC 15010) was used for hairy root induction. The putative transformed roots were grown on PGR-free B5 media. Gene integra­tion was confirmed by polymerase chain reaction. The hairy roots were grown under the influence of different auxins (IAA, IBA and NAA), cytokinins (BA, kinetin and
GUPTA et al. ― Enhanced tanshinone production using plant growth regulators
441
TDZ) and ABA. Three different tanshinones, i.e. crypto-tanshinone and tanshinone I and IIA, were analyzed using HPLC. Different PGRs had inhibitory effect on tanshinone IIA accumulation in hairy roots, whereas it enhanced cryp-totanshinone and tanshinone I accumulation. Tanshinone I content have increased 5-fold (0.451 mg g-1) in the hairy roots grown under the influence of 1.0 mg 1-l ABA, where­as, cryptotanshinone content increased 7.5-fold under the influence of 1.0 mg l-1 TDZ. The present work aims to increase the tanshinone I and cryptotanshinone contents by tissue culture methodologies, which may be helpful to pro­duce cost-effective drugs. The use of different biotic and abiotic elicitors may increase the total tanshinone content and may be the subject of further research.
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植物生長調節劑對丹參毛狀根液態培養累積丹參酮之影響
古普塔書信1 劉人榜2 廖松淵2 詹効松1 蔡新聲1,3
1朝陽科技大學生化科技研究所
2國立中興大學生命科學系
3國立中興大學農藝學系
丹參{Salvia miltiorrhiza Bunge)所含之二萜類丹參酮化合物於現代藥理硏究證實其對許多疾病具有 良好的療效。本硏究以丹參葉片爲材料,建立毛狀根培養系統,並探討植物生長調節劑對丹參毛狀根 生產3種丹參酮(tanshinone-I, tanshinone-IIA, cryptotanshinone)之景》響。以農桿根群菌(Agrobacterium rhizogenes ) BCRC15010菌系感染丹參葉片培植體可誘導毛狀根形成率達78% 。轉殖毛狀根之鑑定則利 用農桿根群菌rolBrolC基因序列爲引子,進行PCR反應並比對其基因片段序列後加以證實。誘導 所得之丹參毛狀根於無光照條件下培養於B5液體培養基中具有生長迅速的特性。丹參毛狀根培養在含 不同植物生長調節劑(auxins, cytokininsabscisic acid)的培養基中均可增加丹參酮類產生;毛狀根培 養在含1 mg/L ABA (abscisic acid)TDZ (thidiazuron)培養基中時'其tanshinone-Icryptotanshinone 含量分別是溫室植株根部含量的57.5倍;添加TDZ, ABABA (benzyladenine)於培養基中可提升 cryptotanshinone之含量,其値分別爲市售藥材之6.3, 5.03.75倍。本硏究結果顯示添加植物生長調節 劑可作爲提升丹參酮產量之有效策略。
關鍵詞:丹參;毛狀根培養;丹參酮。