Introduction

With more than 8000 structures currently known, phenolic compounds are the most abundant secondary metabolites of plants; they are ranging from simple molecules such as phenolic acids to highly polymerized substances such as tannins (Dai and Mumper 2010; Liu et al. 2018; Ho et al. 2019a). Polygonum multiflorum Thunb., an important medicinal herb of Southeast Asia, have been characterized for the content of phenolic compounds including stilbenes, flavonoids, tannin, anthraquinones, and phospholipids, which are responsible for their pharmaceutical and antioxidant properties (Lishuang et al. 2014; Ho et al. 2019a). Various studies have also indicated a much wider role for bioactive compounds of P. multiflorum as anti-cancer, anti-inflammatory, anti-aging, anti-diabetic, anti-microbial, and anti-oxidant (Bounda and Feng 2015). The root extracts of this herb were traditionally used for the treatment of coronary heart disease, hyperlipidemia, neurosis (Wu et al. 2012), hair dye, baldness and hair loss, potent liver and kidney tonic (Han et al. 2015; Liu et al. 2018), and potential prophylactic use for reducing adiposity (Choi et al. 2018). Besides that, chemical constituent, processing mechanism, quality evaluation, and their biological activities have been widely discussed in the previous studies (Thiruvengadam et al. 2014; Ho et al. 2018a, 2019a; Liu et al. 2018). Recently, the rising demand for this plant led to overexploitation of natural habitat; but the content of bioactive compounds in P. multiflorum roots is unstable in the natural environment. Low yields, slow growth cycles, and fluctuations in quality and quantity due to adverse environmental, seasonal, and geographical conditions are several drawbacks by field cultivation (Ho et al. 2019a). Therefore, alternative systems for production of bioactive compounds are extremely important for P. multiflorum root cultures.

Plant cells, tissues, and organs cultures are useful research tools and objects of plant developmental biology (Fehér 2019). The recent advance in adventitious roots, hairy roots, callus, somatic embryogenesis culture, and mass multiplication of medicinal plants represents the best alternative to conventional methods for increasing the yields of bioactive compounds while helping to conserve plant bioresources in their natural habitat (Park and Paek 2014; Nhut et al. 2015). Adventitious roots are attractive sources of phytochemicals, including phenolic compounds, owing to their rapid growth and genetic and biosynthetic stability (Chandra and Chandra 2011; Murthy et al. 2014). They have been successfully used for biomass and bioactive compounds production in numerous medicinal plants such as Oplopanax elatus (Jiang et al. 2015, 2017), Panax ginseng (Paek et al. 2009), Withania somnifera (Praveen and Murthy 2012), Panax vietnamensis (Tam et al. 2013), and Eleutherococcus koreanum (Lee et al. 2015). In case of P. multiflorum, there have several studies on hairy root (Thiruvengadam et al. 2014; Ho et al. 2018b) and adventitious root (Ho et al. 2017, 2018a, 2019b) culture to produce of biomass and bioactive compounds. However, further optimization of culture conditions are needed for the propagation and multiplication in adventitious root of P. multiflorum.

In addition, the main concern in the field of phytochemical production from the cell or root cultures is converting laboratory-scale research to large-scale production (Paek et al. 2009; Baque et al. 2012a; Murthy et al. 2014). Airlift bioreactors appear to be ideal for plant cell and organ cultures, and are suitable for the cultivation of hairy and adventitious roots of various medicinal plants (Murthy et al. 2014, 2016). Compared with bulb-, cone-, and cylinder-type bioreactors, balloon-type bubble bioreactor (BTBB) is the most suitable for large-scale production of biomass and ginsenosides from adventitious root and cell culture of Panax ginseng (Kim et al. 2004; Thanh et al. 2014) and other medicinal plants (Baque et al. 2012a; Cui et al. 2013; Murthy et al. 2016). BTBB provides optimum conditions for root growth and bioactive compounds accumulation by efficiently controlling the culture conditions, foam generation, shear stress, and oxygen supply (Baque et al. 2012a; Murthy et al. 2016).

