Influence of LED light spectra on in vitro somatic embryogenesis and LC-MS analysis of chlorogenic acid and rutin in Peucedanum japonicum Thunb.: a medicinal herb

Abstract

Background

Peucedanum japonicum Thunb, an important medicinal herb is reported to possess pharmacological properties such as anti-obesity, anti-oxidant, anti-inflammatory, anti-bacterial, anti-diabetic and anti-platelet aggregation. The present study aimed to develop an in vitro plant regeneration system of P. japonicum via somatic embryogenesis and to analyse chlorogenic acid and rutin contents in a few commercially available plant products of P. japonicum in Japan and Taiwan markets, and tissue culture plants derived from somatic embryos.

Results

Induction of somatic embryogenesis could be achieved when root derived calli after three subcultures were transferred from Murashige Skoog's salts and vitamins (MS basal) medium with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.1-5 mg/L) to a medium with abscisic acid (ABA) (0.5-4 mg/L), or exposed to eight different light spectra provided by light-emitting diode (LED) sources. Concentrations of ABA and LED light spectra had an influence on number of somatic embryos induced and proliferation of callus. Development of secondary somatic embryos and conversion of embryos to plantlets was achieved on a medium with ABA, or their exposure to red or blue lights in a special incubation chamber. Four months old tissue culture plants derived from somatic embryos showed significantly higher levels of chlorogenic acid (10.5 mg/g dw) compared to commercial product sold in Japanese market (0.55 mg/g dw). However, rutin was absent in tissue culture plants in contrast to commercial sample (0.33 mg/g dw).

Conclusion

In this report, we describe in vitro plant regeneration system in P. japonicum via somatic embryogenesis and production of chlorogenic acid in tissue culture plants. The present study has application in further tissue culture propagation of elite plant material with high chlorogenic acid content, and identification of high yielding plants with the LC-MS method.

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DOI 10.1186/s40529-016-0124-z

Inuence ofLED light spectra oninvitro somatic embryogenesis andLCMS analysis ofchlorogenic acid andrutin inPeucedanum japonicum Thunb.: a medicinal herb

ChiaChen Chen1, Dinesh Chandra Agrawal3, MawRong Lee4, RenJye Lee4, ChaoLin Kuo1, ChiRei Wu1, HsinSheng Tsay3,5* and HungChi Chang2,3*

Background

Peucedanum japonicum Thunb., (Umbelliferae), the longevity herb is a medicinally important perennial plant. The species is distributed in Japan, the Philippines, China, Taiwan, and Korea. Leaves of P. japonicum have

been traditionally consumed in the treatment of cough in the Yaeyama Islands (Okinawa), Japan. The roots of this herb have been used as a folk medicine for cold and neuralgic diseases in Taiwan, (Chen et al. 1996). Several coumarins isolated from roots and whole plant of P. japonicum have been reported to possess pharmacological activities such as anti-obesity (Nugara etal. 2014; Nukitrangsan etal. 2012; Okabe etal. 2011), anti-oxidant (Hisamoto et al. 2003), anti-inammatory, anti-bacterial (Yang etal. 2009), anti-diabetic (Nukitrangsan etal.

*Correspondence: [email protected]; [email protected]

3 Department of GoldenAger Industry Management, Chaoyang University of Technology, Taichung 41349, TaiwanFull list of author information is available at the end of the article

2016 Chen et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/

Web End =http://creativecommons.org/licenses/by/4.0/ ), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

