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Establishment of Tripterygium wilfordii Hook. f. Hairy Root Culture and Optimization of Its Culture Conditions for the Production of Triptolide and WilforineS
Establishment of Tripterygium wilfordii Hook. f. Hairy Root Culture and Optimization of Its Culture Conditions for the Production of Triptolide and WilforineS
Journal of Microbiology and Biotechnology. 2014. Jun, 24(6): 823-834
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : February 21, 2014
  • Accepted : March 19, 2014
  • Published : June 30, 2014
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About the Authors
Chuanshu Zhu
Biopesticide Technology and Engineering Center, Yangling, Shaanxi Province 712100, P. R. China
Guopeng Miao
Research & Development Center of Biorational Pesticides, Northwest A & F University, Yangling, Shaanxi Province 712100, P. R. China
Jia Guo
Research & Development Center of Biorational Pesticides, Northwest A & F University, Yangling, Shaanxi Province 712100, P. R. China
Yanbo Huo
Research & Development Center of Biorational Pesticides, Northwest A & F University, Yangling, Shaanxi Province 712100, P. R. China
Xing Zhang
Biopesticide Technology and Engineering Center, Yangling, Shaanxi Province 712100, P. R. China
Jiahua Xie
Department of Pharmaceutical Sciences, Biomanufacturing Research Institute & Technology Enterprise, North Carolina Central University, Durham, NC 27707, USA
Juntao Feng
Biopesticide Technology and Engineering Center, Yangling, Shaanxi Province 712100, P. R. China
jtfeng@126.com

Abstract
In order to solve the shortage of natural Tripterygium wilfordii Hook. f. plant resource for the production of the important secondary metabolites triptolide and wilforine, hairy roots were induced from its root calli by Agrobacterium rhizogenes . Induced hairy roots not only could be maintained and grown well in hormone-free half-strength Murashige and Skoog medium but also could produce sufficient amounts of both triptolide and wilforine. Although hairy roots produced approximately 15% less triptolide than adventitious roots and 10% less wilforine than naturally grown roots, they could grow fast and could be a suitable system for producing both secondary metabolites compared with other tissues. Addition of 50 μM methyl jasmonate (MeJA) could slightly affect hairy root growth, but dramatically stimulated the production of both triptolide and wilforine, whereas 50 μM salicylic acid had no apparent effect on hairy root growth with slightly stimulatory effects on the production of both secondary metabolites. Addition of precursor nicotinic acid, isoleucine, or aspartic acid at the concentration of 500 μM had varying effects on hairy root growth, but none of them had stimulatory effects on triptolide production, and only the former two had slightly beneficial effects on wilforine production. The majority of triptolide produced was secreted into the medium, whereas most of the produced wilforine was retained inside of hairy roots. Our studies provide a promising way to produce triptolide and wilforine in T. wilfordii hairy root cultures combined with MeJA treatment.
Keywords
Introduction
Tripterygium wilfordii Hook. f. (also known as “Leigongteng” in Chinese) is a climbing vine plant ( Fig. 1 A) native to Eastern and Southern China, Korea, Japan, and Taiwan, and has a long history of use in traditional Chinese herbal medicine [3 , 18] . It has been used medicinally in mixtures for the treatments of kidney diseases [42] , cancers [15] , arthritis [29] , etc . Recent studies showed that there are about 380 secondary metabolites in T. wilfordii , and many of them had their structures and biological activities characterized, such as triptolide ( Fig. 1 B) and wilforine ( Fig. 1 C) [3 , 17 , 36] . Triptolide (C 20 H 24 O 6 ), a diterpenoid epoxide, possesses potent anti-inflammatory, immunosuppressive, antitumor, and antileukemic activities [11 , 15 , 36] as well as insecticidal activity against Mythimna separata [17] . Wilforine, a sesquiterpene pyridine alkaloid, is another important bioactive compound in T. wilfordii plants, and is effective in treating idiopathic pulmonary fibrosis (an inflammatory lung condition) and arthritis, as well as having antifeedant activities against Pieris rapae and Locusta migratoria [3 , 17 , 23] . Purification of triptolide and wilforine from T. wilfordii plants has both biomedical and agricultural significances. However, the major limitations are their slow growth, long period of growth, and low yield of secondary metabolite production [43] , which dramatically affect their supplies for the purification.
