Introduction

Arnebia euchroma (Royle) Johnst, which belongs to family Boraginaceae, is a highly valued and critically endangered medicinal herb that grows on gravel slopes, alluvial fans, grasslands, and meadows in the regions with an altitude ranging from 2500 to 4200 m. A. euchroma is the national secondary protected plant in China, and used to treat various diseases, such as hepatitis, phlebitis, vascular purpura, psoriasis, and burns (Mohsenikia et al. 2017; Fan et al. 2012; Nasiri et al. 2016). The medicinal parts, that is, the roots of A. euchroma, contain naphthoquinones, phenols, benzoquinones, phenolic acids, alkaloids, and sterols (Xin et al. 2012). Meanwhile, the most prominent compound is naphthoquinones, which is not only the main active ingredient of A. euchroma with a variety of physiological activities [i.e., antibacterial (Singh et al. 2015), anti-inflammatory (Kaith et al. 1996) and anticancer (Jeziorek et al. 2018)], but is also widely used as natural pigments in pharmaceutical, cosmetic, and printing industries (Hou et al. 2018; Buchanan et al. 1997). Given its important medicinal efficacy and economic value, A. euchroma has been overexploited. A. euchroma is also difficult to cultivate because its growth environment is extremely strict, and it has a long growth cycle, high seed abortion rate, and low germination rate (Xiong et al. 2009). Thus, the narrowly distributed and sparsely populated wild resources of A. euchroma have been severely damaged, thereby resulting in a rapid decline in the distribution and quantity of its wild species and a serious resource shortage. Therefore, protecting the germplasm resources of A. euchroma and providing a stable and sustainable use of medicinal and industry materials are crucial (Seabrook 2005; Li et al. 2010; Dutta and Jatothu 2013). Plant tissue culture is an important way to solve the lack of resources via the acquisition regenerated plantlets and the produce medicinal active substances. Some studies have shown that plant tissue culture can be used for shoot organogenesis and secondary metabolite accumulation in A. euchroma (Jiang et al. 2005; Zakhlenjuk and Kunakh 1998).

Numerous studies have indicated that different physicochemical factors influence the establishment of regeneration system and accumulation of secondary metabolite of A. euchroma (Manjkhola et al. 2005; Malik et al. 2013). While, among various physicochemical factors, light plays a significant role in regulating the growth, development, and secondary metabolic accumulation of many plants in vitro, such as blue LEDs promoted bigger biomass of Myrtus communis L. (Cioć et al. 2017), red LEDs stimulated the maximum biomass of Rhodiola imbricata Edgew (Kapoor et al. 2018), red LEDs or RB (1:2) stimulated the maximum shoots production of Dendrobium officinal (Lin et al. 2011), B:R (1:1) LEDs stimulated the elongation of shoots and chlorophyll synthesis of Vanilla planifolia (Ramírez-Mosqueda et al. 2017), and the highest Ginsenoside-Rg1 and Ginsenoside-Rb1 contents of Panax vietnamensis were found under fluorescent light (Nhut et al. 2015). light is considered as an important abiotic which can affect the metabolite production (Akula and Ravishankar 2011), and there has research that naphthoquinones, compound of A. euchroma, its derivative can be sensitive to changed light conditions and can induced chemical reactions by light (Tasdelen and Yagci 2013). At present, although some factors, such as plant growth regulator temperature, sucrose, and pH, affect A. euchroma tissue culture, studies on the regeneration system and accumulation of the secondary metabolite of A. euchroma under different light conditions are lacking.

Therefore, the objectives of this work were to examine how the regeneration (including shoot formation, proliferation, growth, and development) and naphthoquinone accumulation of A. euchroma were affected under different light conditions, including, darkness, fluorescent lamp (Fl), white LED, blue LEDs, red LEDs, and a combination of 25% red, 25% blue, and 50% white LEDs [RWB (1:1:2)]. Then, suitable light quality for plantlet formation and naphthoquinones accumulation, which can contribute to the propagation and commercial production of this overexploited and valuable medicinal plant, can be obtained.