Furthermore, by the simplicity and speed of sample preparation and analysis, Fourier transformation infrared (FT-IR) spectroscopy combines with multivariate analysis have been used to rapidly discriminate of metabolic equivalent in herbal medicine resources (Kwon et al. 2014; Kim et al. 2015). Although the previous research indicated that FT-IR spectroscopic analysis enabled discrimination in metabolites accumulation between hairy root and adventitious root cultures in P. multiflorum (Ho et al. 2018b), no study has reported for discriminated adventitious root cultivated under different culture conditions and in pilot-scale bioreactors. Hence, the current study was carried out not only used a BTBB to optimize the culture conditions (auxin concentration, salt strength, and sucrose concentration) for maximizing the production of biomass and bioactive compounds (phenolics and flavonoids); but also used FT-IR spectroscopy to rapidly determine their metabolite equivalence in whole-cell extracts prepared from various adventitious root sources of P. multiflorum (Table 1).

Table 1 List of P. multiflorum samples used for FT-IR analysis
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Materials and methods

Adventitious root culture

Adventitious root cultures of Polygonum multiflorum Thunb., which were induced in Floriculture and Biotechnology laboratory, Chungbuk National University, Republic of Korea, were maintained in liquid MS medium (Murashige and Skoog 1962) supplemented with 4.92 µM indole-3-butyric acid (IBA) and 3% sucrose. Cultures were grown at 24 ± 1 °C under dark conditions in a BTBB, an air-lift bioreactor (Fig. 1), and subcultured regularly at 4-week intervals.

Fig. 1
figure 1

Adventitious root culture in an air-lift bioreactor. A Schematic diagram of a balloon-type bubble bioreactor (BTBB). (a) air compressor, (b) air flow meter, (c) membrane filter, (d) glass sparger, (e) medium sampling port, (f) Rubber cap. Arrows indicate air flow. B Adventitious root culture of Polygonum multiflorum in BTBB

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Optimization of culture conditions

To optimize the culture conditions, adventitious roots of P. multiflorum were cultivated for 4 weeks in a 3-L BTBB (Fig. 1) containing 2 L of MS medium (initial pH = 5.7–5.8) at different auxin concentrations (0.5, 1.0, 2.0, and 4.0 mg·L−1 of IBA or 1-naphthalene acetic acid [NAA]). The MS medium at different strengths (0.25, 0.5, 0.75, 1.0, 1.5, and 2.0Χ) and sucrose concentrations (0%, 1.5%, 3%, 5%, 7%, and 10%) were also applied. Experiments were performed in sequence in the dark at 24 ± 1ºC and 0.1 vvm (air volume flow per unit of culture volume per minute) dissolved oxygen.

Determination of root biomass

After 4 weeks of culture, roots were harvested and washed with distilled water. The fresh weight (FW) of roots was measured after removing excess surface water with a tissue paper, and dry weight (DW) of roots was recorded after drying the roots to a constant weight at 60 °C for 48 h. The growth index (GI) and overall productivity (P) were calculated as follows:

$${\text{GI}}\;{ = }\;{\text{(Final}}\;{\text{DW}}\; - \;{\text{Initial}}\;{\text{DW)/}}\;{\text{Initial}}\;{\text{DWA}}$$
(1)
$$P\; = \;Metabolite\;yield\; \times \;Volumetric\;root\;biomass\;yield$$
(2)

where, the final and initial DW are estimated in grams; P is estimated in mg·L−1 (w/v); metabolic yield is estimated as mg·g−1 root DW; volumetric root biomass yield is estimated as g·L−1 DW.

Determination of total phenolic and flavonoid contents

Preparation of root extract

The root extractions were prepared according to the reports of Ho et al. (2017, 2019b). Dried roots (0.5 g) were refluxed (LS-2050-S10, LS-TECH, Korea) with 15 mL of 80% methanol at 80 °C for 1 h. Samples were then filtered through a filter paper (Advantec 110 mm, Toyo Rosihi Kaisha Ltd., Tokyo, Japan).