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The plant of T. wilfordii (A) and the structures of triptolide (B) and wilforine (C).
Plant cell and tissue cultures are an ideal alternative for the production of secondary metabolites for slow-growing plants, with many additional advantages such as independence of geographical and seasonal variations, and uniform quality and yield [28] . Previously, we established cell suspension cultures for T. wilfordii to produce triptolide and wilforine [21] . However, its low production in cell suspension cultures led us to develop adventitious root cultures for producing these natural products [22] . Nevertheless, the challenge of adventitious root cultures is their slow growth with low biomass. Therefore, it is necessary to exploit alternative approaches for the production of these secondary metabolites.
Hairy roots induced by Agrobacterium rhizogenes harboring the Ri plasmid have been considered to be a suitable way to produce secondary metabolites because of their stability and high productivity in hormone-free culture conditions [4 , 7 , 9 , 31 , 33] . Plant hairy root culture is an alternative method for the production of phytochemicals used in pharmaceuticals, cosmetics, and food additives [7 , 33] . Hairy roots have been successfully induced by A. rhizogenes in many medicinal plants to be used to produce a number of high-value secondary metabolites, such as tropane alkaloids in Brugmansia candida [27] , ajmalicine and ajmaline in Rauvolfia micrantha [32] , camptothecin in Camptotheca acuminate [16] , artemisinin in Artemisia annua [34] , saponin in Panax ginseng [41] , and tanshinone in Salvia miltiorrhiza [37 , 38] . In T. wilfordii , hairy roots were induced from leaf explants by A. rhizogenes transformation and used to extract several terpenoids [25] . However, there is no systematic study on the production of secondary metabolites in T. wilfordii hairy roots.
The addition of elicitors and precursors has been welldocumented to enhance metabolite production in plant cell and hair y r oot cultur es [1 , 2 , 5 , 10 , 12 , 13 , 19 , 30 , 35 , 40] . Both methyl jasmonate (MeJA) and/or salicylic acid (SA) have been previously applied in hairy root cultures as elicitors to improve the production of antioxidant compounds in P. ginseng [12] , asiaticoside in Centella asiatica [13] , sesquiterpene lactone in Cichorium intybus [19] , and gossypol and methylated gossypol in cotton [5] . In addition, applying precursors of secondary metabolites in hairy root cultures has also been proven to efficiently promote the secondary metabolite production. This has been demonstrated by feeding precursors to enhance the productions of glucotropaeolin in Tropaeolum majus [35] and phytoestrogenic isoflavone in Psoralea corylifolia [30] . In the metabolism of plant alkaloids, many amino acids and small molecules are precursors for alkaloid biosynthesis [3 , 16 , 20 , 32] . Based on the structure of wilforine, nicotine acid, isoleucine, and aspartic acid were chosen as precursors to increase the accumulation of this and related secondary metabolites in T. wilfordii [3 , 14] .
In the present study, we established the hairy root cultures in T. wilfordii in order to efficiently produce triptolide and wilforine. Two A. rhizogenes strains and two induction media were tested for inducing hairy roots. The effects of two elicitors and three precursors on the production of these secondary metabolites in hairy root cultures were investigated. This study provides a promising system for producing these two secondary metabolites, which could not only solve the limitation of natural plant resource but also improve the low production yield.