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2012), tyrosinase inhibition (Hisamoto et al. 2004), and anti-platelet aggregation (Chen etal. 1996). Antibacterial activities of hydro-distilled essential oils of whole parts of P. japonicum indicating anti-inammatory and potential health benets on human skin have been reported (Yang et al. 2009). Furthermore, several in vitro studies have demonstrated the occurrence of hypolipidaemic compounds in leaves of P. japonicum (Hsu and Yen 2007; Li etal. 2006). While studying the radical scavenging activity of P. japonicum, (Hisamoto et al. 2003) isolated 17 compounds from the leaves, of which several compounds played an important role in the potent anti-oxidant activity. Nugara etal. (2014) demonstrated that partially puried hexane phase (HP) had a crucial role in regulating lipid metabolism-related gene expression and energy expenditure invitro, inferring that alterations in lipid metabolism rather than inhibition of lipid absorption may be the primary mechanism for the antiobesity activity of P. japonicum. In two other reports, the ethanol extract of aerial parts of P. japonicum ameliorated the symptom of diabetes in animal model by increasing the insulin sensitivity in liver, adipose, and muscle tissues (Nukitrangsan et al. 2011, 2012). Of the genes studied, P. japonicum could prevent the obesity via the up-regulation of FXR that plays a pivotal role in the obesity related lipid metabolism (Nukitrangsan etal. 2011, 2012).

In somatic embryogenesis, a plant or embryo is derived from somatic cell(s) and somatic embryos are formed from plant cells that normally are not involved in the development of embryos (Bajaj 1995). Applications of somatic embryogenesis include: clonal propagation of genetically uniform plant material; elimination of viruses; provision of source tissue for genetic transformation; generation of whole plants from single cells called protoplasts; development of synthetic seeds (Bajaj 1995). Also, somatic embryogenesis has served as a model to understand the physiological and biochemical events that occur during plant development (Dudits et al. 1995). A large number of reports are available on somatic embryogenesis in a highly diverse taxonomic group of plants and studies have been periodically reviewed (Bajaj 1995; Dudits et al. 1995; Thorpe 1995; Jain and Gupta 2005; Mujib and Samaj 2006; Gutirrez-Mora etal. 2012). The present study was carried out to develop a plant regeneration system of P. japonicum via somatic embryogenesis and also to carry out LCMS analysis of chlorogenic acid and rutin contents in a few commercial products of the species marketed in Japan and Taiwan, and tissue culture plants derived from somatic embryos. Chlorogenic acid, a phenolic compound and an antioxidant has been reported to be more potent for body weight reduction and regulation of lipid metabolism than caeic acid (Cho

et al. 2010). It has been demonstrated that chlorogenic acid inhibited preadipocyte population growth, which may provide a proposed mechanism for reducing obesity (Hsu etal. 2006). Rutin, a common natural avonoid has potential anti-tumor efficacy and anti-inammatory eects (Deschner etal. 1991), Rutin acts as an eective inhibitor of lipid peroxidation (Yang etal. 2008) and contributes to the antibacterial (Arima etal. 2002; Watt and Pretorius 2001) and antioxidant (Ibtissem et al. 2012) properties of the plant. To the best of our knowledge, no published report has examined the eects of LED light spectra on in vitro induction of somatic embryo-genesis and production of chlorogenic acid and rutin in P. japonicum. The present study will not only facilitate further in vitro propagation, genetic transformation of this medicinally important plant species, but also help in selecting the elite plant materials of P. japonicum by LC MS analysis.

Methods

Plant material

Seeds of P. japonicum used in the present study were collected from Penghu Island in Taiwan (233618.0N 1193108.7E). Commercial samples of P. japonicum from Japan and Taiwan were obtained from the authorized sources.

Establishment ofaseptic seedlings

Seeds of P. japonicum were disinfected by washing several times with sterile distilled water, followed by dipping in 70% ethanol (v/v) for 10s, then immersing in a solution of 0.5% (v/v) sodium hypochlorite containing 1 drop of Tween-20 for 5min and the step repeated three times. Final washing step carried out in a laminar ow cabinet consisted of 3 rinses of 5 min each with sterile distilled water. Thereafter, disinfected seeds were inoculated in pre-sterilized petridishes (90 mm dia). Each dish contained 20mL of 1Murashige and

Skoogs (1962) salts and vitamins, hereinafter referred as MS basal medium (MSBM). Gellan Gum powder (GPP) (0.4%) purchased from PhytoTechnology Laboratories was used as a gelling agent and 3 % sucrose was supplemented to each medium. The pH of all the media was adjusted to 5.7 0.1, prior addition of

GPP, and before autoclaving for 15min under 1.05kg/ cm at 121C. The petridishes were incubated in a culture room at 252C, a light and dark cycle of 16/8h and an illumination intensity of 34 mol/m2/s. After 14 days, germinated seeds were transferred to glass bottles (650 mL capacity) each containing 100 mL of MS basal medium supplemented with 3% sucrose and 0.4% GPP.