Materials and methods

Plant material and light conditions

Arnebia euchroma seeds were collected from Wenquan County, Xinjiang. The seeds were washed under running tap water for 30 min, treated with 75% (v/v) ethanol for 15 s followed by 15% (v/v) H2O2 for 24 min, and then washed five times with sterile distilled water. The sterilized seeds were inoculated on 1/2 MS medium (Murashige and Skoog 1962) and supplemented with 0.05 g/L activated carbon and 7 g/L agar. The pH of the medium was adjusted to 5.8 before autoclaving at 121 °C for 25 min. The seeds were incubated for approximately 15 days in the culture room maintained at 25 ± 1 °C and under the dark for germination. The sterile seedling cotyledons were placed on modified LS medium (825 mg/L NH4NO3), supplemented with 40 g/L sucrose, 7 g/L agar, 0.05 g/L casein hydrolysate, 0.08 mg/L AgNO3, and 0.8 mg/L thidiazuron to attain callus, which was used for the next experiment.

The cultures were maintained under different lighting conditions, including (1) darkness, (2) Fl (with a wavelength of 420 nm), (3) white LEDs (with a wavelength of 410–416 nm), (4) blue LEDs (with a wavelength of 450–455 nm), (5) red LEDs (with a wavelength of 650–660 nm), and (6) the combination of red, blue, and white LEDs at the percentages of 25, 25, and 50%, respectively. All the treatments were cultured at 60 μmol m−2 s−1, 25 ± 1 °C temperature, 14/8 h photoperiod (day/night), and 50% relative humidity.

Formation and proliferation

Callus was transferred into the modified LS medium, supplemented with 40 g/L sucrose, 7 g/L agar, 0.05 g/L casein hydrolysate, 0.08 mg/L AgNO3, 0.01 mg/L naphthylacetic acid (NAA), 0.5 mg/L kinetin (KT), and 2 mg/L 6-benzylaminopurine (6-BA) to obtain cluster bud after 30 days of culture. After culturing, the germination rate, fresh weight (FW), dry weight (DW), and shoot number were recorded. The cluster bud was cut into individual shoots and then inoculated into the same medium as the induced buds for proliferation culture. The proliferation rate was recorded. The germination and the proliferation rate were calculated according to the method of Wang et al. (2016) as follows:

$$\begin{aligned} {\text{Germination}}\;{\text{rate}} & = {\text{number}}\;{\text{of}}\;{\text{callus}}\;{\text{with}}\;{\text{differentiated}}\;{\text{buds}}/{\text{total}}\;{\text{number}}\;{\text{of}}\;{\text{inoculated}}\;{\text{callus}}, \\ {\text{Proliferation}}\;{\text{rate}} & = {\text{number}}\;{\text{of}}\;{\text{proliferating}}\;{\text{buds}}\;{\text{after}}\;{\text{culture}}/{\text{total}}\;{\text{number}}\;{\text{of}}\;{\text{inoculated}}\;{\text{single}}\;{\text{buds}}. \\ \end{aligned}$$

Growth and development

After proliferation, the buds were cut into single shoot, inoculated into the modified LS medium, and then supplemented with 40 g/L sucrose, 7 g/L agar, 0.05 g/L casein hydrolysate, and 0.08 mg/L AgNO3. After 30 days of culture, the growth index (Kokotkiewicz et al. 2014), plant height, and leaf number were recorded:

$${\text{GI}} = \left[ {\left( {G_{1} - G_{0} } \right)/G_{0} } \right],$$

where GI is the growth index, G1 is the plant fresh weight at the end of cultivation, and G0 is the fresh weight of the inoculum.

The chlorophyll (Chl) and carotenoids’ (Car) concentrations were measured via spectrophotometry by referring to the method of Wang and Huang (2015). Chl and Car were extracted from the leaves, and the leaf (0.2 g FW) samples were placed into a mortar with quartz sand, CaCO3, and 2–3 mL of 95% ethanol and then ground into homogenate. Then, the sample was ground in 10 mL of ethanol until the leaf tissue turned white. The absorbance was measured using UV-1600PC at the wavelengths of 470, 649, and 665 nm, as follows:

$$\begin{aligned} &{\text{Chl}}\,{\text{a}}\;{\text{concentration}}:\;{\text{Chl a }}\left( {{\text{mg}}/{\text{L}}} \right) = 1 3. 9 5 {\text{A}}_{ 6 6 5} - 6. 8 8 {\text{A}}_{ 6 4 9} , \\ &{\text{Chl}}\,{\text{b}}\;{\text{concentration}}:\;{\text{Chl b }}\left( {{\text{mg}}/{\text{L}}} \right) = 2 4. 9 6 {\text{A}}_{ 6 4 9} - 7. 3 2 {\text{A}}_{ 6 6 5} , \\ &{\text{Total}}\;{\text{chlorophyll}}\;{\text{oncentration}}\;{\text{Chl}} = {\text{Chl a}} + {\text{Chl b}}, \\ &{\text{Car}}\;{\text{concentration}}:\;{\text{Car }}\left( {{\text{mg}}/{\text{L}}} \right) = \left( { 1000{\text{A}}_{ 4 70} - 2.0 5 {\text{ Chl a}} - 1 1 4. 8 {\text{ Chl b}}} \right)/ 2 4 5. \\ \end{aligned}$$

Rooting culture

The plantlets were transferred into the rooting medium-containing modified LS medium, 20 g/L sucrose, 7 g/L agar, 0.05 g/L casein hydrolysate, 0.05 mg/L NAA, and 0.05 mg/L indole-3-butyric acid to induce rooting. After 40 days of culture, the rooting rate was measured, as follows:

$${\text{Rooting}}\;{\text{rate}} = {\text{number}}\;{\text{of}}\;{\text{plantlet}}\;{\text{with}}\;{\text{rooting}}/{\text{total}}\;{\text{number}}\;{\text{of}}\;{\text{inoculated}}\;{\text{roots}}.$$

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Naphthoquinone quantification using HPLC

The euphylla obtained via seed germination was inoculated in the modified LS medium and supplemented with 30 g/L sucrose, 7 g/L agar, 0.05 g/L casein hydrolysate, 0.5 mg/L NAA, and 0.1 mg/L KT for 20 days to achieve adventitious roots. Then, collect adventitious roots and dry it at 50 °C for 5 h. In addition, naphthoquinones were extracted from the adventitious roots (0.5 g DW), which were ultrasonicated with 20 mL of methanol (150 w, 40 kHz) for 50 min. After the extracted solution was cooled to room temperature, and the lost weight via volatilization was made up with methanol, the solution was filtered with 0.45 μm microporous membrane, and the filtrate for the next analysis. Naphthoquinones (shikonin and β′β-dimethylacrylalkannin) were quantified using HPLC (Agilent 1260) with InertSustain C18 column (250 × 4.6 mm, 5 μm). The mobile phases of solvents A (acetonitrile) and B (water:formic acid = 100:0.5) were used as gradient elution. The condition for the gradient is as follows (v/v): 0–10 min, 70% A; 10–20 min, 90% A; 20–25 min, 100% A; 25–27 min, 70% A. The flow rate was 1 mL/min at the temperature of 30 °C, and the detection wavelength was 516 nm.

Data collection and analysis

All treatments were in triplicates, and each replicate had 30 explants. Data were scored and analyzed for variance at the end of cultivation. All statistical data were analyzed using SPSS software (version 23.0) and subjected to one-way ANOVA. Means were compared using the LSD multiple test, and the difference was considered statistically significant at P < 0.05.

Results and discussion

Effect of light quality on A. euchroma regeneration

Effect of light quality on formation and proliferation

The shoot formation and proliferation of A. euchroma from callus were significantly affected by different lights (Table 1, Figs. 1 and 2). As shown in Table 1, the maximum FW and DW were obtained under RBW (1:1:2) LEDs, and the minimum FW and DW were obtained under dark conditions. These results were consistent with those reported by Kim et al. (Kim et al. 2004), who observed the high FW and DW of Chrysanthemum under mixed LEDs. The highest germination rate and number of shoots were also recorded under RBW (1:1:2) LEDs, and the minimum values were observed in the dark. This finding was consistent with those reported in the study of Nhut et al. (2015), who observed the highest germination rate and number of shoots on Panax vietnamensis under mixed LEDs, whereas the lowest number of shoots was obtained in the dark. Meanwhile, Fig. 1 shows that the bud was white and slender in the dark (Fig. 1a) but green and healthy under Fl (Fig. 1b) and LEDs (Fig. 1c–f). When the bud was cut into individual shoots and cultured for 30 days to proliferate, the maximum proliferation index (14.66 ± 0.84, P < 0.05) was observed under RBW (1:1:2) LEDs (Fig. 2). However, the minimum proliferation index (2.33 ± 0.33, P < 0.05) was obtained in the dark (Fig. 2).