Materials and Methods
- Materials and Chemicals
Roots were collected from T. wilfordii plants grown in the greenhouse of Northwest A&F University, China. A. rhizogenes strain ATCC 15834 was purchased from the American Type Culture Collection (Manassas, USA) and A. rhizogenes A4 strain was purchased from the Chinese Culture Collection Center (Beijing, China). Acetosyringone, yeast mannitol broth (YMB), and standard compounds triptolide and wilforine were purchased from Sigma Aldrich (St. Louis, USA). The DNA extraction kit and PCR kit were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China). Primers were synthesized by Shanghai Sangon Biotechnological Company (Shanghai, China).
- Hairy Root Induction and Maintenance
T. wilfordii roots were sterilized in 70% ethanol for 20 sec, and then rinsed four times with sterilized H 2 O. They were then sterilized in 1g/l HgCl 2 for 4 min. After rinsing with sterilized H 2 O four times, the sterilized roots were used for callus induction. Calli were induced on Murashige and Skoog (MS) medium [24] with 2,4-dichlorophenoxyacetic acid (1.0 mg/l), kinetin (0.1 mg/l), agar (7 g/l), and sucrose (30 g/l). The pH of the medium was adjusted to 5.8 with 1M NaOH solution prior to autoclaving at 121℃ for 30 min. After culturing for 30 days at 25℃ in the dark, the new growing calli were subcultured once a month three times under the same conditions before they were used for inducing hairy roots. Two A. rhizogenes ATCC 15834 and A4 strains were tested to induce hairy roots. Agrobacterium culture was initiated in 100 ml of YMB medium. After incubating for 24 h at 28℃ in the dark on a rotary shaker at 225 rpm, 2 ml of overnight culture was re-inoculated into 25 ml of fresh YMB medium until the OD 600 was 0.6-0.8. Prior to bacterial infection, acetosyringone (100 mM stock solution in dimethyl sulfoxide) was added to the Agrobacterium culture to the final concentration of 100 μM. The calli were wounded using a preparative needle before they were inoculated with either A. rhizogenes strain and swirled for 20 min. Inoculated explants were blotted dry on sterile filter paper to remove excess bacterial solution. Gently wounded calli without bacterial infection were used as a control. After inoculation, the calli were transferred to MS or half strength of MS salts (1/2MS) medium without any growth regulators and co-cultured for 3 days in the dark. To eliminate A. rhizogenes , the calli were transferred to fresh MS or 1/2MS medium containing 100 ml/l coconut milk and 0.5 g/l cefotaxime (Sangon Biotech Co., Ltd., Shanghai, China). All the bacteria-free hairy roots were maintained on 1/2MS medium with 30 g/l sucrose and 100 ml/l coconut milk without any growth regulators or antibiotics at 25 ± 1℃ in darkness. Subcultures were made every four weeks in the same conditions.
- PCR Confirmation of Transgenic Hairy Roots
To confirm induced hairy roots resulted from A. rhizogenes transformation, the T-DNA fragment in the hairy root genome was detected by PCR amplification. Hairy roots induced by A4 were randomly selected and subjected to PCR analysis. Total genomic DNA was isolated from hairy roots as well as adventitious roots prepared by Miao et al . [22] (as a negative control) using the MiniBEST Universal Genomic DNA Extraction Kit (Dalian, China). Plasmid DNA from A. rhizogenes A4 strain extracted by the E.Z.N.A. Plasmid Mini kit (Omega Bio-Tek Inc., Norcross, USA) was used as a positive control. All three genes rol B, rol C, and aux 1 inside of the Ri T-DNA region were analyzed, whereas two genes ( vir D2 and vir G) outside of the Ri T-DNA region were used as negative controls. Each gene-specific primer is listed in Table 1 . Each 25 μl PCR mixture contained 50 ng of DNA templates, 2.5 μl of 10× PCR buffer, 1 unit of Taq DNA polymerase (TaKaRa Biotechnology Co., Ltd.), a final concentration of 0.5 μM of each primer, 2 mM MgCl 2 , and 0.2 mM dNTPs. PCR amplification was performed in a C1000 thermal cycler (Bio-Rad Laboratories Pty Ltd., California, USA). The reaction began with the initial denaturation at 94℃ for 3 min and was followed by 29 amplification cycles of denaturation at 94℃ for 1 min, annealing at 52.0℃ for 1 min, and elongation at 72℃ for 1 min, plus the final elongation at 72℃ for 6 min. The PCR products were resolved on 1% (w/v) agarose gel (Sangon Biotech Co., Ltd.) along with a 2,000 bp DNA ladder marker (TaKaRa Biotechnology Co. Ltd.). The results were recorded by the GelDoc-It imaging system (Bio-Rad Laboratories Pty Ltd.).