Determination of total phenolic content (TPC)

TPC was analyzed spectrophotometrically from the root ethanolic extract using the Folin-Ciocalteu colorimetric method (Wu et al. 2006). Methanolic extracts (0.05 mL) were mixed with 2.55 mL of distilled water. Then, 0.1 mL of 2 N Folin-Ciocalteu reagent was added to each sample. After 5 min, 20% Na2CO3 (0.5 mL) was added and thoroughly mixed. The reactions were incubated in the dark at room temperature. Color change was recorded after 30 min by measuring the absorbance at 760 nm using a spectrophotometer (Optizen POP, Mecasys Co., Ltd, Daejeon, Korea). The measurements were compared with a standard curve obtained for gallic acid (Sigma Chemical Co., St. Louis, MO, USA) and expressed as mg gallic acid equivalent (GAE) per g DW adventitious roots.

Determination of total flavonoid content (TFC)

TFC was determined colorimetrically according to the method of Wu et al. (2006). Methanolic root extracts and (+)-catechin (Sigma Chemical Co.) standard (0.25 mL) were mixed with 1.25 mL of distilled water. Then, 0.075 mL of 5% NaNO2 was added to each sample and shaken vigorously. After 6 min, 0.15 mL of 10% AlCl3 was added, and the sample was incubated at room temperature for 5 min. Absorbance was measured at 510 nm using a spectrophotometer (Optizen POP, Mecasys Co., Ltd, Korea). The results were expressed as mg (+)-catechin equivalents per g DW adventitious roots.

FT-IR analysis

The method for FT-IR spectroscopy analysis, processing of spectral data, and multivariate analysis were carried out as described previously by Ho et al. (2018b). In this study, samples from various adventitious root sources of P. multiflorum were used to rapidly determine their metabolite equivalence in whole-cell extracts. Besides the samples in the current work, the other samples were done in the condition at the previous studies of Ho et al. 2017, and 2018a were also collected (list at Table 1). After grinding the dried roots, 20 mg of root powder was combined with 200 µL of 20% (v/v) methanol in a 1.5 mL microfuge tube and mixed vigorously for extraction. A Tensor 27 FT-IR spectrometer (Bruker Optics GmbH, Ettlingen, Germany), equipped with a deuterated triglycine sulfate (DTGS) detector, was used for infrared measurements. The pre-processed FT-IR spectral data after a second differentiation were imported into the R statistical analysis program (version 2.15.0; R Development Core Team) for pre-processing the spectra of the data baseline correction, normalization, and mean centering of the FT-IR spectra. PCA and PLS-DA were performed using the R program (Ho et al. 2018b).

Statistical analysis

The experiment was conducted in a completely randomized design, with at least three replicates. Significant differences were determined by Duncan’s multiple range test using the SAS software (version 9.4; SAS Institute, USA).

Results and Discussion

Effect of IBA and NAA on adventitious root growth and bioactive compounds production

Adventitious root growth

Optimum auxin concentration is critical for root growth and bioactive compounds accumulation in adventitious root cultures. To determine the optimum type and concentration of auxin required for P. multiflorum adventitious root cultures, we supplemented the MS medium with different concentrations of IBA or NAA. The highest FW (80.48 g·L−1), DW (10.12 g·L−1), and GI (19.25) were obtained with 2 mg·L−1 of IBA, meanwhile the highest percent DW (18.69%) was observed with 0.5 mg·L−1 of IBA, (Table 2; Fig. S1). IBA was more effective than NAA for later root induction and root growth. IBA produced numerous slender and elongated roots, whereas NAA produced short and thick roots (Fig. S1). Selection of the auxin type and concentration affected root differentiation or dedifferentiation, root biomass, and bioactive compounds production. In the absence of exogenous phytohormone supply, root explants show no growth (Zhang et al. 2012). The positive and negative effects of IBA and NAA, respectively, on adventitious root induction and growth in P. multiflorum are consistent with previous studies (Martinez-Bonfil et al. 2014; Ghimire et al. 2018; Zhang et al. 2020; Zhao et al. 2020).

