Low-intensity monochromatic red LED light can improve shikonin productivity in long-term cultivated Lithospermum erythrorhizon Sieb. et Zucc. calli

Low-intensity monochromatic red LED light can improve shikonin productivity in long-term cultivated Lithospermum erythrorhizon Sieb. et Zucc. calli | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Low-intensity monochromatic red LED light can improve shikonin productivity in long-term cultivated Lithospermum erythrorhizon Sieb. et Zucc. calli Galina N Veremeichik, Slavena A Silantieva, Valeria P. Grigorchuk, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7030114/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Nov, 2025 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted 4 You are reading this latest preprint version Abstract Currently, global challenges require the search for alternative sources of phytochemicals used by Mankind throughout its history. Plant cell culture technology can provide efficient and sustainable sources of phytochemicals with reduced energy and carbon footprints. The first patented industrial process of cultivating calluses as sources of phytochemicals was based on the calli of Lithospermum erythrorhizon Siebold & Zucc (Boraginaceae). The high pharmacological activity of L. eryrthrorhizon is due to the content of the naphthoquinone shikonin. Despite the development of LED lighting technology, in-depth studies of the effects of light on the biosynthesis of shikonin in cell cultures have not been carried out. In the present work, the impact of artificial monochromatic and bichromatic LED light at wide ranges of intensities (50, 100, and 300 µmol m -2 s -1 ) on the growth and biosynthesis of caffeic acid derivatives (CADs) as well as shikonin and shikonofurans in long-term continuously cultivated L. erythrorhizon calli was investigated for the first time. Red light has the greatest growth-stimulating effect regardless of intensity. The most effective treatment for CADs productivity is red/blue and green light treatment. The most effective way to produce shikonin in long-term cultivated L. erythrorhizon calli is to use red light with an intensity of 50 µmol m -2 s -1 and increase the inductor concentration. It may also be assumed that the blue and green components of white light have a negative effect on shikonin biosynthesis because of the light-dependent shift in the biosynthesis of CADs. Artificial light rosmarinic acid long-term cultured calli Lithospermum erythrorhizon Siebold & Zucc shikonin derivatives rabdosiin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Throughout history, plants have been the main source of phytochemical compounds used by Mankind for food, medicinal, and cosmetic purposes (Jamshidi-Kia et al., 2018 ). Currently, global climate change, limited fresh water and food supplies, achievements in sustainable development and increasing energy needs are among the most important global challenges facing humanity. Plant cell culture technology can address some of these issues by providing efficient and sustainable sources of phytochemicals with reduced energy and carbon footprints (Krasteva et al., 2021 ). In vitro plant cultures have been successfully used for the production of metabolites as well as for the biotransformation of organic compounds (Georgiev et al ., 2008). These pioneering studies served as a fundamental basis for subsequent studies on the large-scale cultivation of plant cells as bioreactor systems with ever-increasing working volumes (Pantchev et al., 2018 ). Notably, the first patented industrial process of cultivating cell culture as a source of phytochemicals was based on the callus line of Purple Gromwel, also known as Zicao, Lithospermum erythrorhizon Siebold & Zucc, which belong to the Boraginaceae family (Curtin, 1983 ). L. eryrthrorhizon is one of the main plants in traditional Chinese medicine and has been actively studied in recent decades (Sun et al., 2022 ). The high pharmacological activity of L. eryrthrorhizon is due to the content of specific polyphenols and mainly derivatives of the naphthoquinone shikonin. Shikonin has a variety of therapeutic effects, including anti-inflammatory, anticancer, cardiovascular, antimicrobiome, analgesic, antiobesity, and brain protection effects. These effects are caused primarily by the regulation of the NF-κB, PI3K/Akt/MAPK, Akt/mTOR, TGF-β, GSK3β, and TLR4/Akt signalling pathways; the NLRP3 inflammasome; reactive oxygen stress; Bax/Bcl-2; and other pathways. The pharmacokinetics of shikonin include unfavourable oral bioavailability, a plasma protein binding rate of 64.6%, and an increase in the levels of several metabolic enzymes, especially cytochrome P450. With respect to toxicological effects, shikonin has the potential to produce skin allergies and nephrotoxicity (Sun et al., 2022 ; Yadav et al., 2022 ). The main source of shikonin in wild-grown L. erythrorhizon has faced the risk of extinction in recent years. Alternative approaches such as cultivation or chemical biosynthesis do not provide cost-effective sources for the industrial production of this compound (Yazaki, 2017 ). Since the 1970s, L. erythrorhizon cell cultures have been actively studied as sources of shikonin. The results of numerous studies have made it possible to develop and optimize a two-stage system for the industrial production of shikonin on the basis of cell cultures. Compared with farm-based production, cell culture-based shikonin production increases yield by up to 800 times (Yazaki, 2017 ). Over decades of active research into optimizing L. erythrorhizon cell culture productivity, a list of shikonin inducers and repressors has been compiled (Sun et al., 2022 ). Various components of the nutrient medium, including agar, plant growth regulators, and various elicitors, are capable of both inducing or increasing, as well as completely blocking, the biosynthesis of shikonin. The least studied effector is light. In 2002, it was shown that exposure to light has a negative effect on the biosynthesis of shikonin. The experiment was conducted as follows: the culture flask was covered with a commercially available colored cellophane sheet and irradiated with white light (10000 lx) from fluorescent lamps. The light intensities in the blue, red, and green cellophane-covered flasks were 600, 1000, and 900 lx, respectively. White and blue light were shown to have strongly negative effects, whereas red and green light slightly reduced shikonin accumulation (Yamamoto et al., 2002 ). However, despite the development of lighting technology and the possibility of regulating the intensity and spectral composition of light, studies on the effects of light on the biosynthesis of shikonin in cell cultures have not been carried out. However, such studies are of at least general interest, since in addition to shikonin, a number of other compounds are synthesized in cell cultures that accompany the shikonin biosynthetic pathway. A detailed definition of the shikonin biosynthesis pathway allows for a more informed approach to the issue of its regulation. While free shikonin cannot be detected, it is present in living plant cells as esters of low-molecular-weight fatty acids such as acetate. Shikonin biosynthesis occurs through the phenylpropanoid and mevalonate pathways. The byproduct geranylhydroquinone is a precursor to both shikonin and shikonofurans. In addition to shikonin esters and shikonofurans, phenylpropanoid pathway products such as caffeic acid derivatives have been found in large quantities in L. erythrorhizon cell cultures. Moreover, their biosynthesis competes with shikonin biosynthesis at the coumaroyl-CoA stage (Sun et al., 2022 ). Thus, in addition to shikonin esters, L. erythrorhizon cell cultures contain shikonofurans and CAD derivatives such as LAB, RA, and rabdosiin. The approach to the activation of shikonin biosynthesis must be based on differential regulation of the biosynthesis of the entire spectrum of compounds. Modern methods for modulating light exposure allow reproducible experiments on the influence of light factors on the secondary metabolism of plants to be conducted (Cavallaro and Rosario Muleo 2022). The effects of light exposure on the growth parameters of plant cell cultures are no less interesting. Thus, red light stimulates the growth of undifferentiated cultures