The primers used for PCR identification ofT. wilfordiihairy roots.
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The primers used for PCR identification of T. wilfordii hairy roots.
- Screening of Suitable Medium for Hairy Root Culture
To select a suitable medium for hairy root culture, transgenic hairy roots (0.3 g fresh weight) confirmed by PCR were subcultured on MS, 1/2MS, Woody Plant medium (WPM) [32] , or Gamborg’s B5 (B5) medium [6] with 7 g/l agar, 30 g/l sucrose, and 100 ml/l coconut milk without antibiotics or growth regulators at 25 ± 1℃ in the dark for 50 days. Then, the dry weights (DW) of the cultures were measured for comparative analysis.
- Preparation of Hairy Roots for Triptolide and Wilforine Analysis
Hairy roots (0.3 g FW) were cultivated in a 250 ml shake flask containing 100 ml of liquid 1/2MS medium without growth regulators at 28℃ in the dark on a rotary shaker at 100-110 rpm. The same amount of adventitious roots (0.3 g FW) was cultivated under the same conditions as a control. After both types of roots were cultured for 50 days, they were harvested for analyzing the contents of triptolide and wilforine. Untransformed calli and naturally grown roots (collected from Taining, Fujian Province, China) were also used as controls. All tissue samples were dried in 45℃ in a constant-temperature oven. The same amount of dried tissues of each sample type was subjected to metabolite analysis.
- Preparation of Hairy Roots Treated by Various Chemicals for Triptolide and Wilforine Production
To improve the production of triptolide and wilforine in hairy roots, two elicitors and three precursors were tested by the addition of each chemical into the hairy root culture. The concentration of 50 μM was used for the elicitors salicylic acid (SA) and methyl jasmonate (MeJA), whereas 500 μM was used for the precursors nicotinic acid, isoleucine, and aspartic acid. Each chemical was applied separately into the 7-day-old liquid hairy root cultures described above. The control was treated with an equal volume of distilled water. The hairy roots and corresponding medium from the same culture were collected after treatments for 3, 6, 9, 1 2, and 1 5 days, respectively. After harvesting, the hairy roots were dried in an oven at 45℃ to constant weight. The dry weight of each hairy root culture was determined. The culture medium was extracted three times using an equal volume of ethyl acetate, and the combined organic liquid was evaporated in a rotary evaporator, and then dissolved in 60% acetonitrile-water for measuring the metabolites. All treatments were performed in triplicate.
- Extraction and Quantification of Triptolide and Wilforine
The procedures of extraction and HPLC analysis of triptolide and wilforine were the same as described previously [21] . Triptolide was quantified based on peak areas at 219 nm, whereas wilforine was analyzed at 230 nm.
- Statistical Analysis
All data presented are the mean values of three replicates, which were statistically analyzed using Duncan’s multiple range test at p < 0.05 in SPSS (IBM, USA). Every experiment was repeated twice.
Results
- Hairy Root Induction and Maintenance
The hairy roots appeared within 30 days from infected calli by either Agrobacterium strain when they were cultured on either MS or 1/2MS medium in the dark ( Fig. 2 A). However, there was no hairy root formed on the callus without Agrobacterium infection. These results indicate both strains can induce hairy roots. After 50 days of culture, the induction rate of hairy roots was recorded for each treatment. The results showed that different strains and media had different induction rates ( Table 2 ). Strain A4 had a higher induction rate than that of strain ATCC 15834, whereas 1/2MS medium had more hairy roots than the full MS. The highest induction rate of hairy roots was approximately 80% in the treatment with 1/2MS medium and A. rhizogenes strain A4. The induction rates of other combinations of strains and media were 73.0% for MS and A4, 62.0% for 1/2MS and ATCC 15834, and 46.0% for MS and ATCC 15834 ( Table 2 ). When hairy roots were subcultured onto solid and liquid media, they grew well ( Figs. 2 B- 2 F), which would be used for downstream analysis.