Table 2 Effect of IBA and NAA on the biomass production of P. multiflorum adventitious roots after 4 weeks of culture
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Phenolic compounds production

IBA significantly increased the TPC of P. multiflorum adventitious root culture (p ≤ 0.05) but did not affect the TFC (Fig. 2). The highest contents of phenolics and flavonoids (44.99 and 21.34 mg g−1 DW, respectively) were obtained at 2 mg L−1 IBA (Fig. 2a, b). The productivity of bioactive compounds in the bioreactor culture was calculated based on root DW. MS medium supplemented with 2 mg L−1 IBA showed the highest productivity of both total phenolics (455.29 mg L−1 medium) and total flavonoids (215.92 mg L−1 medium). Auxins in the culture medium have a major impact on the growth, multiplication, and metabolic activities of adventitious roots (Murthy et al. 2016; Zhao et al. 2020). Previously, medium supplemented with IBA or NAA was established for comparing growth and bioactive compounds in Panax gingseng (Jeong et al. 2009), Morinda citrifolia (Baque et al. 2012a), Eurycoma logifolia (Lulu et al. 2015); Panax notoginseng (Zhao et al. 2020); these studies also showed that IBA was flavor for root biomass and bioactive compounds accumulation. Tam et al. (2013) showed that IBA (5 mg L−1) was more effective than NAA and indole-3-acetic acid (IAA) in increasing the growth and ginsenoside content of Panax vietnamensis adventitious root culture. Our results indicated that 2 mg L−1 IBA was suitable for biomass production and bioactive compounds accumulation in the bioreactor system of P. multiflorum adventitious root cultures.

Fig. 2
figure 2

Effect of IBA and NAA on bioactive compound accumulation in adventitious root cultures of P. multiflorum after 4 weeks. a Total phenolic content (TPC). b Total flavonoid content (TFC). c, d Productivity of total phenols (c) and total flavonoids (d). Data represent mean ± standard error (SE; n = 3). Different lowercase letters in each graph indicate significant differences (p ≤ 0.05; Duncan’s multiple range test)

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Effect of MS medium salt strength on adventitious root growth and bioactive compounds accumulation

Adventitious root growth

Salt strength of the growth medium such as MS (Murashige and Skoog 1962), Gamborg B5 (Gamborg et al. 1968), LS (Linsmaier and Skoog 1965), and SH (Schenk and Hildebrandt 1972) has a major impact on biomass production and secondary metabolites accumulation in plant cell and organ cultures of several plant species (Murthy et al. 2016). For example, half-strength MS medium is effective in increasing the biomass and bioactive compounds content of adventitious roots of M. citrifolia in a bioreactor culture (Baque et al. 2013). Tam et al. (2015) showed that the SH medium is suitable for adventitious root culture of Panax vietnamensis. In Panax ginseng, both half- and full-strength MS media increased the production of adventitious roots, whereas full-strength MS medium maximized the ginsenoside content (Yu et al. 2000). In this study, salt strength of the MS medium significantly affected biomass and bioactive compounds production in P. multiflorum adventitious root culture. The FW, DW, and GI of P. multiflorum adventitious root culture increased with the increase in MS salt concentration up to full-strength MS, and decreased thereafter (Table 3; Fig. S2). The FW of adventitious root cultures showed no significant differences at MS salt concentrations ranging from 0.75–2X. Notably, the highest FW (87.56 g·L−1), DW (10.92 g·L−1), %DW (12.48%), and GI (20.83) of P. multiflorum adventitious root cultures were achieved at full-strength MS medium (p ≤ 0.05). In vitro cultures of plant cells, tissues, and organs consume water and minerals from the culture media to maintain growth and development (Murthy et al. 2016). Salt strength of the MS medium affects water potential, which affects the uptake of water and nutrients by the adventitious roots (Baque et al. 2010; Zhang et al. 2020). In this study, MS salt strength greater than 1X may decrease the water potential, which inhibited the absorption of water and mineral nutrients from the culture medium, thus inhibiting root growth. By contrast, at low salt strength, especially 0.25X MS, the adventitious root culture showed no increase in biomass production because of the lack of nutrients required for anabolism (Cui et al. 2010; Lee and Paek 2012).