Table 1 Effect of light quality on A. euchroma formation
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Fig. 1
figure 1

Effects of light quality on plant formation of A. euchroma after cultivating callus on bud induction medium for 30 days. a Buds incubated in the dark. b Buds incubated under fluorescent lamp. c Buds incubated under white LEDs. d Buds incubated under blue LEDs. e Buds incubated under red LEDs. f Buds incubated under RBW (1:1:2) LEDs

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Fig. 2
figure 2

Effect of light quality on A. euchroma proliferation. Values followed by different letters denote statistically significant differences (P<0.05). Data represent mean±SE

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In addition to providing energy source, light also plays an important role in the in vitro culture as a signal received by photoreceptors, thereby regulating growth, differentiation, and metabolism and affecting plant photomorphogenesis (Seabrook 2005; Ouyang et al. 2003). The results showed that the higher FW and DW levels, germination rate, shoot number, and proliferation index were observed under all LED treatments than those in the dark and Fl treatments. Specifically, RBW (1:1:2) LEDs were highly advantageous in promoting A. euchroma shoot formation and proliferation. Combined with previous evidence, the reason why RBW (1:1:2) LEDs are more advantageous in promoting the shoot formation and proliferation of A. enchroma may be attributed to the increase in the amount of Pfr, which can regulate the expression of photoreactive genes (Zhu et al. 2000).

Effect of light quality on growth and development

The growth and development of A. euchroma were significantly affected by different light conditions (Table 2 and Fig. 3). As shown in Table 2, the A. euchroma growth index, shoot height, leaf number, and photosynthetic pigment concentration, including those of Chl a, Chl b, Chl (a + b), and Car, were the highest under red LEDs. The growth index, shoot height, and leaf number showed significant difference with other treatments (P < 0.05). The contents of photosynthetic pigments are an important indicator of many processes taking place in the plant body (Münzbergová and Haisel 2019). The Chl (a + b) and Car concentration had insignificant differences among blue, red, and RBW (1:1:2) LED treatments. Meanwhile the minimum values of the growth index, shoot height, leaf number, and photosynthetic pigment concentration were observed in the dark, and the leaves became yellow and thin (Fig. 3a). The growth was slow and deformed compared with those under other conditions. These results showed that red LEDs significantly promoted the growth index and shoot height and increased the leaf number of A. euchroma and the photosynthetic pigment content. These results confirmed those of the previous studies on Oncidium, wherein that red light spectrum enhances induction, proliferation, and the carbohydrate contents of PLBs, as well as subsequent plantlet lengths (Liu et al. 2011).

Table 2 Effects of light quality on A. euchroma growth and development
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Fig. 3
figure 3

Effect of light quality on A. euchroma growth and development after 30 days of culture. a Cultivated in the dark. b Cultivated under fluorescent light. c Cultivated under white LEDs. d Cultivated under blue LEDs. e Cultivated under red LEDs. f Cultivated under RBW (1:1:2) LEDs

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Light can affect plant cell division, growth, and differentiation (Walter 2008). Schuerger et al. (1997) reported that red light is important for the development of the plant photosynthetic apparatus, and light increases starch accumulation by increasing net photosynthetic rate. Kim et al. (2004) obtained greatest stem elongation and internode length under R and RFr in Chrysanthemum. However, Cybularzurban et al. (2007) reported that red light promotes leaf growth, but decreased the Chl and Car contents of plantlets in vitro. Hahn et al. (2000) reported a reverse effect of red light, thereby suggesting that red light inhibits shoot growth, because monochromic red light causes the imbalance of light energy distribution available for photosystems I and II. The growth, stem elongation, leaf number, and photosynthetic pigment concentration may be promoted or inhibited using red light according to species. Meanwhile, our results showed that red LEDs were advantageous in enhancing the growth and development of A. euchroma.