without damaging secondary metabolism (Sobhani Najafabadi et al., 2019 ; Veremeichik et al., 2024b ). Therefore, in the present work, we investigated the effects of different types of light exposure on the growth and phytochemical content of long-term continuously cultivated calli of L. eryrthrorhizon . The Callus line BK-39 of L. eryrthrorhizon was obtained in 1993, while the first phytochemical analysis of this calli was performed in 2001 (Bulgakov et al., 2001 ). At that time, the 8-year-old L. eryrthrorhizon callus line contained 7 shikonin derivatives: acetylshikonin, propionylshikonin, isobutyrylshikonin, dimethyacrylshikonin, isovalerylshikonin, hydroxyisovalerylshikonin, and methylbutyrylshikonin. There are no published data about any other phytochemical compounds produced by the callus line BK-39 of L. eryrthrorhizon until 2005. In 2005, the 12-year-old callus line BK-39 of L. eryrthrorhizon produced approximately 1% DW of both rabdosiin and RA (Bulgakov et al., 2005 ). A more detailed analysis performed in 2021 (Shkryl et al., 2021 ) revealed the presence of caffeic acid derivatives (rabdosiin and RA), shikonofuran derivatives (shikonofuran D, E, and C), and shikonin derivatives (hydroxyisovalerylshikonin, acetylshikonin, isobutyrylshikonin, and isovalerylshikonin) in the 28-year-old callus line BK-39 of L. eryrthrorhizon . However, quantitative analysis of these compounds in the 28-year-old callus line BK-39 of L. eryrthrorhizon has not been performed. 2. Materials and methods 2.1. Plant materials, growth conditions, and experimental design The callus line BK-39 of L. erythrorhizon was obtained previously, in 1993, from stem explants [ Bulgakov et al., 2001 ]. The modified W media (ammonium nitrate content was reduced to 400 mg/l) was supplemented with the following components (mg/l): nicotinic acid (0.5), thiamine-HCl (0.2), mesoinositol (100), peptone (100), pyridoxine-HCl (0.5), sucrose (25000), agar (6000), kinetin (2), indole-3-acetic acid (0.2), and CuSO4 (0.3). L. erythrorhizon calli were continuously cultivated in the same solid medium in the dark at 24°C for more than 30 years and subcultured once every 30 days. The study employs specimens (strains) deposited in the Bioresource Collection of the Federal Scientific Centre of East Asia Terrestrial Biodiversity of the Far East Branch of the Russian Academy of Sciences (reg. number 2797657). For the experiments, after inoculation (2 g of inoculant per 50 mL of solid medium in Erlenmeyer flasks), the calli were immediately transferred under different light treatments for 30 days. As a control, we used calli growing in the dark. Thirty-day-old L. erythrorhizon calli growing under different light conditions were photographed, harvested, weighed, and dried for chemical analysis. For a technical replication, 10 jars containing 2 grams of inoculant were exposed to each variant of LED light. Three separate experiments were conducted as biological replicates. The four-section growing chambers (100 × 50 × 50 cm) with light sources and a photoperiod (light/dark) of 16–8 hours were designed and manufactured at the IACP FEB RAS. The light source matrices were made up of 24 three-watt LEDs (CHANZON, China) of various colours, creating one integrated light source as described previously (Veremeichik et al ., 2024). The reflective aluminum foil was used to diffuse light. The temperature (24°C) and air humidity (70%) were supported via an FFB1212SH 12025 exhaust fan. The spectrum and intensities in each divided section were modulated according to the experimental design. In this study, in addition to warm white light (designated “W”), red (660 nm, designated “R”) and green (520 nm, designated “G”) monochromatic light as well as bichromatic red and blue (designated “RB”) monochromatic light were used. Each section of the chamber was equipped with LED lamps with different light intensities: 50, 100, or 300 µmol m − 2 s − 1 . Each segment had 1–10 light-emitting matrices, which produced the requisite level of photosynthetic photon flux density (PPFD). The intensity of the light in each portion of the chamber was adjusted by altering the supply current for each matrix. The spectra were measured with a PG200N spectrophotometer (UPRtek, Taiwan). Currents in the driver supply system were managed by a UT61A digital multimeter (Uni-T, China). 2.2. Phenolic acid extraction and HPLC-DAD-ESI-MS/MS) conditions Chemicals Analytical standard of rosmarinic acid was obtained from Aldrich (Germany). Analytical standard of rabdosiin with a purity of 98.5% was obtained previously from a callus culture of Eritrichium sericeum (Fedoreyev et al., 2005 ). A standard sample of shikonin with a purity of 98.5% was obtained previously from the roots of the plant L. erythorhizon collected in Primorsky Krai (Fedoreev et al., 1979 ). All the eluents and extraction solutions were prepared with ultrapure water (Millipore, Bedford, MA, USA). All solvents were of analytical grade. Sample preparation for analytical chromatography Polyphenol extraction was performed according to a previously published protocol [Veremeichik et al ., 2024]. Briefly, ultrasonic extraction of dried and powdered callus material in 80% aqueous methanol was performed. The extracts were purified using a 0.45-µm membrane (Millipore, Bedford, MA, USA) before HPLC analysis. Analytical chromatography and mass- spectrometry The polyphenol extracts were studied at the Instrumental Centre of Biotechnology and Gene Engineering of IBSS FEB RAS using an 1260 Infinity analytical HPLC system (Agilent Technologies, Santa Clara, California, USA) interfaced with an ion trap mass spectrometer (Bruker HCT ultra PTM Discovery System, Bruker Daltonik GmbH, Bremen, Germany). An analytical Zorbax C18 column (150 mm, 2.1 mm i.d., 3.5 µm, Agilent Technologies, USA) for polyphenol separation was applied at 40°C. The mobile phase consisted of a gradient elution of ultrapure water (A) and acetonitrile (B) with 0.1% formic acid added in both cases. The following linear gradient at a flow rate of 0.2 mL/min was used: 0 min, 5% B; 20 min, 30% B; and 30 min, 100% B. A photodiode array detector was employed in the range between 200 and 600 nm to obtain UV‒Vis spectra. Chromatograms for quantification were recorded at a wavelength of 325 nm. The MS instrument was operated in electrospray ionization (ESI) mode, and negative ions were detected. The following settings were used: the range of m/z detection was 100–650, the drying gas (N 2 ) flow rate was 8.0 L/min, the nebulizer gas (N 2 ) pressure was 25 psi, the ion source potential was − 3.8 kV, and the drying gas temperature was 325°C. Tandem mass spectra were acquired in Auto-MS 2 mode (smart fragmentation) by increasing the collision energy. The fragmentation amplitude was set to 1 V. The productivity of the polyphenols was calculated as follows: Productivity (mg/l) = Content × DW, where Content denotes the content of an individual compound (mg/g DW) and DW denotes the dry weight (g) of the callus biomass per liter of medium (g/l). 2.3. Determination of shikonin ester content The content of shikonin esters in the cells was determined using a UV-1800 spectrophotometer (Shimadzu USA Manufacturing Inc., Oregon, USA). For extraction, 2.0 ml of ethanol was added to 50 mg of dried crushed calli, which were subsequently placed in an ultrasonic bath at 45°C for 10 min and left for 2 hours at room temperature. The resulting extract was filtered through a 0.45 µm syringe filter. The optical density of the extracts was measured at a wavelength of 526 nm. The content of shikonin derivatives in the sample was calculated using a calibration curve created from a solution of shikonin standard in ethanol (y = 0.0199577x – 0.0035186, R 2 = 0.9994). 2.4. Statistical analysis The STATISTICA software package (StatSoft, Inc., USA) was used for the statistical analysis. All values are presented as the mean ± standard error (SE). Student's t test was employed for the statistical assessment to compare two independent groups. Analysis of variance (ANOVA) was used, together with a multiple comparison approach, to compare several datasets. The cut-off point for statistical significance was fixed at p < 0.05. 