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Induction ofcallus

For induction of somatic embryogenesis in P. japonicum, initial experiments were carried out to nd out a suitable culture medium and an explant. Root, leaf blade and petiole parts of 35days old invitro raised seedlings of P. japonicum were used as explants. These were separately inoculated in pre-sterilized petridishes (90 mm). Each petridish contained 20mL of MS basal medium supplemented with a range of concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) (0.15.0mg/L). Each medium was supplemented and 3% sucrose and 0.4% GPP. Cultures were incubated in a culture room at 252C in dark. After 35 days, induction of callus in each was recorded. Calli induced were subcultured for three cycles, on MSBM with same 2,4-D concentration. At the end of each subculture cycle of 35days, fresh weight and morphological changes in the calli were recorded. Out of three explants, root showed the maximum callus proliferation, hence root callus was used for further experiments for induction of somatic embryos.

Inuence ofABA oninduction ofsomatic embryos

To investigate the inuence of ABA on induction of somatic embryos, callus was cultured in petridishes (90mm) containing MS basal medium with abscisic acid (ABA) (0.5, 1.0, 2.0, 4.0 mg/L), 3 % sucrose and 0.4 % GPP. Five clumps of 100mg callus was inoculated in each petridish. These were incubated in a culture room at 252C in dark. After 60days of incubation, morphological changes and induction of somatic embryos in the calli were recorded.

Inuence ofdierent light spectra oninduction ofsomatic embryos andcallus proliferation

This experiment was performed to evaluate the inuence of dierent light spectra on induction of somatic embryos, proliferation of callus (fresh weight) and conversion of somatic embryos into plantlets. Also, the inuence of dierent light spectra on chlorogenic acid and rutin contents in callus was analyzed. Callus cultures were incubated in a specially designed plant growth chamber equipped with eight dierent LED-lights (Nano Bio Light Technology Co., Ltd., Taiwan: http://www.nanobiolight.com/en/

Web End =http://www. http://www.nanobiolight.com/en/

Web End =nanobiolight.com/en/ ). The chamber has two tiers of sections where pertidishes can be kept and exposed to different light spectra by specially designed LED-lids. There were total eight LED-lids (CW-5000 K, WW-2700 K, 8R1B, 7R1G1B, 3R3B3IR, 6R, 6B and 6IR). These LED-lids consisted of single or combinations of four dierent light spectra emitting blue (450 nm), green (525 nm), red (660 nm), and far-red (730 nm) lights. LED-lids CW-5000K and WW-2700K represents cool and warm white light while, 5000 K and 2700 K represents color

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temperature, respectively. Eight LED-lids consisted of the following ratio of light spectra in the order of blue (B): green (G): red (R): infra-red (IR); CW-5000 K (28:43:29:0), WW-2700 K (8:46:46:0), 8R1B (16:0:84:0), 7R1G1B (17:9:74:0), 3R3B3IR (57:0:43:37), 9R (0:0:100:0), 9B (100:0:0:0), 9IR (0:0:0:100) (Personal communication with the Nano Bio Light Technology Co., Ltd., Taiwan). Number (9, 7, 3, 1) in each LED-lid code represents the number of LED chips used for a particular lid. Light intensity among these eight lids varied in the range of 5464mole/m2/s depending upon the type of the LED-lid. Calli were inoculated in petridishes (90mm) which were kept in horizontal sections in the LED chamber.

Induction ofsecondary somatic embryos andconversion toplantlets

Vitried somatic embryos obtained in the LED light experiment were cut into tiny pieces forming an embryo-genic mass. This mass (200mg/each petridish) was inoculated on a fresh MS basal medium with ABA (0.5, 1.0, 2.0, 4.0mg/L), 3% sucrose and 0.4% GPP and incubated in a culture room at 252C in dark. Observations were recorded after 60days of incubation.