Effect of light quality on rooting

The effect of light quality on A. euchroma rooting rate is shown in Fig. 4. The highest rooting rate in A. euchroma plantlets was obtained under red LEDs (89.29% ± 2.06%), and the difference in the rooting rate under blue LEDs (84.74 ± 0.26%, P > 0.05) was insignificant. Given that the plantlets cannot normally grow and develop and even die in the dark, the rooting rate cannot be recorded and absent under the dark condition. For other treatments, the lowest rooting rate was observed under Fl treatment (65.43% ± 1.56%) and was significantly lower than that under LED treatments (P < 0.05).

Fig. 4
figure 4

Effect of light quality on A. euchroma rooting rate. Values followed by different letters denote statistically significant differences (P<0.05). Data represent mean±SE

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Understanding how plants respond to changes in light quality is difficult, because studies only compare specific ratios in many different species, and their responses are often contradictory (Długosz et al. 2017). A previous study validated that Tripterospermum japonicum rooting is promoted under red light (Moon et al. 2006), which is consistent with our results. However, a high number of Rehmannia glutinosa roots were obtained under blue LEDs (Manivannan et al. 2015). In the present study, A. euchroma rooting rate can be enhanced under red LEDs.

Effect of light quality on naphthoquinones (shikonin and β′β-dimethylacrylalkannin) accumulation

The previous studies on A. euchroma cell suspension cultures demonstrated that shikonin derivative production can be influenced by some physicochemical factors (Malik et al. 2008, 2013). Naphthoquinones (i.e., shikonin and β′β-dimethylacrylalkannin) are used as indicators of drug eligibility in the Chinese pharmacopeia due to their high biological and pharmacological activities. Therefore, the systematic investigation of the effect of different light qualities on naphthoquinone accumulation in A. euchroma is necessary. Considering that the roots are the medicinal parts of A. euchroma, we also obtained adventitious roots in the euphylla as explants. The naphthoquinone content of A. euchroma adventitious roots cultured for 20 days under different light conditions was analyzed using HPLC, and the results are shown in Fig. 5. The shikonin content ranged from 65.01 μg/g to 121.77 μg/g DW, and the maximum shikonin accumulation (121.77 ± 6.68 μg/g DW) was observed in the dark, which was significantly higher than that under blue (89.81 ± 4.93, P < 0.05), red (87.66 ± 7.29 μg/g DW, P < 0.05), RBW (86.06 ± 3.20), and white LEDs (65.01 ± 1.89 μg/g DW, P < 0.05). The minimum shikonin content (70.67 ± 3.71 μg/g DW) was observed under Fl. Similarly, the maximum β′β-dimethylacrylalkannin content was observed in the dark (619.18 ± 15.71 μg/g DW), and the minimum content was observed under Fl (224.31 ± 11.90 μg/g DW). These results were consistent with those of Gupta et al. (2014), who found that A. euchroma cell suspension cultures and napthoquinones’ production increased in the dark. Thus, the dark condition is beneficial to naphthoquinones accumulation, whereas the light condition was attributed to the inhibition of naphthoquinone accumulation. Specifically, shikonin and β′β-dimethylacrylalkannin contents that was cultured under LEDs was significantly lower than that under dark culture (P < 0.05), and the fluorescence light severely inhibited naphthoquinones accumulation. Some naphthoquinones’ production characterized specific light sensitivity, and the o-naphthoquinone methide/thiol click chemistry has a unique reversible and provides a light-directed release or replacement of the immobilized substances (Tasdelen and Yagci 2013). Thus, we suspect in this study that shikonin and β′β-dimethylacrylalkannin may be sensitive to light.

Fig. 5
figure 5

Effect of light quality on naphthoquinone accumulation in the adventitious roots of A. euchroma. Values followed by different letters denote statistically significant differences (P<0.05). Data represent mean±SE

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Conclusion

RBW (1:1:2) LEDs promoted plant formation and proliferation, and red LEDs stimulated the maximum growth, development, and rooting during the A. enchroma tissue culture. Dark condition was also beneficial to naphthoquinones (shikonin and β′β-dimethylacrylalkannin) accumulation in the roots when the euphylla was used to attain the adventitious roots in vitro. Therefore, this research may contribute to the protection of the overexploited medicinal plant. This work may also set a foundation for further studies on the efficient and large-scale commercial production of A. euchroma by searching for a suitable light quality to promote plant regeneration system and naphthoquinones (shikonin and β′β-dimethylacrylalkannin) accumulation.