Table 3 Effect of MS medium salt strength on adventitious root biomass production in P. multiflorum after 4 weeks of culture
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Phenolic compounds production

MS medium salt strength affected not only the biomass of P. multiflorum adventitious root cultures but also bioactive compounds accumulation in these cultures (Fig. 3). The highest TPC (56.24 mg·g−1 DW) were observed at the lowest salt strength (0.25X) and tended to decrease with increase in salt strength, while the TFC presented an insignificant difference in 0.25X up to 1,0X MS. Previous studies reported that the effect of salt strength on the highest root biomass and highest bioactive compounds accumulation in adventitious root culture were differed in various plant species. For example, Cui et al. (2010) showed that full-strength MS was optimal for biomass production; however, the highest TPC and TFC were recorded at 0.25X MS in Hypericum perforatum. In bioreactor culture of M. citrifolia, adventitious roots showed the highest biomass at 0.5X MS, whereas highest content of bioactive compounds was obtained at 1.5X MS (Baque et al. 2013). Therefore, to determine the MS salt strength optimal for both biomass and bioactive compounds production, we calculated the total productivity of bioactive compounds (mg·L−1). Previous studies showed that 0.5X MS medium was suitable for biomass and metabolite production in adventitious root cultures of Echinacea angustifolia (Wu et al. 2006), Eleutherococcus koreanum (Lee and Paek 2012), and M. citrifolia (Baque et al. 2013). Whereas the highest ginsenosides accumulation was achieved at 0.5X MS medium without NH4NO3 in P. notoginseng (Zhao et al. 2020). These results suggested that low salt strength improves the interaction among nutrients in the medium, causing an enhanced supply of ions to root. On the other hand, high salt strength strongly inhibited root biomass and deleterious effect on the bioactive compounds production (Ghimire et al. 2018). However, in the present study, the highest productivity of bioactive compounds was achieved at full-strength MS medium (TPC, 548.61 mg·g−1 DW; TFC, 227.96 mg·g−1 DW). This is consistent with a previous study, the highest biomass and withanolide-A accumulation on Withania somnifera, biomass and celastrol accumulation on Tripterygium wilfordii were resulted in full-strength MS (Praveen and Murthy 2012; Zhang et al. 2020, respectively). Our results suggest that full-strength MS medium is the most effective for adventitious root culture in P. multiflorum.

Fig. 3
figure 3

Effect of MS medium salt strength on bioactive compound production in adventitious root cultures of P. multiflorum after 4 weeks. a Content of bioactive compounds (mg·g−1 DW). b Productivity of bioactive compounds (mg·L−1 medium). Data represent mean ± SE (n = 3). Different lowercase letters in each graph indicate significant differences (p ≤ 0.05; Duncan’s multiple range test)

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Effect of sucrose concentration on adventitious root growth and bioactive compounds production