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Hairy root induction and subculture in solid and liquid media of T. wilfordii. The hairy roots appeared from infected calli after culture for 30 days (A); hairy roots were subcultured on solid 1/2MS medium for 15 (B) and 30d ays (C); hairy roots were subcultured in liquid 1/2MS medium (D); hairy roots were subcultured in liquid 1/2MS medium for 15 (E) and 30 days (F).
Effects of media andA. rhizogenesstrains on hairy root inductions from leaf calli ofT. wilfordiiHook. f.
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Effects of media and A. rhizogenes strains on hairy root inductions from leaf calli of T. wilfordii Hook. f.
- PCR Confirmation of Transgenic Hairy Roots
To confirm that the hairy roots resulted from the transformation of A. rhizogenes , PCR analysis was used to confirm the insertion of the T-DNA region derived from the Ri plasmid. Five hairy root lines were analyzed by PCR amplification. One adventitious root line was used as a negative control, whereas plasmid DNA from A. rhizogenes A4 strain was used as a positive control. Hairy root lines resulted from true A. rhizogenes transformation would have positive PCR results for all three pairs of primers for rol B, rol C, and aux 1 genes, but negative for either pair of primers of vir D2 or vir G gene. Non-transformed adventitious roots would show no PCR product for all five sets of primers, whereas roots contaminated with Agrobacterium would have PCR products for all five sets of primers. The results with three pairs of primers for rol B, rol C, and aux 1 showed that all five hairy root lines had about 500, 500, and 800 bp PCR products as predicted sizes ( Fig. 3 ). With two primer sets for vir D2 and vir G genes, no amplification was observed in either of these hairy root lines ( Fig. 3 ). In contrast, all five pairs of primers could amplify PCR pr oducts in the plasmid DNA, but did not amplify any product in adventitious root sample ( Fig. 3 ). These results suggest that all five hairy root lines are true transgenic roots and that they are free of Agrobacterium contamination.
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PCR analysis of genomic DNA isolated from T. wilfordii hairy root transformed by A. rhizogenes strain A4. Lane 1: positive control of Ri plasmid DNA; lanes 2-6: genomic DNA isolated from five different lines of T. wilfordii hairy roots; lane 7: negative control of genomic DNA of adventitious roots; M: DL 2,000 bp DNA marker.
- Contents of Triptolide and Wilforine in Hairy Roots
The contents of triptolide and wilforine were analyzed in hairy roots after they were cultured in 1/2MS liquid medium for 50 days. Their contents in calli, adventitious roots, and naturally grown roots were also measured. The results showed that all types of tissue samples had detectable levels of these two metabolites ( Figs. 4 and 5 ). The contents of triptolide in hairy roots, calli, adventitious roots, and naturally grown roots were 39.98, 1.76, 47.86, and 21.4 μg/g, respectively ( Fig. 5 A), and those of wilforine were 234.61, 77.11, 49.16, and 259.06 μg/g, respectively ( Fig. 5 B). These results showed that hairy roots could produce sufficient amounts of both metabolites even though they produced approximately 15% less triptolide than adventitious roots and 10% less wilforine than naturally grown roots.
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Typical chromatograms of triptolide and wilforine in the standards (A) and sample (B).
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Contents of triptolide (A) and wilforine (B) in hairy roots, untransformed calluses, adventitious roots, and naturally grown roots of T. wilfordii. Values are means of three triplicates ± SD. Different letters on the top of bars show significant difference at p < 0.05 according to Duncan’s multiple range test.