3. Results 3.1. Phytochemical composition of the 32-year-old callus line BK-39 of L. eryrthrorhizon In the present work, we first analysed phytochemical compounds in the 32-year-old L. eryrthrorhizon callus line BK-39 and compared the obtained results with known data to determine the influence of continuous long-term cultivation on the productivity of the L. eryrthrorhizon callus line BK-39. The HPLC–PDA-ESI-HR-MS/MS2 method was used to determine the phenolic compounds in the crude aqueous-metabololic extracts of L. erythrorhizon cells. First, we ensured that all previously identified components [Shkryl et al. ,2021] were preserved in the studied cell culture. The chromatographic profile (Fig. 1 ) of the control sample demonstrated the presence of several peaks divided into three groups. The two major peaks were identified as caffeic acid derivatives due to their full similarity with available standards: rabdosiin ( 1 ) and rosmarinic acid ( 2 ) (Fig. 1 , a). We were also able to identify minor components via identification carried out earlier [Shkryl et al. ,2021]. The next three peaks were assigned as shikonofurans: shikonofuran D ( 3 ), shikonofuran E ( 4 ) and shikonofuran C ( 5 ) (Fig. 1 , a). Red naphthoquinone pigments (shikonin derivatives) were also detected: hydroxyisovalerylshikonin ( 6 ), acetylshikonin ( 7 ), isobutyrylshikonin ( 8 ) and isovalerylshikonin ( 9 ) (Fig. 1 , b). All information about the determined phenolic compounds is summarized in Table 1 . The quantitative measurement of rabdosiin ( 1 ) was performed via the external standard method with a research-grade standard sample of previously isolated rabdosiin. Rosmarinic acid ( 2 ) and shikonofurans ( 3–5 ) were quantified on the basis of four-point regression curves built with the reference commercial standard of rosmarinic acid. Table 1 List of phenolic compounds produced by L. erythrorhizon calli grown under control dark conditions. Peak number Compound Rt, min UV max, nm ESI-MS, [M-H] − , m/z Content, % DW 1 Rabdosiin 15.0 253, 285, 346 717 1.06 ± 0.003 2 Rosmarinic acid 15.8 287, 327 359 1.04 ± 0.138 3 Shikonofuran D 25.8 269, 323 343 0.02 ± 0.003 4 Shikonofuran E 26.4 271, 328 355 0.02 ± 0.002 5 Shikonofuran C 26.9 270, 325 357 0.02 ± 0.003 6 Hydroxyisovalerylshikonin 27.8 272, 518 387 0.12 ± 0.002 7 Acetylshikonin 29.0 274, 516 329 0.04 ± 0.003 8 Isobutyrylshikonin 31.9 273, 517 357 0.45 ± 0.037 9 Isovalerylshikonin 33.0 273, 517 371 0.34 ± 0.024 The results of the quantitative analysis (Table 1 ) revealed that the contents of the major compounds, rabdosiin and RA, did not change beginning in 2005 during continuous long-term cultivation and reached 1% DW. However, the content of shikonin derivatives was significantly lower than that in 2001. Only four of the seven compounds were detected in the 32-year-old callus line BK-39 of L. eryrthrorhizon . Interestingly, these four shikonin derivatives were predominant in the 8-year-old callus line BK-39 of L. eryrthrorhizon . While minor compounds were completely absent, the compositions of these four major shikonin derivatives changed. In 2001, the major compounds were isobutyrylshikonin, acetylshikonin, isovalerylshikonin, and hydroxyisovalerylshikonin (up to 38, 27, 12, and 9% of shikonin derivatives, respectively). In 2025, the major compounds were isobutyrylshikonin, isovalerylshikonin, and hydroxyisovalerylshikonin (48, 35, and 12% of shikonin derivatives, respectively), while the content of acetylshikonin was reduced to 4% of that of shikonin derivatives. Unfortunately, we cannot assess changes in the content of shikonofurans since there are no earlier data. The total content of shikonofurans in the 32-year-old callus line BK-39 was not greater than 0.06% DW. 3.2. The growth of L. eryrthrorhizon calli cultivated under different light treatments Previously, we showed that different LED treatments can improve the productivity of long-term cultivated calli (Veremeichik et al., 2024a ). On the basis of previous studies, we can also conclude that the most appropriate intensities of LED light exposure are 100 and 300 µmol m − 2 s − 1 . In 2002, light exposure was shown to block shikonin biosynthesis (Yamamoto et al., 2002 ). However, despite significant advances in photobiology, similar studies using finely tuned lighting have not been carried out. In the present study, we investigated the phytochemical content of the 32-year-old callus line BK-39 of L. eryrthrorhizon grown for 30 days under monochromatic red, green, bichromatic red and blue LED light at intensities of 100 and 300 µmol m − 2 s − 1 : R100 and R300; G100 and G300; RB100 and RB300. Dark conditions (D) were used as positive controls; and warm white LED light with an intensity of 100 µmol m − 2 s − 1 (W100) was used as a negative control. First, we investigated the effects of different light conditions on the growth of L. eryrthrorhizon callus culture. L. eryrthrorhizon calli were cultivated once under different light condition for 30 days. In our work, we investigated the effects of two main variables of LED lighting on the productivity of L. eryrthrorhizon cell cultures. These are the spectral composition and the illumination intensity, expressed in PPFD. The four-section chambers are equipped with adjustable lamps. The LEDs are located on one board and combined into one matrix (Fig. 2 , a). Each board is powered by a current driver of the DS-EUM-075S105DG type, with which the level of irradiation can be adjusted by changing the current for the LEDs. Warm white (W) and monochromatic, red (R) and green (R), and bichromatic red and blue (RB) lighting options (Fig. 2 , b-e) with different characteristics were used (Fig. 2 , f). The light intensities chosen for the experiments were 100 and 300 µmol m − 2 s − 1 . Calli grown in the dark were used as a control. We analysed the effects of warm white light, red, green and a combination of red and blue light with intensities of 100 and 300 µmol m − 2 s − 1 . Compared with the dark-grown control, all the light treatments had no negative effect on the growth of the L. eryrthrorhizon callus culture. However, warm white, green and a combination of red and blue light resulted in the loss of color in the culture. While L. eryrthrorhizon calli grown in the dark are rich in crimson color, the calli grown under light conditions are devoid of color. However, the calli grown under red light remained colored (Fig. 3 ). 3.3. Phytochemical contents in L. eryrthrorhizon calli cultivated under different light treatments First, we were interested in how different LED light treatments affect the contents of shikonin derivatives. As shown in Table 2 , all the light treatments completely blocked the biosynthesis of the shikonin derivatives in L. eryrthrorhizon calli, despite red light of both intensities (100 and 300 µmol m 2 s 1 ). Red light treatment led to a 20-fold reduction in the contents of the major compounds isobutyrylshikonin and isovalerylshikonin. Biosynthesis of minor compounds (hydroxyisovalerylshikonin and acetylshikonin) as well as the biosynthesis of shikonofurans were blocked in red light-treated L. eryrthrorhizon calli. Table 2 HPLC analysis of shikonin derivatives in L. eryrthrorhizon calli cultivated for 30 days under various light treatments: D, darkness; warm white, monochromatic, and bichromatic sources designated W, R, G, and RB, respectively, with intensities of 100 and 300 µmol m − 2 s − 1 . Light treatment, µmol m − 2 s − 1 D W100 R100 R300 G100 G300 RB100 RB300 Shikonins, mg/g DW Hydroxyisovalerylshikonin 1.18 ± 0.021 ND ND ND ND ND ND ND Acetylshikonin 0.39 ± 0.028 ND ND ND ND ND ND ND Isobutyrylshikonin 4.53 ± 0.366* ND 0.193 ± 0.001 0.235 ± 0.003 ND ND ND ND Isovalerylshikonin 3.35 ± 0.236* ND 0.225 ± 0.001 0.215 ± 0.002 ND ND ND ND Shikonofurans, mg/g DW Shikonofuran D 0.17 ± 0.028 ND ND ND ND ND ND ND Shikonofuran E 0.16 ± 0.024 ND ND ND ND ND ND ND Shikonofuran C 0.18 ± 0.028 ND ND ND ND ND ND ND ND, not detected. The mean ± standard error of the mean is used to show the data from three separate studies with ten biological replicates. * above the error indicates statistically significant differences (ANOVA, p ˂0.05). We analysed the effects of different light treatments on the contents of the major polyphenolic compounds, rabdosiin and RA. Warm white light with an intensity of 100 µmol m − 2 s − 1 did not affect the biosynthesis or growth of either caffeic acid derivative compared with the control dark-grown L. eryrthrorhizon calli (Fig. 4 ). Interestingly, whole green and red‒blue light treatments with an intensity of 100 µmol m − 2 s − 1 led to a 1.5- and 2-fold increase in the rabdosiin content, respectively, whereas red light treatment resulted in an almost twofold rabdosiin content compared with that of the control dark-grown L. eryrthrorhizon calli (Fig. 4 , a). Increasing the light treatment intensity to 300 µmol m − 2 s − 1 did not have positive effects on the rabdosiin content. Considering the impact of light treatments on the growth of L. eryrthrorhizon calli, the productivity of rabdosiin in G100 and RB100 light-treated L. eryrthrorhizon calli was greater than 1.5 times greater than that in the control dark-grown L. eryrthrorhizon calli (Fig. 4 , a). At that time, no light treatment had any positive effect on the biosynthesis or productivity of RA (Fig. 4 , b). 