Adventitious root growth

The type and concentration of carbon substrates supplied to the plant material during in vitro culture affect its growth, development, and secondary metabolite yield (Murthy et al. 2014). In comparison with other sugars (glucose, fructose, and maltose), sucrose is the most effective for the growth of adventitious root and hairy root cultures (Praveen and Murthy 2012; Shilpha et al. 2015; Murthy et al. 2016). In this study, the effect of various sucrose concentrations (0%, 1.5%, 3%, 7%, and 10%) was tested on root biomass and bioactive compounds accumulation in P. multiflorum adventitious root culture after 4 weeks. Sucrose concentrations of up to 5% had a positive effect on root biomass (Table 4; Fig. S3). The maximum FW (98.46 g·L−1), DW (13.46 g·L−1), and GI (25.91) were reached at 5% sucrose (p ≤ 0.05). The vigorus root growth related to the more sucrose consumer. However, the increase of sucrose concentration led to increase of the osmolarity of culture media, which was the reason for dehydration and promotes the diffusion of metabolites from tissues into media, could cause the loss of cell viability, reduced growth, modified the root morphology, and inhibited lateral branching (Thiruvengadam et al. 2014; Jiao et al. 2015; Murthy et al. 2016). The current study was also showed that root biomass of P. multiflorum decreased dramatically upon a further increase in initial sucrose concentration to 7% or 10%. By contrast, roots did not grow and die thereafter in the culture medium without sucrose (0%) (Table 4).

Table 4 Effect of sucrose concentration on the adventitious root biomass of P. multiflorum after 4 weeks of culture
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Phenolic compounds production

Similar to root biomass, the TPC and TFC of P. multiflorum adventitious root cultures were significantly affected (p ≤ 0.05) by the initial sucrose concentration (Fig. 4). The highest TPC (53.08 mg·g−1 DW) and TFC (25.10 mg·g−1 DW) were achieved at 5% sucrose. In addition, significantly higher productivity of total phenolics and flavonoids was observed at 5% sucrose (714.39 and 337.79 mg·L−1 medium, respectively). Recently, in addition to proving energy, sucrose has been recognized as a signaling molecule that affects the growth, development, and metabolism of in vitro plant cultures (Praveen and Murthy 2012; Murthy et al. 2014; Zhao et al. 2020). The sucrose-specific regulation of biosynthesis of various bioactive compounds such as saponin, anthocyanin, phenolics, flavonoids, anthraquinone, etc., have previous been reported (Baque et al. 2012a, b). Generally, 3% sucrose is used for the growth and development of plant cell and organ cultures (Murthy et al. 2014; Shilpha et al. 2015; Yoon et al. 2015). However, an increase in bioactive compounds accumulation is observed at higher sucrose concentrations, possibly because of higher osmotic stress at these concentrations (Cui et al. 2010; Baque et al. 2012b). The osmotic stress is responsible for the induction of reactive oxygen species (ROS), which is subsequently responsible for inducing of signal transduction, genes expression, followed by the accumulation of bioactive compounds. Sucrose concentrations of 5% and 7% led to the highest accumulation of biomass and metabolites in adventitious roots of Echinacea angustifolia (Wu et al. 2006). Paek et al. (2009) observed that 5% initial sucrose concentration was suitable for biomass and ginsenoside production in the bioreactor culture of P. ginseng adventitious roots. Meanwhile, the total yield of three ginsenosides (Rg1, Re and Rb1) was highest in root cultures with 3% sucrose in Panax notoginseng (Zhao et al. 2020). The ability to absorb and process carbon source metabolism varies depend on each plant species; in the present study, the highest root biomass and bioactive compounds accumulation were achieved at 5% sucrose concentration, indicating that 5% sucrose is suitable for P. multiflorum adventitious root culture in a bioreactor.

Fig. 4
figure 4

Effect of sucrose concentration on bioactive compound production in P. multiflorum adventitious root cultures. a Content of bioactive compounds. b Productivity of bioactive compounds. Data represent mean ± SE (n = 3). Different lowercase letters in each graph indicate significant differences (p ≤ 0.05; Duncan’s multiple range test)

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Rapid discrimination among adventitious root in various culture condition sources based on FT-IR spectroscopy and multivariate analysis