Acclimation andsurvival ofsomatic embryosderived plantlets

For further growth, small plantlets developed in the LED light experiment were transferred to glass bottles (650mL capacity) each containing 100mL of MS basal medium supplemented with 3% sucrose and 0.4% GPP and incubated in a culture room at 25 2 C, a light and dark cycle of 16/8h and an illumination intensity of 34mol/m2s. When these plantlets grew about 56cm in length, these were carefully taken out from the bottles, gently washed to remove adhering medium and then transplanted to plastic pots (90 mm dia) containing a mixture of peat soil: perlite: vermiculite (2:1:1). Each pot was covered with a transparent plastic sachet to maintain the humidity. These pots along with sachets were kept in the University greenhouse. After 2 weeks, sachets were removed. Survival rate was recorded after 5 weeks of transplanting.

LC/MS analysis oftissue culture plants andcommercially available samples ofP. japonicumPreparation ofHPLC/MS standard andsamples

Standard samples of chlorogenic acid (ChromaDex) and rutin (Fluka Analytical) were purchased from Sigma-Aldrich Co. LLC. Each standard (10mg) was taken into a separate 10mL volumetric ask, and then added methanol to the mark; diluted 10 times into 100ppm with puried water; took 0.1mL respectively and further diluted to 10ppm. The diluted standard solution was ltered with

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0.22m lter Millipore (Millipore, USA), and then 5L was injected for LC/MS analysis. Samples of calli (from the LED light spectra experiment) for the LC/MS analysis were collected from the petridishes and their fresh weights were recorded. All the samples were then freeze-dried for 24h and their dry weights were recorded. Fraction (100mg) of each dried sample was crushed into ne powder and dissolved in 10mL of ethanol. It was ultrasonicated for 10min and the supernatant was collected after centrifugation at 5000rpm for 10min. This process was repeated three times for each sample. After ltration, ethanol extracts were evaporated to dryness with the help of a rotary evaporator. The residue was dissolved in 10mL ethanol and ltered through a 0.22m (Millipore, USA) membrane before LCMS analysis.

Instrument andthe conditions

The analysis was carried out using a Surveryor HPLC MS pump with an auto sampler. The solution was separated on an Xbridge C18 LC column (2.1 150 mm, 5m) from Waters (Waters Corp., Milford, MA, USA) at a room temperature. The mobile phases were pure water (Millipore, USA) containing 0.1 % acetic acid (A) and acetonitrile/methanol (9/1, v/v) (B). The gradient was initialized at 80% A held for 2min, then increased linearly from 80% A to 60% A in 7min, increased to 10% A in the following 3min, further increased to 5% A in the following 5min, and then decreased to 80% A over 1min. The column was then re-equilibrated at 80% A for 4min. The ow rate was 0.2 mL/min. Mass Spectra analyses were performed using a TSQ Quantum ultra EMR tandem Mass Spectrometer (Thermo Electron, San Jose, CA, USA), equipped with an atmospheric pressure ionization (API) interface. The spectra were obtained in positive ESI mode. The spray voltage was 4.5kV, the capillary temperature was 275C, the sheath gas pressure was 35 arbitrary units, and the auxiliary gas was 5 arbitrary units. The mass scan was ranged from m/z 200 to 1000.

Statistical analysis

Software SAS 9.1 was used for statistical analysis. Data were subjected to the least signicant dierence (LSD) tested at 5 % probability level (p > 0.05) wherever possible. Each treatment had minimum 20 replicates. The experiments were repeated three times except LCMS analysis.

Results anddiscussion

Induction ofcallus andsomatic embryogenesis

All three explants of P. japonicum, leaf blade, petiole and root induced callus at all the concentrations of 2,4-D tested (0.55.0mg/L), though the quantity and quality of callus varied depending upon explant and concentration

of 2,4-D (data not shown). However, there was no induction of callus on the medium devoid of 2,4-D. Induction of somatic embryos could not be observed even after 3 subcultures on 2,4-D medium. Therefore, further experiments were carried out with a range of ABA concentrations and dierent LED light spectra for induction of somatic embryogenesis. Since callus derived from root explants proliferated easily, hence was used for further experiments. ABA concentration in the culture medium had an inuence on callus fresh weight and induction of somatic embryos. The maximum percentage of callus clumps (30%) induced somatic embryos on medium with ABA at 4.0mg/L, however, it induced a fewer number of somatic embryos (13) of globular stage, and the least callus proliferation per clump (172mg). In contrast to it, a medium with 1.0 mg/L of ABA induced the maximum (44) globular stage somatic embryos, and an average 329mg callus per clump (Table1).