3.4. The growth and phytochemical content of L. eryrthrorhizon calli cultivated under low-intensity red and blue light treatments Since exposure to red light did not block shikonin biosynthesis in L. eryrthrorhizon calli, we next tested the effect of red light at a reduced intensity of up to 50 µmol m − 2 s − 1 (R50). In addition, we studied the effects of supplementation with 1/5 (R40B10) or half (R25B25) blue light on the overall intensity of 50 µmol m − 2 s − 1 . We showed that all three light treatments had a strongly positive effect on callus growth, with an increase of more than 15% (Fig. 5 , a). However, when the calli were exposed to pure red light, the color of the culture was no less intense than that of the dark-grown control. When blue light was supplemented, the color of the calli visually became less saturated, which indicates a decrease in the accumulation of shikonin (Fig. 5 , b). Surprisingly, R50 treatment led to a significant increase in shikonin biosynthesis. As shown by HPLC analysis, the contents of hydroxyisovalerylshikonin and isovalerylshikonin were increased approximately 2- and 1.4-fold, respectively, compared with those in the control dark-grown L. eryrthrorhizon calli (Table 3 ). The contents of isobutyrylshikonin and acetylshikonin did not change. At that time, the content of shikonofuranes was reduced approximately 2-fold in R50-treated L. eryrthrorhizon calli compared with the control dark-grown L. eryrthrorhizon calli (Table 3 ). Supplementation of red light with blue light led to a dramatic decrease in the shikonin derivative content and total blockade of shikonofurane biosynthesis. Interestingly, none of these light treatments led to significant changes in the rabdosiin content; however, R50 treatment led to a significant decrease in the RA content (Fig. 6 , a). However, considering the impact of these light treatments on growth, the productivity of both rabdosiin and RA was more than 20% greater in R40B10-treated L. eryrthrorhizon calli than in control dark-grown R40B10-treated calli (Fig. 6 , b). Table 3 HPLC analysis of shikonin derivatives in L. eryrthrorhizon calli cultivated for 30 days under various light treatments: D, darkness; monochromatic, and bichromatic sources designated R and RB, respectively, with intensities of 50 µmol m − 2 s − 1 . Light treatment, µmol m − 2 s − 1 D R50 R25B25 R40B10 Shikonins, mg/g DW Hydroxyisovalerylshikonin 1.18 ± 0.021 b 2.23 ± 0.637 a 0.13 ± 0.003 c 0.11 ± 0.001 c Acetylshikonin 0.39 ± 0.028 a 0.46 ± 0.035 a ND ND Isobutyrylshikonin 4.53 ± 0.366 a 4.45 ± 0.371 a 0.24 ± 0.098 b 0.36 ± 0.001 b Isovalerylshikonin 3.35 ± 0.236 b 4.17 ± 0.457 a 0.28 ± 0.136 c 0.49 ± 0.003 d Shikonofurans, mg/g DW Shikonofuran D 0.17 ± 0.028 a 0.07 ± 0.006 b ND ND Shikonofuran E 0.16 ± 0.024 a 0.11 ± 0.005 b ND ND Shikonofuran C 0.18 ± 0.028 a 0.10 ± 0.004 b ND ND ND, not detected. The mean ± standard error of the mean is used to show the data from three separate studies with ten biological replicates. The different letters above the error indicate statistically significant differences (ANOVA, p ˂0.05). 3.5. The impact of inductors and low-intensity red light treatment on the productivity of shikonin in the L. eryrthrorhizon calli Copper ions are the most effective inducers of shikonin biosynthesis in the callus cultures of L. eryrthrorhizon (Sun et al., 2022 ). In the present study, L. eryrthrorhizon calli were stably grown on solid media supplemented with copper glycerate (0.3 mg/l). We investigated the combined effects of low-intensity red LED light and increased concentrations of inducers on shikonin productivity in L. eryrthrorhizon calli. L. eryrthrorhizon calli were grown for one passage (30 days) in the control dark conditions (D) and under low-intensity (50 µmol m − 2 s − 1 ) red LED light (R50). For cultivation, solid medium supplemented with 0.3 copper glycerate was used, and the concentrations were increased to 1.2 and 2.4 (mg/L). Increasing the copper glycerate concentration to 1.2 mg/L did not suppress growth and increased the content and production of shikonin esters by 20% when the samples were grown in the dark (Fig. 7 ). When grown in the dark, increasing the copper glycerate concentration to 2.4 mg/L had an inhibitory effect on growth (Fig. 7 , a). However, the content of shikonin esters did not increase compared with that in the 1.2 cultivar (Fig. 7 , b). Moreover, the production of shikonin increased by 20% with the addition of 1.2 and insignificantly with the addition of 2.4 (Fig. 7 , c). When L. eryrthrorhizon calli were grown under red light with an intensity of 50 µmol m − 2 s − 1 , we did not find a negative effect of increasing the concentration of copper glycerate on the growth of the culture. Increasing the concentration of copper glycerate to 1.2 mg/L and growing L. eryrthrorhizon calli under red light with an intensity of 50 µmol m − 2 s − 1 allowed us to increase the productivity of the culture by more than 2 times (Fig. 7 ). 4. Discussion It is believed that with long-term perennial cultivation, the ability of calli not only to regenerate but also to produce secondary metabolites decreases (Liu et al., 2009 ). In the present work, we analysed the growth and biosynthetic characteristics of the L. eryrthrorhizon callus line BK-39 obtained in 1993 after 32 years of cultivation (more than 380 passages). Thus, the growth of the callus line BK-39 was approximately 19 g/L DW in 2001, after 8 years of continuous cultivation. As we showed in the present work, after 32 years of continuous cultivation, the growth of the L. eryrthrorhizon callus line BK-39 was approximately 5 g/L DW. We suggest that long-term cultivation may be the reason for this significant (approximately fourfold) decrease in growth. The effects of over a decade of continuous cultivation on the growth of a callus culture are poorly understood. In a recent study, we demonstrated that long-term cultured calli did not experience a decline in growth when their secondary metabolite biosynthesis decreased (Veremeichik et al., 2024b ; Veremeichik et al ., 2025). We assumed that the youthful culture was characterized by explosive growth, which eventually levelled off to a comfortable level. In this study, we also examined phytochemicals in the 32-year-old L. eryrthrorhizon callus line BK-39. To determine the impact of continuous long-term cultivation on the production of the L. eryrthrorhizon callus line BK-39, we compared the results with existing data. The chromatographic profile of the L. eryrthrorhizon callus line BK-39 demonstrated the presence of two major peaks, rabdosiin and RA, and minor components such as shikonofurans and shikonin derivatives. The Callus line BK-39 of L. eryrthrorhizon was obtained in 1993, while the first phytochemical analysis of this callus was performed in 2001 (Bulgakov et al., 2001 ). In 2005, the 12-year-old callus line BK-39 of L. eryrthrorhizon produced approximately 1% DW of both rabdosiin and RA (Bulgakov et al., 2005 ). Quantitative analysis revealed that the contents of the major compounds, rabdosiin and RA, did not change beginning in 2005 during continuous long-term cultivation and reached 1% DW. However, the content of shikonin derivatives was significantly lower than that in 2001. Only four of the seven compounds were detected in the 32-year-old callus line BK-39 of L. eryrthrorhizon . We previously demonstrated that various LED treatments can increase the productivity of Mertensia maritima calli cultivated for a