Owing to its simplicity and speed of sample preparation and analysis, FT-IR spectroscopy has been used to evaluate whole-cell extracts (Kim et al. 2009; Kwon et al. 2014; Song et al. 2016). In this study, FT-IR spectroscopy, followed by multivariate analysis, was used to discriminate among adventitious root cultures of P. multiflorum based on metabolic equivalence (Table 1). Comparison of FT-IR spectra of various samples showed the biggest differences within the ranges of 1500–1700 cm−1 (region of amino acids and proteins including amides I and II), 1300–1500 cm−1 (region of phosphodiester group of nucleic acids and phospholipids), 900–1200 cm−1 (region of carbohydrates including monosaccharides and polysaccharides) (Fig. 5a). PCA showed that PC1 and PC2 explained 16.0% and 11.9% (total 27.9%) of the variation, respectively (Fig. 5b). The FT-IR data of most of the adventitious root samples overlapped with each other; however, only the natural root was classified. These results suggested that the qualitative and quantitative patterns of FT-IR spectra-based metabolites of whole-cell extracts were very similar across adventitious root samples grown under different culture conditions. Previously, Ho et al. (2018b) showed significant variation in adventitious root and hairy root cultures of P. multiflorum within the 800–850 cm−1 region, which reflect the structure of polyphenol molecules containing a CH ring (Schulz and Baranska 2007). However, in this study, the results of PCA loading value analysis showed the biggest difference within the FT-IR spectral ranges of 1500–1700 cm−1 and 900–1200 cm−1 (Fig. 5c). This implies that regions of amino acids, proteins (including amides I and II), and carbohydrates played an important role in the discrimination among P. multifolorum adventitious root samples. Consistent with our results, Kwon et al. (2014) reported that the polysaccharide (1050–1150 cm−1) and protein amide (1550–1650 cm−1) regions are important for discriminating among adventitious root cultures derived from different ages and types of ginseng cultivars.

Fig. 5
figure 5

Representative FT-IR spectra of P. multiflorum root samples. a Two-dimensional PCA scores. Each line represents the FT-IR spectra of an adventitious root sample cultured under different conditions. b Loading values of PCA plot. c PCA scores. d PLS-DA score plot. Dotted ellipses represent clusters of P. multiflorum root samples. P, ex vitro root; P1–P11, Adventitious root samples grown in different culture conditions (listed in Table 1)

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Furthermore, P. multiflorum samples could be distinguished more clearly using PLS-DA than using PCA (Fig. 5d). Ex vitro root samples are distributed on the left side of the PLS-DA score plot, whereas all adventitious root samples are distributed on the right side. In contrast to PCA results, PLS-DA results showed that adventitious root samples formed a separate group at the bottom of the plot. PLS-DA analysis of the FT-IR spectral data showed that all 11 P. multiflorum root samples clustered into three major groups. The first group consisted of ex vitro root samples; the second group consisted of adventitious root samples grown in a 500-L tank bioreactor; and the third group consisted of adventitious root samples treated with sucrose (3% and 5%) and elicitors (50 and 100 µM MeJA; 50 and 100 µM SA), and those grown in a flask (0.25-L), 5-L bioreactor, and 20-L bioreactor. Kim et al. (2009) showed that PLS-DA results in discrete clusters in cultivation strawberry leaves and fruits. Similarly, Song et al. (2016) showed that PLS-DA separated five Citrus lines into three distinct clusters. The results of the current study showed that the qualitative and quantitative patterns of total metabolites could be discriminated by FT-IR spectrum analysis of whole-cell extracts of P. multiflorum root samples.

Conclusions

In this study, we optimized the culture conditions for P. multiflorum by carried out the experiments in sequence. The highest root biomass and bioactive compounds production were achieved on full-strength MS medium supplemented with 2 mg·L−1 IBA and 5% sucrose after 4 weeks of culture. Our results showed that FT-IR analysis can rapidly discriminate among root samples in various culture condition sources based on total metabolite equivalence. Overall, this study suggests that adventitious root cultures could be scaled up to provide materials for commercial purposes.