Induction of somatic embryogenesis (SE) occurs either directly when embryos develop from explant tissue without any intervening callus phase, or indirectly when an explant rst produces callus which later dierentiate into somatic embryos as observed in the present study. Innumerable reports have been published demonstrating both types of somatic embryogenesis in diverse taxonomic groups of plants (Bajaj 1995; Thorpe 1995; Jain and Gupta 2005; Mujib and Samaj 2006; Gutirrez-Mora etal. 2012; Lema-Rumiska etal. 2013). Somatic embryogenesis is the developmental process by which somatic cells in a plant develop into bipolar structures without vascular connection to the parental tissue, akin to zygotic embryos. Plant growth regulators (PGRs), especially auxins play very important role in the induction of somatic embryogenesis in plants. Among dierent auxins, 2,4-dichlorophenoxyacetic acid (2,4-D) is one of the most

Table 1 Inuence of abscisic acid (ABA) on induction ofsomatic embryos incallus ofP. japonicum

ABA (mg/L)

Percentage ofcal lus clumps showing somatic embryo

Total number ofsomatic embryos

Average callus fresh weight/ clump (mg)a

0 15 10 (t) 447 48 a

0.5 15 27 (g) 336 16 b

1.0 20 44 (g) 329 42 b

2.0 25 17 (g) 212 27 c

4.0 30 13 (g) 172 12 d

Culture medium: MS basal medium+3% sucrose+0.4% GPP. Total number

of callus clumps=20 (10 in each petridish); each callus clump=100mg;

Observations at 60days of culture

g globular, t torpedo

a Meanstandard error. Means within each column followed by the same

letter(s) are not signicantly dierent at 5% level by Fishers protected LSD test

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common plant growth regulator applied for induction of somatic embryogenesis. 2,4-D, a synthetic and auxinic herbicide acts not only as an exogenous auxin analogue, but also as an eective stressor (Gaj 2004). In our study, both ABA and light spectra experiments, control calli also showed somatic embryos indicating that medium with 2,4-D initiated the necessary physiological changes in the calli during subcultures, however, withdrawal of 2,4-D from the medium was necessary for the development of somatic embryos. It has been observed earlier that in some cases withdrawal of auxins from the culture medium promotes induction of somatic embryogenesis in calli (Gutirrez-Mora etal. 2012; Lema-Rumiska etal. 2013). As observed in our study, the process of induction of somatic embryos occurs through an orderly series of characteristic embryological stages like globular, heart and torpedo.

Inuence ofLED light spectra onsomatic embryogenesis

Results on the inuence of eight LED light spectra on development of somatic embryos, vitrication, callus proliferation (fresh weight) and callus texture is given in the Table2. The maximum number of somatic embryos (329) in globular and torpedo stages was recorded with the light spectra 8R1B, however, the maximum callus proliferation (fresh weight) (821 mg/callus clump) was obtained with the 9IR treatment. The maximum percentage of vitried somatic embryos (31) were recorded with the light spectrum WW. The callus texture from compact to friable, and dry to succulent varied depending upon the light spectrum it was exposed (Fig.1cj) Newly proliferated calli under light spectra CW and 9B showed light pink and purple color, respectively, while callus under 9IR developed deep golden color. Four light spectra