- Selection of Optimal Medium for Hairy Root Growth
In order to select an optimal medium for hairy root growth, four different media, including MS, 1/2MS, WPM, and B5, were tested. After hairy roots (0.3 g FW for the initial culture) were cultured in different media for 50 days, the dry weight of each culture was measured. The results showed that the dry weights were 0.41, 0.49, 0.07, and 0.35 g, respectively ( Fig. 6 ). The dry weight indicates that both MS and 1/2MS are suitable media for hairy root growth, but the 1/2MS medium is better with a potentially low cost compared with full MS medium. Therefore, the latter one was used for all following studies.
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Dry weight (DW) of T. wilfordii hairy roots after culture in MS, 1/2MS, WPM, or B5 medium for 50 days. Values are means of three triplicates ± SD. Different letters on the top of bars show significant difference at p < 0.05 according to Duncan’s multiple range test.
- Effects of Elicitors and Precursors on Hairy Root Growth
The dry weights were measured after hairy roots were treated with 50 μM SA and MeJA, and 500 μM nicotinic acid, isoleucine, and aspartic acid, respectively, for 3, 6, 9, 12, and 15 days. SA treatment had no apparent effect on dry weight during the 15 day culture period ( Fig. 7 ). MeJA showed inhibitory effects on the hairy root growth during the whole culture period with the reduction rates of 9% in the initial 3 days of culture and had significant inhibition with the reduction rates of 19.6-24.8% after culture for 6-15 days ( Fig. 7 ). Two out of three precursors showed beneficial effects on hairy root growth. Both nicotinic acid and aspartic acid could promote the hairy root growth with slightly higher dry weights than untreated control ( Fig. 7 ). However, isoleucine inhibited hairy root growth ( Fig. 7 ).
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Dry weight (DW) of T. wilfordii hairy roots in each flask after adding 50 μM SA, 50 μM MeJA, 500 μM nicotinic acid (Nico), 500 μM isoleucine (Ile), 500 μM aspartic acid (Asp), or an equal volume of distilled water as the control (CK) for 3, 6, 9, 12, and 15 days. Each chemical was added after hairy roots were subcultured for 7 days. Values are means of three triplicates ± SD. Different letters on the top of bars show significant difference at p < 0.05 according to Duncan’s multiple range test.
- Effects of Elicitors and Precursors on the Production of Triptolide and Wilforine in Hairy Roots
To investigate the effects of elicitors and precursors on the production of triptolide and wilforine, their contents in cultured hairy roots from the elicitor and precursor treatments and corresponding media were measured every 3 days until 15 days. The total contents of triptolide in hairy roots and liquid medium showed that MeJA could promote its production significantly after 6 days of culture and reached the highest yield with 1,448.43 μg per flask after 15 days ( Fig. 8 A). At that time, MeJA treatment had nearly 2-fold higher triptolide content than untreated control. SA inhibited triptolide production during the initial 3 and 6 days of cultures. It then began to benefit triptolide production significantly during 9 and 12 days of cultures, but its beneficial effects during this period were still much less than those of MeJA. However, none of the three precursors, nicotinic acid, isoleucine and aspartic acid had stimulatory effects on its production ( Fig. 8 A). By comparing the contents of produced triptolide in hairy roots ( Fig. 8 B) and liquid medium ( Fig. 8 C), the results showed that 60-90% of triptolide could be secreted into medium. The secretion rate was independent from culture time and treated chemical.
For wilforine, the addition of MeJA could enhance its production significantly during the whole culture period ( Fig. 9 A). After 15 days of culture, the yield of its production in MeJA treatment could reach 3,851.42 μg per flask, which was 6.7-fold higher than untreated control. SA treatment also had stimulatory effects on the production of wilforine during the whole culture period ( Fig. 9 A), but its stimulatory effects were much less than those of MeJA. Among the three precursors, nicotinic acid could promote the wilforine production during the culture period of 9-15 days while isoleucine could also benefit the wilforine production during 9-12 days of cultures; but their beneficial effects were much less than MeJA ( Fig. 9 A). No stimulatory effect on its production was observed in aspartic acid treatment. Unlike triptolide, most of the produced wilforine did not secrete into the medium, but was kept inside of hairy roots instead ( Figs. 9 B and 9 C). Moreover, wilforine released in the medium began to degrade after 9 days of cultures treated by MeJA, which is consistent with our previous study [22] .