long period of time (Veremeichik et al., 2024a ). We may infer from earlier research that the ideal range for LED light exposure intensity is between 100 and 300 µmol m − 2 s − 1 . Shikonin production was demonstrated to be blocked by light exposure in 2002 (Yamamoto et al., 2002 ). However, comparable investigations with precisely calibrated lighting have not been conducted, despite notable advancements in photobiology. First, we compared the phytochemical content of a 32-year-old L. eryrthrorhizon callus line grown for 30 days under monochromatic red and green light and bichromatic red and blue LED light treatment to that of calli grown under warm white light as a negative control and dark conditions as a positive control. Compared with that of the dark-grown control, the growth of the L. eryrthrorhizon callus culture was unaffected by any of the light treatments. However, the color of the culture was lost as a result of warm white, green, and a mix of red and blue light. The calli of L. eryrthrorhizon that are grown in the dark have a deep crimson hue, but those that are grown in light have no colour at all. The color of the calli that were exposed to red light, however, was maintained. Therefore, regardless of intensity, red light has the strongest growth-stimulating effect. Growth is not adversely affected by the impacts of green light. This pattern is typical of cell cultures in general. The information gathered for this study is consistent with earlier findings for plants and callus cultivation. Accordingly, cultivation of Hypericum perforatum callus cultures under red light and dark conditions resulted in much greater biomass accumulation, whereas cultivation of the cultures under blue light had the opposite effect (Najafabadi et al., 2019 ). Notably, cardoon seedlings grow 60–100% faster under red light than they do in a greenhouse, whereas blue light inhibits their growth (Rabara et al., 2017 ). According to our earlier findings, blue light at an intensity of 100 µmol m − 2 s − 1 completely inhibited the growth of M. maritima , whereas red and green light had no detrimental effects on cell culture growth (Veremeichik et al., 2024b ). First, we wanted to determine how the content of shikonin derivatives was affected by various LED light treatments. Despite red light, all light treatments completely prevented the production of shikonin compounds in L. eryrthrorhizon calli. The amount of the main shikonin derivatives was reduced by 20 times when the samples were exposed to red light at intensities of 100 and 300 µmol m − 2 s − 1 . We examined how the various light treatments affected the levels of rabdosiin and RA, two important polyphenolic chemicals. Compared with that of the control dark-grown L. eryrthrorhizon calli, the production of both caffeic acid derivatives was unaffected by warm white light. Intriguingly, the rabdosiin content increased 1.5- and 2-fold in response to the green and red–blue light treatments, but it decreased nearly twofold-fold in response to the red light treatment compared with that in the control dark-grown L. eryrthrorhizon calli. Raising the light treatment intensities to 300 µmol m − 2 s − 1 did not have the same beneficial effect on the amount of rabdosiin. Compared with that in the control dark-grown L. eryrthrorhizon calli, the productivity of rabdosiin in the G100 and RB100 light-treated L. eryrthrorhizon calli was more than 1.5 times greater, considering the effects of the light treatments on growth. None of the light treatments had any beneficial effects on RA biosynthesis or output. It was previously demonstrated that blue light suppressed the formation of shikonin while increasing the level of RA (Gaisser and Heide, 1996 ). These findings imply that Lithospermum cells have two routes, one that leads to the biosynthesis of polyphenols and the other to the creation of shikonin, both of which share an early biosynthetic sequence. Importantly, the pattern of RA accumulation in the aerial and underground sections of the whole plant differed; that is, shikonin was found only in the underground tissues, whereas RA was essentially undetectable in the root tissues. Nevertheless, it is unknown whether light regulates the biosynthesis of these caffeic acid oligomers in the same way that it governs the biosynthesis of shikonin (Yamamoto et al., 2000 ). However, effective approaches for the regulation of RA biosynthesis include the use of RA, which is one of the most frequently occurring caffeic acid esters in the plant kingdom in addition to chlorogenic acid. RA has numerous biological and pharmacological activities (Petersen 2013 ). In plants, RA is believed to serve as a preformed defence compound against pathogens and herbivores (Petersen and Simmonds, 2003). Moreover, caffeic acid esters can act as UV protectants (Cle´ et al. , 2008). Numerous pharmacological and biological activities, such as anti-inflammatory, antioxidative, antidiabetes, antivirus, antitumour, neuroprotective, and hepatoprotection effects, of RA and related compounds have been described (Guan et al., 2022 ). Antiviral activity was shown for extracts from Melissa officinalis against Herpes simplex infections (Astani et al., 2012 ). Owing to their high productivity, plant cell cultures are a potential source of RA. Thus, Salvia officinalis suspension culture produce approximately 36% (Hippolyte et al., 1992 ). The regulation of rhabdosin biosynthesis is of no less interest. The RA dimer rabdosiin belongs to the lignans, constituting an abundant class of phenylpropanoids and having a number of medically important biological activities, such as antitumor, antimitotic, and antiviral properties (Umezawa, 2003 ). Rabdosiin was detected in Rabdosia japonica (Lamiaceae) (Agata et al., 1988 )d erythrorhizon (Boraginaceae) (Yamamoto et al., 2000 ). Recently, it was shown that rabdosiin has anticancer (Flegkas et al., 2019 ), antiallergic (Ito et al., 1998 ), and nefroprotective activities (Inyushkina et al., 2007 ). Since exposure to red light did not block shikonin biosynthesis in L. eryrthrorhizon calli, we tested the effect of red light at a reduced intensity of up to 50 µmol m − 2 s − 1 . In addition, we studied the effect of supplementation with 1/5 and half blue light on the overall intensity of 50 µmol m − 2 s − 1 . These light treatments had a strongly positive effect on callus growth, with an increase of more than 15%. R50 treatment led to a significant increase in shikonin biosynthesis, whereas blue light supplementation led to a decrease in the accumulation of shikonin and total blockade of shikonin biosynthesis. Interestingly, none of these light treatments led to significant changes in the rabdosiin content; however, the R50 treatment led to a significant decrease in the RA content. However, considering the impact of these light treatments on growth, the productivity of both rabdosiin and RA was more than 20% greater in R40B10-treated L. eryrthrorhizon calli than in the dark-grown control. Copper ions are the most effective inducers of shikonin biosynthesis in the callus cultures of L. eryrthrorhizon (Sun et al., 2022 ). We investigated the combined effects of low-intensity red LED light and increased concentrations of inducers on shikonin productivity in L. eryrthrorhizon calli. Increasing the copper glycerate concentration to 1.2 mg/L did not suppress growth and increased the content and production of shikonin esters by 20% when the samples were grown in the dark. When the mixture was grown in the dark, the production of shikonin increased by 20% with the addition of 1.2, but the increase was not significant with the addition of 2.4. When L. eryrthrorhizon calli were grown under R50, we did not find a negative effect of increasing the concentration of copper glycerate on the growth of the culture. Increasing the concentration of copper glycerate to 1.2 mg/l allowed us to increase the productivity of the culture by more than 2 times. In summary, we propose the following scheme for the light-dependent differential regulation of the biosynthesis of shikonin and CADs in L. erythrorhizon calli (Fig. 8 ). Shikonin and shikonofurans are byproducts of geranylhydroquinone. Two key precursors of geranylhydroquinone, geranyl diphosphate (GPP), are derived via the mevalonate pathway, and p -hydroxybenzoic acid (PHB) is derived via the phenylpropanoid pathway. Biosynthesis of the aromatic intermediate PHB derived from coumaroyl-CoA, which is a key precursor for RA biosynthesis derived from tyrosine. The main derivatives of RA in L. erythrorhizon are lithospermic acid B (LAB) and rabdosiin (Sun et al., 2022 ). As we showed in the present work, monochromatic green and bichromatic red and blue LED light treatments shifted biosynthesis from the coumaroyl-CoA stage to the RA stage, whereas low-intensity red light treatments, in contrast, shifted biosynthesis to the shikonin stage. Moreover, low-intensity red light treatment shifted the biosynthesis to the side of shikonin in the geranylhydroquinone stage. 