3R3B3IR, 9IR, 9R and 9B did not induce any somatic embryos (Table 2). Light quantity, quality and duration are known to control morphogenesis, growth and dierentiation of plant cells, tissues and organ cultures (Moshe and Dalia 2007). Generally, uorescent lamps, metal halide lamps, high-pressure sodium lamps or incandescent lamps are used as a source of light in tissue culture. Some of these light sources emit non-essential wavelengths of low quality (Kim etal. 2004) aecting plant growth and development. More advance light-emitting diode (LED) light sources have several advantages. LED light systems are much smaller in size, are more durable, have wavelength specicity and provide emitting surfaces relatively cool. Also, one can determine their spectral composition.LED light sources enable wavelengths to be matched to plant photoreceptors to provide more optimal production and to inuence plant morphology and metabolic composition (Bourget 2008; Massa et al. 2008; Morrow 2008). Due to these advantages, LED light sources have already been used for invitro cultivation of several commercially important plants such as upland cotton, banana, strawberry, grape, potato, maize, birch, Cymbidium, Lilium, chrysanthemum and Phalaenopsis (Saebo etal. 1995; Nhut etal. 2003; Li etal. 2010). The integration, quality, duration and intensity of red-, infrared-, blue- and ultraviolet-light can have a profound inuence on plants by activating or deactivating physiological reactions and controlling their growth and development (Briggs etal. 2001; Briggs and Olney 2001; Clouse 2001).These studies have demonstrated that LED light is more suitable for plant growth than uorescent lamps. Similar to our results, benecial eects of LED light sources on induction of embryogenesis in Oncidium have been reported (Chung et al. 2010). Now LED light system is

Table 2 Inuence ofdierent light spectra oninduction ofsomatic embryos andcallus growth inP. japonicum

Light spectrum Total number ofsomatic embryos

Percentage of vitried somatic embryos

Average callus fresh weight (mg/clump)a

Callus texture

CW5000 K 110 (g) 20 732 37 b Compact, dry, pink color

WW2700 K 90 (g + t) 31 644 14 de Compact, succulent

7R1G1B 191 (g + t) 20 635 18 def Compact, succulent

8R1B 329 (g + t) 20 555 14 ef Compact, light golden color

3R3B3IR 0 0 721 16 bc Compact, dry, with white cells

9IR 0 0 821 15 a Compact, deep golden color

9R 0 0 631 6 def Compact, light creamish color, succulent

9B 0 0 681 11 bcd New callus friable, purple color, succulent

Culture medium: MS basal medium+3% sucrose+0.4% GPP. Total number of callus clumps each petridish=20; each callus clump=100mg; observations at

60days of culture

g globular, t torpedo

a Meanstandard error. Means within each column followed by the same letter(s) are not signicantly dierent at 5% level by Fishers protected LSD test

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being increasingly used to boost horticulture industry in Taiwan and several other countries (Fang etal. 2011). More recently, studies on the inuence of LED lighting on plant growth, physiology and secondary metabolism have been reviewed (Ouzounis etal. 2015; Olle and Versile 2013). However, the exact mechanism of how LED lights sources eect production of secondary metabolites at a molecular level is not yet known.

Secondary somatic embryogenesis

Secondary somatic embryogenesis (SSE) is the phenomenon whereby new embryos are initiated from somatic embryos. Thus, an unlimited number of secondary somatic embryos can be generated in a cyclic manner from a culture of primary embryos (Raemakers et al. 1995). In our study, the embryogenic mass of vitried

(hyperhydric) somatic embryos obtained in the LED light experiment when cultured on fresh MS basal medium supplemented with ABA (0.54 mg/L), or incubated under eight dierent LED-lids induced a varying number of secondary somatic embryos and developed plantlets (Fig.1k) (data not shown). In literature, there are several contradictory reports on role of ABA in the induction of secondary somatic embryogenesis in caraway embryo-genic cell clumps (Ammirato 1987), celery cell cultures (Nadel et al. 1990) and carrot cell cultures (Iida et al. 1992) indicating an interplay of several factors in aecting the secondary somatic embryogenesis.

Survival oftissue culture plants

Conversion of somatic embryos to plantlets was observed in light spectra 9R and 9B (Fig. 1k). Plantlets could

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successfully be established after their transfer to plastic pots containing soil mix (peat soil:perlite:vermiculite (2:1:1) with a survival rate of 73% in the University greenhouse.