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Contents of triptolide in T. wilfordii hairy roots and medium (A), hairy roots (B), and cultured medium (C) after adding 50 μM SA, 50 μM MeJA, 500 μM nicotinic acid (Nico), 500 μM isoleucine (Ile), 500 μM aspartic acid (Asp), or an equal volume of distilled water as the control (CK) for 3, 6, 9, 12, and 15 days. Each chemical was added after hairy roots were subcultured for 7 days. Values are means of three triplicates ± SD. Different letters on the top of bars show significant difference at p < 0.05 according to Duncan’s multiple range test.
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Contents of wilforine in T. wilfordii hairy roots and medium (A), dried hairy roots (B), and cultured medium (C) after adding 50 μM SA, 50 μM MeJA, 500 μM nicotinic acid (Nico), 500 μM isoleucine (Ile), 500 μM aspartic acid (Asp), or an equal volume of distilled water as the control (CK) for 3, 6, 9, 12, and 15 days. Each chemical was added after hairy roots were subcultured for 7 days. Values are means of three triplicates ± SD. Different letters on the top of bars show significant difference at p < 0.05 according to Duncan’s multiple range test.
Discussion
The medicinal plant T. wilfordii produces a large number of bioactive secondary metabolites [3 , 18] . Many of them, for example, wilforine and triptolide, are important for both agriculture and the pharmaceutical industry. Like most of other medicinal plants [4 , 12 , 16] , however, the natural resource of T. wilfordii plants is limited for mass production of these natural products because of their slow growth, and long period of growth, along with low yield [3 , 21 , 22 , 43] . An alternative pathway to solve above problems is needed. In this study, we have demonstrated that hairy root cultures combined with MeJA treatment could efficiently produce both triptolide and wilforine to meet the modern agricultural and pharmaceutical needs.
The hairy root culture has been considered as one of the suitable ways for the production of secondary metabolites in many medicinal plants in recent years [1 , 2 , 5 , 8 , 12 , 13 , 20 , 30] . This is also true for the production of triptolide and wilforine in T. wilfordii . For the induction o f hair y r oots, A. rhizogenes strain A4 had a higher induction rate than ATCC 15834, although both could induce hairy roots. This result is consistent with previous reports that different types of Agrobacterium strains of A. rhizogenes would have a different efficiency in inducing hairy roots [8 , 20] . For both inducing and maintenance of hairy roots in T. wilfordii , we found that 1/2MS was better than the other media. It has been reported that sucrose and/or auxin supplementation could affect hairy root production [26 , 39] , but it is not clear at the moment why 1/2MS medium with the half strength of basal salts can benefit T. wilfordii hairy root growth. Further study is needed to understand the benefit of low salt medium for hairy root growth.
When we analyzed the contents of triptolide and wilforine in hairy roots, untransformed calli, adventitious roots, and naturally grown roots, we found that the highest content of triptolide was in adventitious roots ( Fig. 5 A), whereas that of wilforine was in naturally grown roots ( Fig. 5 B). The contents of triptolide and wilforine in hairy roots were about 10% and 15% lower than in adventitious roots and naturally grown roots, respectively. Since hairy roots grow much faster than other types of tissues, the total production of triptolide and wilforine would be much higher in hairy roots than in other tissues. Therefore, T. wilfordii hairy root culture could be suitable for largescale production of these two natural products as demonstrated in other medicinal plant species [4 , 7 , 9 , 31] .