5. Conclusion Research on light sources and environmental factors that can increase the sustainability and profitability of PFALs has become increasingly important in recent years (Orsini et al., 2020 ). In this study, the impact of artificial monochromatic and bichromatic LED light at wide ranges of intensities (50, 100, and 300 µmol m − 2 s − 1 ) on the growth and biosynthesis of caffeic acid derivatives as well as shikonin and shikonofurans in long-term cultivated L. erythrorhizon callus cultures was investigated for the first time. In general, the following conclusions can be drawn: i) In long-term cultivated L. erythrorhizon callus cultures, the content of shikonin decreased after more than 30 years of cultivation, whereas the content of CADs did not significantly change. ii) Red light has the greatest growth-stimulating effect regardless of intensity. It can also be assumed, on the basis of literary data, that this pattern is characteristic of cell cultures in general. iii) The most effective treatment for CAD productivity (both RA and rabdosiin) is red/blue and green light treatment. iv) The most effective way to produce shikonin in long-term cultivated L. erythrorhizon calli is to use red light with an intensity of 50 µmol m − 2 s − 1 and increase the copper glycerate concentration. v) It may also be assumed that the blue and green components of white light have a negative effect on shikonin biosynthesis because of the light-dependent shift in the biosynthesis of CADs. Declarations Acknowledgements The analyses described in this work were performed via equipment from the Instrumental Centre for Biotechnology and Gene Engineering at the Federal Scientific Centre of East Asia Terrestrial Biodiversity of the Far East Branch of the Russian Academy of Sciences within the state assignment of the Ministry of Science and Higher Education of the Russian Federation (0207-2024-0022) via lightning equipment from the Institute of Automation and Control Processes, Far Eastern Branch of the Russian Academy of Sciences within the state assignment of the Ministry of Science and Higher Education of the Russian Federation (FWFW-2024-0004). The spectrophotometric analysis was performed within the state assignment of the G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Science (agreement No. 075-03-2025-231). CRediT author statement G.N. Veremeichik: Conceptualization, Data curation, Project administration, Supervision, Validation, Visualization, Writing – original draft. G.N. Veremeichik; V.P. Grigorchuk; S.A. Silantieva; E.P. Subbotin; E.V. Brodovskaya, G.K. Tchernoded; O.A. Tikhonova; S.A. Fedoreyev; N.P. Mishchenko; E. A. Vasileva; A.A. Khopta; S.O. Kozhanov: Investigation, Methodology, Formal Analysis. V.P. Bulgakov; Y.N. Kulchin; Siberev A.S.: Resources, Funding acquisition. Funding This research was funded by a grant from the Ministry of Science and Higher Education of the Russian Federation for large scientific projects in priority areas of scientific and technological development (subsidy identifier 075-15-2024-540). Data availability The datasets generated during and/or analysed during the current study are available from the corresponding author upon reasonable request. Code availability Not applicable. Ethics approval Not applicable. Consent to participate Not applicable. Consent for publication All the authors whose names appeared on the submission approved the version to be published and agreed to be accountable for all aspects of the work in ensuring that the questions related to the accuracy of integrity of any part of the work were appropriately investigated and resolved. Conflict of interest The authors declare that they have no competing interests . References Agata I, Hatano T, Nishibe S, Okuda T (1988) Rabdosiin, a new rosmarinic acid dimer with a lignan skeleton, from Rabdosia japonica . Chem Pharm Bull 36:3223–3225. https://doi.org/10.1248/cpb.36.3223 Astani A, Reichling J, Schnitzler P (2012) Melissa officinalis extract inhibits attachment of herpes simplex virus in vitro. Chemother 58:70–77. https://doi.org/10.1159/000335590 Bulgakov VP, Kozyrenko MM, Fedoreyev SA, Mischenko NP, Denisenko VA, Zvereva LV, Pokushalova TV, Zhuravlev YuN (2001) Shikonin production by p-fluorophenylalanine resistant cells of Lithospermum erythrorhizon . 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7030114","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":481673248,"identity":"d93dda29-974f-4caf-b268-e2058902c725","order_by":0,"name":"Galina N Veremeichik","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYFAC5gYIzQ6kEwqI0sII1cJzAKjFgCQtEglAghgt8u4HGz8wttnk8898nfjhgQFDvsEBAloMzyQ2SzC2pVnOuJ27WQLoMMsNBLU0JDZI/zlz2IDhdu4GkBYDwrb0P2z+wXDmv4H8zbObfxClRV4isU2CoeKAgcEN3m3E2WIg8bDNgqEi2cDwTO42iwQDCQNJgrb0Jx++wWBgZyB3/Ozmmz8qbAz4CNqCpkCCgHqQLQ2E1YyCUTAKRsFIBwBFYkBlWFsmkAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-0549-2801","institution":"Federal'nyj naucnyj centr bioraznoobrazia nazemnoj bioty Vostocnoj Azii Dal'nevostocnogo otdelenia Rossijskoj akademii nauk","correspondingAuthor":true,"prefix":"","firstName":"Galina","middleName":"N","lastName":"Veremeichik","suffix":""},{"id":481673249,"identity":"2a7e65ee-7e13-4458-8fa7-5d97df4d1c4e","order_by":1,"name":"Slavena A Silantieva","email":"","orcid":"","institution":"Institute of Automation and Control Processes FEB RAS: FGBUN Institut avtomatiki i processov upravlenia Dal'nevostocnogo otdelenia Rossijskoj akademii nauk","correspondingAuthor":false,"prefix":"","firstName":"Slavena","middleName":"A","lastName":"Silantieva","suffix":""},{"id":481673250,"identity":"0af5096e-3c11-43aa-97c0-49ea8f1c1d9b","order_by":2,"name":"Valeria P. 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Tchernoded","email":"","orcid":"","institution":"Federal Scientific Center of the East Asia Terrestrial Biodiversity of the Russian Academy of Sciences Far Eastern Branch: FGBUN FNC Bioraznoobrazia nazemnoj bioty Vostocnoj Azii Dal'nevostocnogo otdelenia Rossijskoj akademii nauk","correspondingAuthor":false,"prefix":"","firstName":"Galina","middleName":"K.","lastName":"Tchernoded","suffix":""},{"id":481673255,"identity":"5d546ee6-2e9b-4d01-82c1-8e0b44018ffe","order_by":7,"name":"Sergei A. Fedoreyev","email":"","orcid":"","institution":"G B Elyakov Pacific Institute of Bioorganic Chemistry: FGBUN Tihookeanskij institut bioorganiceskoj himii imeni G B Elakova Dal'nevostocnogo otdelenia Rossijskoj akademii nauk","correspondingAuthor":false,"prefix":"","firstName":"Sergei","middleName":"A.","lastName":"Fedoreyev","suffix":""},{"id":481673256,"identity":"a0dc1b0e-c8dc-4612-a01d-13ceabe2b1a6","order_by":8,"name":"Natalia P. Mishchenko","email":"","orcid":"","institution":"G B Elyakov Pacific Institute of Bioorganic Chemistry: FGBUN Tihookeanskij institut bioorganiceskoj himii imeni G B Elakova Dal'nevostocnogo otdelenia Rossijskoj akademii nauk","correspondingAuthor":false,"prefix":"","firstName":"Natalia","middleName":"P.","lastName":"Mishchenko","suffix":""},{"id":481673257,"identity":"b592001e-f89e-47ca-8bce-256fb91e0d36","order_by":9,"name":"Elena A. Vasileva","email":"","orcid":"","institution":"G B Elyakov Pacific Institute of Bioorganic Chemistry: FGBUN Tihookeanskij institut bioorganiceskoj himii imeni G B Elakova Dal'nevostocnogo otdelenia Rossijskoj akademii nauk","correspondingAuthor":false,"prefix":"","firstName":"Elena","middleName":"A.","lastName":"Vasileva","suffix":""},{"id":481673258,"identity":"13cd0bd5-255f-40a1-8fa2-38cddf308644","order_by":10,"name":"Anastasia A. Khopta","email":"","orcid":"","institution":"Institute of Automation and Control Processes FEB RAS: FGBUN Institut avtomatiki i processov upravlenia