LCMS analysis

Results of LCMS analysis of chlorogenic acid and rutin contents in the commercial samples, tissue culture plants and calli from LED light spectra experiment are presented in Table 3. The maximum chlorogenic acid content (10.5 mg/g dw) was recorded in tissue culture plants derived from somatic embryos. However, rutin could not be detected in tissue culture plants and calli obtained in LED light spectra experiment. Among the three sources, leaf powder samples of Taiwan showed the maximum chlorogenic acid content (0.60 mg/g dw) and rutin (1.01mg/g dw), respectively. While leaf powder samples of two dierent Japanese companies showed more or less similar contents of Chlorogenic acid (0.55 and 0.48 mg/g dw, respectively) and rutin (0.33 and 0.29mg/g dw, respectively). Petiole powder sample from Taiwan also contained chlorogenic acid (0.27 mg/g dw) and rutin (0.31mg/g dw), however root powder sample of Taiwan did not have chlorogenic acid, but showed negligible amount of rutin (0.01mg/g dw). Calli proliferated in LED light spectra experiment contained only chlorogenic acid in varying quantities but no rutin (Table3). The maximum chlorogenic acid (3.44 mg/g dw) was recorded in callus exposed to light spectra 3R3B3IR. Callus cultured in light spectrum 9R did not contain any of the two compounds.

Similar to the present study, production of higher contents of secondary metabolite compounds by tissue culture plants compared to wild plants and commercial samples available in market has been reported earlier in medicinal plants such as Glossogyne tenuifolia (Chen etal. 2014), Saussurea involucrata (Kuo etal. 2015).

Conclusions

In vitro induction of somatic embryogenesis and plant regeneration system in P. japonicum has been developed. Concentrations of ABA and dierent LED light spectra had a signicant inuence on somatic embryogenesis in P. japonicum. In contrast to samples obtained from markets in Japan and Taiwan, tissue culture plants had signicantly higher amounts of chlorogenic acid. The study has application in micropropagation and selection of elite materials of P. japonicum by LCMS method.

Abbreviations

ABA: abscisic acid; 2,4D: 2,4dichlorophenoxyacetic acid; LED: lightemitting diode (LED); MS basal medium: Murashige and Skoogs macro, micro salts and vitamins.

Authors contributions

CCC carried out the experimental work; ADC wrote the manuscript; LMR and LRJ provided LCMS laboratory facilities and helped in LCMS analytical work; CLK identied the wild plant materials and provided other signicant inputs during the study; WCR helped in the data analysis; THS provided laboratory facilities and important inputs for the manuscript preparation; CHC provided funding and designed the experiments. All authors read and approved the nal manuscript.

Author details

1 Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung 40402, Taiwan. 2 Departmentof GoldenAger Industry Management, Chaoyang University of Technology, Taichung 41349, Taiwan. 3 Department of GoldenAger Industry Management, Chaoyang University of Technology, Taichung 41349, Taiwan. 4 Departmentof Chemistry, National ChungHsing University, Taichung 40227, Taiwan.

5 Department of Agronomy, National ChungHsing University, Taichung 40227, Taiwan.

Acknowledgements

Research Grants (NSC 1012313B324002 and 1022313B324001MY3) from the Ministry of Science and Technology (MOST), R.O.C. is gratefully acknowledged.

Competing interests

The authors declare that they have no competing interests.

Received: 10 January 2016 Accepted: 25 February 2016

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Table 3 LC-MS analysis ofchlorogenic acid andrutin indifferent plant materials ofP. japonicum

Plant material Secondary metabolites

Chlorogenic acid (mg/g dw)

Rutin (mg/g dw)

Leaf powder1a from Japan 0.55 0.33 Leaf powder2a from Japan 0.48 0.29 Leaf powder from Taiwan 0.60 1.01 Petiole powder from Taiwan 0.27 0.31 Root powder from Taiwan 0 0.01 Tissue culture plants (4 months old) 10.5 0 CallusCW 0.44 0 CallusWW 0.07 0 Callus7R1G1B 0.20 0 Callus8R1B 0.08 0 Callus3R3B3IR 3.44 0 Callus9IR 0.19 0 Callus9R 0 0 Callus9B 0.09 0

a Two leaf powders were purchased from two dierent companies in Japan

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