MeJA and SA are the important signal regulators of many biological processes, including biotic stress, abiotic stress, and secondary metabolites biosynthesis in plant cells [10 , 19 , 27 , 40] . In this study, MeJA showed strong stimulatory effects on the production of triptolide and wilforine, and SA also had slightly positive effects on their productions ( Figs. 8 A and 9 A). These results are consistent with our previous study [22] and other reports for various metabolites ( e.g. , tropane alkaloids [27] , glucotropaeolin [35] , asiaticoside [13] , gossypol [5] , and phytoestrogenic isoflavones [30] ). The stimulatory mechanism of MeJA has not been fully characterized yet, but it could relate to the stress response of hairy roots. In higher plants, there are two independent biosynthetic pathways involved in terpenoid biosynthesis [3 , 36] . Triptolide, a diterpenoid, is biosynthesized in the mevalonate pathway localized in the cytosol. Wilforine, a sesquiterpene, is biosynthesized in the methylerythritol phosphate pathway in the plastids [3 , 14 , 36 , 37 , 38] . It has been reported that MeJA likes water stress inducers polyethylene glycol and abscisic acid, having stress effects on hairy root cultures and subsequently enhancing secondary metabolite production [37 , 38] . They also discovered that both transcript levels and enzyme activities of 3-hydroxy-3-methylglutaryl co-enzyme A reductase (HMGR) in the mevalonate pathway and 1-deoxy-D-xylulose 5-phosphate synthase (DXS) in the methylerythritol phosphate pathway were induced by these treatments in hairy root cultures [37 , 38] . The stimulatory effects of MeJA on secondary metabolite production in T. wilfordii hairy root cultures could relate to induced expression of these genes in both the mevalonate and methylerythritol phosphate pathways.
Precursor feeding strategy was used to enhance the biosynthesis of secondary metabolites in hairy root cultures of Psoralea corylifolia and Tropaeolum majus [30 , 35] . Wilforine is a sesquiterpene pyridine alkaloid. In nature, the biosynthesis of sesquiterpene pyridine alkaloids requires the formation of evoninic acid from nicotinic acid and isoleucine, which is configured with the sesquiterpene moiety [14] . Therefore, it is understandable that both nicotinic acid and isoleucine could benefit the wilforine roduction. As for slight or no effects of precursors on hairy oot cultures, the appropriate concentration of precursors and time of addition of precursors are crucial factors [30 , 35] . At the same time, the feedback inhibition to the etabolic pathway and metabolic enzyme activities due to excess precursors are necessary for consideration during the optimization of production. In future, it would be worth it to try to combine MeJA and nicotinic acid or isoleucine treatment together, which may further improve secondary metabolite production.
After analyzing the contents of triptolide and wilforine in hairy roots and corresponding media, it was found that triptolide was mainly secreted into the culture medium ( Figs. 8 B and 8 C), whereas wilforine was mainly retained inside of the hairy roots ( Figs. 9 B and 9 C). These results are similar to our previous observation in adventitious root cultures [22] . This difference of the accumulating sites of triptolide and wilforine would facilitate their separation if the same batch of hairy root cultures is used to produce both compounds. Secretion of the majority of produced triptolide into the culture medium will also benefit downstream large-scale production and purification if the hairy root cultures are just for triptolide. We can purify this compound only by collecting the cultured medium from the bioreactor.
In conclusion, hairy roots were successfully induced by A. rhizogenes in T. wilfordii . They could be maintained and grown well in hormone-free 1/2MS liquid medium for the production of triptolide and wilforine. MeJA could dramatically stimulate the production of both triptolide and wilforine. The majority of the produced triptolide was found to be secreted into the medium, which allows to conveniently purify triptolide from the culture medium. Our studies suggest that hairy root cultures combined with MeJA treatment could be a promising way to produce triptolide and wilforine as well as other potential bioactive secondary metabolites in T. wilfordii .
Acknowledgements
This project was supported by the National Natural Science Foundation of China (No. 31272110) and the Special Fund for Agro-scientific Research in the Public Interest (No. 200903052).
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