Dal'nevostocnogo otdelenia Rossijskoj akademii nauk","correspondingAuthor":false,"prefix":"","firstName":"Anastasia","middleName":"A.","lastName":"Khopta","suffix":""},{"id":481673259,"identity":"ab352e6d-0191-440a-af04-15928fe4d87b","order_by":11,"name":"Sergei O. Kozhanov","email":"","orcid":"","institution":"Institute of Automation and Control Processes FEB RAS: FGBUN Institut avtomatiki i processov upravlenia Dal'nevostocnogo otdelenia Rossijskoj akademii nauk","correspondingAuthor":false,"prefix":"","firstName":"Sergei","middleName":"O.","lastName":"Kozhanov","suffix":""},{"id":481673260,"identity":"f5412e58-cfc8-4fb5-9b86-1f7c69e330df","order_by":12,"name":"Aleksei I. Sibirev","email":"","orcid":"","institution":"Federal Scientific Agroengineering Center VIM: FGBNU Federal'nyj naucnyj agroinzenernyj centr VIM","correspondingAuthor":false,"prefix":"","firstName":"Aleksei","middleName":"I.","lastName":"Sibirev","suffix":""},{"id":481673261,"identity":"1f5c8c3e-e9ea-4ec3-bc1d-25fec1d50b4a","order_by":13,"name":"Yuri N. Kulchin","email":"","orcid":"","institution":"Institute of Automation and Control Processes FEB RAS: FGBUN Institut avtomatiki i processov upravlenia Dal'nevostocnogo otdelenia Rossijskoj akademii nauk","correspondingAuthor":false,"prefix":"","firstName":"Yuri","middleName":"N.","lastName":"Kulchin","suffix":""},{"id":481673262,"identity":"3127a46b-17fb-4ff7-b2c6-3bfc1ffce4ca","order_by":14,"name":"Victor P. Bulgakov","email":"","orcid":"","institution":"Federal Scientific Center of the East Asia Terrestrial Biodiversity of the Russian Academy of Sciences Far Eastern Branch: FGBUN FNC Bioraznoobrazia nazemnoj bioty Vostocnoj Azii Dal'nevostocnogo otdelenia Rossijskoj akademii nauk","correspondingAuthor":false,"prefix":"","firstName":"Victor","middleName":"P.","lastName":"Bulgakov","suffix":""}],"badges":[],"createdAt":"2025-07-02 14:04:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7030114/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7030114/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11240-025-03280-3","type":"published","date":"2025-11-05T15:57:07+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86325871,"identity":"50cf46fa-856b-4d6a-8fff-a1e9e06ae41c","added_by":"auto","created_at":"2025-07-09 10:44:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":61515,"visible":true,"origin":"","legend":"\u003cp\u003eHPLC profiling of phenolic compounds detected in the crude extract obtained from \u003cem\u003eL.\u003c/em\u003e \u003cem\u003eerythrorhizon\u003c/em\u003e calli grown under control conditions in the dark. Chromatograms were recorded at 325 nm (\u003cstrong\u003ea\u003c/strong\u003e) and 517 nm (\u003cstrong\u003eb\u003c/strong\u003e). The peak numbers correspond to those listed in \u003cstrong\u003eTable 1\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7030114/v1/1839530492e09e61abbf9a00.png"},{"id":86325872,"identity":"401adb92-7d52-4d38-8401-1b2cf523966d","added_by":"auto","created_at":"2025-07-09 10:44:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":431468,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth chambers and characteristics of light. The appearance of the lamp, consisting of 24 LEDs (\u003cstrong\u003ea\u003c/strong\u003e); growing chambers (\u003cstrong\u003eb-e\u003c/strong\u003e) with artificial lighting variations (left to right): warm white and monochromatic sources such as red and green, and bichromatic sources combining red and blue. The normalized spectral characteristics of the light emission levels of warm white and monochromatic light sources as a function of wavelength (nm) are presented (\u003cstrong\u003ef\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7030114/v1/b57581a183184b2eee28972e.png"},{"id":86325876,"identity":"ccc30545-6607-4212-8b6f-0d8951a470c5","added_by":"auto","created_at":"2025-07-09 10:44:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":68470,"visible":true,"origin":"","legend":"\u003cp\u003eDevelopment of a callus culture of \u003cem\u003eL.\u0026nbsp;eryrthrorhizon\u003c/em\u003e grown under various light conditions. Morphology (\u003cstrong\u003ea\u003c/strong\u003e) and biomass accumulation (\u003cstrong\u003eb\u003c/strong\u003e, g/L) of 30-day-old\u003cem\u003e L.\u0026nbsp;eryrthrorhizon\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003ecalli (2 g FW inoculants per 50 ml of solid medium) cultivated for 30 days under various light treatments: D, darkness; warm white, monochromatic red and green, and bichromatic sources of combinations of red and blue light treatments are designated W, R, G, and RB, respectively. Listed light variants were used with intensities of 100 and 300 µmol m\u003csup\u003e-2\u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e. The mean ± standard error of the mean is used to show the data from three separate studies with ten biological replicates.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7030114/v1/d726b7911efc78333e23a403.png"},{"id":86325873,"identity":"fd160765-6340-4f63-b726-25f6bf40ced5","added_by":"auto","created_at":"2025-07-09 10:44:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":89963,"visible":true,"origin":"","legend":"\u003cp\u003eContents and productivity of rabdosiin and rosmarinic acid in \u003cem\u003eL.\u0026nbsp;eryrthrorhizon \u003c/em\u003ecalli grown under different lighting conditions. Contents (\u003cstrong\u003eupper panel\u003c/strong\u003e) and productivity (\u003cstrong\u003elower panel\u003c/strong\u003e) of rabdosiin (\u003cstrong\u003ea\u003c/strong\u003e) and rosmarinic acid (\u003cstrong\u003eb\u003c/strong\u003e) in \u003cem\u003eL.\u0026nbsp;eryrthrorhizon \u003c/em\u003ecalli (mg/g DW) cultivated for 30 days under various light treatments: D, darkness; warm white, monochromatic red and green, and bichromatic sources of combinations of red and blue light treatments are designated W, R, G, and RB, respectively. Listed light variants were used with intensities of 100 and 300 µmol m\u003csup\u003e-2\u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e. The data obtained from three independent experiments with ten biological replicates are presented as the mean ± standard error of the mean, and different letters above the error bars indicate statistically significant differences (ANOVA, p ˂0.05).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7030114/v1/da4e919384fe5a8df97a6ccc.png"},{"id":86325877,"identity":"b0bbc693-58cf-400b-9911-bc55f2c0cb5d","added_by":"auto","created_at":"2025-07-09 10:44:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":223418,"visible":true,"origin":"","legend":"\u003cp\u003eDevelopment of a callus culture of \u003cem\u003eL.\u0026nbsp;eryrthrorhizon\u003c/em\u003e grown under various light conditions. Biomass accumulation (\u003cstrong\u003ea\u003c/strong\u003e, g/L) and morphology (\u003cstrong\u003eb\u003c/strong\u003e) of 30-day-old\u003cem\u003e L.\u0026nbsp;eryrthrorhizon\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003ecalli (2 g FW inoculants per 50 ml of solid medium) cultivated for 30 days under various light treatments: D, darkness; R, red light with intensities of 50 µmol m\u003csup\u003e-2\u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e, and red light supplemented with blue light with intensities of 40 and 10 µmol m\u003csup\u003e-2\u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e (R40B10) and with intensities of 25 and 25 µmol m\u003csup\u003e-2\u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e (R25B25). The mean ± standard error of the mean is used to show the data from three separate studies with ten biological replicates. Asterisks above the error bars indicate statistically significant differences (ANOVA, p ˂0.05).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7030114/v1/0d1574d8220755a2fb1066b7.png"},{"id":86325880,"identity":"8e456e0c-262c-4cd3-bc8b-da23de6ba367","added_by":"auto","created_at":"2025-07-09 10:44:35","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":43194,"visible":true,"origin":"","legend":"\u003cp\u003eContents and productivity of rabdosiin and rosmarinic acid in \u003cem\u003eL.\u0026nbsp;eryrthrorhizon \u003c/em\u003ecalli grown under different lighting conditions. Contents (\u003cstrong\u003ea\u003c/strong\u003e) and productivity (\u003cstrong\u003eb\u003c/strong\u003e) of rabdosiin and rosmarinic acid acids in \u003cem\u003eL.\u0026nbsp;eryrthrorhizon \u003c/em\u003ecalli (mg/g DW) cultivated for 30 days under various light treatments: D, darkness; R, red light with intensities of 50 µmol m\u003csup\u003e-2\u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e, and red light supplemented with blue light with intensities of 40 and 10 µmol m\u003csup\u003e-2\u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e (R40B10) and with intensities of 25 and 25 µmol m\u003csup\u003e-2\u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e (R25B25). The mean ± standard error of the mean is used to show the data from three separate studies with ten biological replicates. Different letters above the error bars indicate statistically significant differences (ANOVA, p ˂0.05).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7030114/v1/f34eeaa67c67a474aecf9562.png"},{"id":86325888,"identity":"70fd9475-d947-4ddd-8bd0-2af4a213c181","added_by":"auto","created_at":"2025-07-09 10:44:35","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":64452,"visible":true,"origin":"","legend":"\u003cp\u003eCombined effect of red light and copper glycerate on growth and shikonin content in \u003cem\u003eL.\u0026nbsp;eryrthrorhizon \u003c/em\u003ecalli. Biomass accumulation (\u003cstrong\u003ea\u003c/strong\u003e, g/l DW) and shikonin content (\u003cstrong\u003eb\u003c/strong\u003e) and productivity (\u003cstrong\u003ec\u003c/strong\u003e) were calculated for \u003cem\u003eL.\u0026nbsp;eryrthrorhizon \u003c/em\u003ecalli grown under control and experimental conditions. Spectrophotometric analysis of shikonin derivatives in \u003cem\u003eL.\u0026nbsp;eryrthrorhizon \u003c/em\u003ecalli cultivated for 30 days under D, darkness and monochromatic R with intensities of 50 µmol m\u003csup\u003e-2\u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e. Calli of \u003cem\u003eL.\u0026nbsp;eryrthrorhizon \u003c/em\u003ewere grown on culture media supplemented with copper glycerate at a standard concentration (0.3 mg/L) and increased in concentration to 1.2 and 2.4 mg/L. The mean ± standard error of the mean is used to show the data from three separate studies with ten biological replicates. Asterisks or different letters above the error bars indicate statistically significant differences (ANOVA, p ˂0.05).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7030114/v1/16f00e5dc7ce96e25671d316.png"},{"id":86325887,"identity":"84a923be-4672-4f1f-96a6-b025d5d3854d","added_by":"auto","created_at":"2025-07-09 10:44:35","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":96378,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of monochromatic and bichromatic LED light treatments on secondary metabolism in \u003cem\u003eL. erythrorhizon \u003c/em\u003ecalli. Shikonin and shikonofurans are byproducts of geranylhydroquinone. Two key precursors of geranylhydroquinone, geranyl diphosphate (GPP), are derived \u003cem\u003evia\u003c/em\u003e the mevalonate pathway, and \u003cem\u003ep\u003c/em\u003e-hydroxybenzoic acid (PHB) is derived \u003cem\u003evia\u003c/em\u003e the phenylpropanoid pathway. Biosynthesis of the aromatic intermediate PHB derived from coumaroyl-CoA, which is a key precursor for RA biosynthesis derived from tyrosine. The main derivatives of RA in \u003cem\u003eL. erythrorhizon\u003c/em\u003e are lithospermic acid B (LAB) and rabdosiin (Sun \u003cem\u003eet al\u003c/em\u003e., 2022). As we showed in the present work, monochromatic green and bichromatic red and blue LED light treatments shifted biosynthesis from the coumaroyl-CoA stage to the RA stage, whereas low-intensity red light treatments, in contrast, shifted biosynthesis to the shikonin stage. Moreover, low-intensity red light treatment shifted the biosynthesis to the side of shikonin in the geranylhydroquinone stage.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7030114/v1/26a457be46fcfc08d3ade0bb.png"},{"id":95563939,"identity":"0bd700a2-7b77-4bdf-b2f7-e077ef0c6f29","added_by":"auto","created_at":"2025-11-10 16:04:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2313906,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7030114/v1/eba60f2a-9173-4547-a9ca-49eba793c987.pdf"}],"financialInterests":"","formattedTitle":"Low-intensity monochromatic red LED light can improve shikonin productivity in long-term cultivated Lithospermum erythrorhizon Sieb. et Zucc. calli","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThroughout history, plants have been the main source of phytochemical compounds used by Mankind for food, medicinal, and cosmetic purposes (Jamshidi-Kia et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Currently, global climate change, limited fresh water and food supplies, achievements in sustainable development and increasing energy needs are among the most important global challenges facing humanity. Plant cell culture technology can address some of these issues by providing efficient and sustainable sources of phytochemicals with reduced energy and carbon footprints (Krasteva et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cem\u003eIn vitro\u003c/em\u003e plant cultures have been successfully used for the production of metabolites as well as for the biotransformation of organic compounds (Georgiev \u003cem\u003eet al\u003c/em\u003e., 2008). These pioneering studies served as a fundamental basis for subsequent studies on the large-scale cultivation of plant cells as bioreactor systems with ever-increasing working volumes (Pantchev et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Notably, the first patented industrial process of cultivating cell culture as a source of phytochemicals was based on the callus line of Purple Gromwel, also known as Zicao, \u003cem\u003eLithospermum erythrorhizon\u003c/em\u003e Siebold \u0026amp; Zucc, which belong to the Boraginaceae family (Curtin, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1983\u003c/span\u003e). \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e is one of the main plants in traditional Chinese medicine and has been actively studied in recent decades (Sun et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The high pharmacological activity of \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e is due to the content of specific polyphenols and mainly derivatives of the naphthoquinone shikonin.\u003c/p\u003e\u003cp\u003eShikonin has a variety of therapeutic effects, including anti-inflammatory, anticancer, cardiovascular, antimicrobiome, analgesic, antiobesity, and brain protection effects. These effects are caused primarily by the regulation of the NF-κB, PI3K/Akt/MAPK, Akt/mTOR, TGF-β, GSK3β, and TLR4/Akt signalling pathways; the NLRP3 inflammasome; reactive oxygen stress; Bax/Bcl-2; and other pathways. The pharmacokinetics of shikonin include unfavourable oral bioavailability, a plasma protein binding rate of 64.6%, and an increase in the levels of several metabolic enzymes, especially cytochrome P450. With respect to toxicological effects, shikonin has the potential to produce skin allergies and nephrotoxicity (Sun et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yadav et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The main source of shikonin in wild-grown \u003cem\u003eL. erythrorhizon\u003c/em\u003e has faced the risk of extinction in recent years. Alternative approaches such as cultivation or chemical biosynthesis do not provide cost-effective sources for the industrial production of this compound (Yazaki, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Since the 1970s, \u003cem\u003eL. erythrorhizon\u003c/em\u003e cell cultures have been actively studied as sources of shikonin. The results of numerous studies have made it possible to develop and optimize a two-stage system for the industrial production of shikonin on the basis of cell cultures. Compared with farm-based production, cell culture-based shikonin production increases yield by up to 800 times (Yazaki, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOver decades of active research into optimizing \u003cem\u003eL. erythrorhizon\u003c/em\u003e cell culture productivity, a list of shikonin inducers and repressors has been compiled (Sun et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Various components of the nutrient medium, including agar, plant growth regulators, and various elicitors, are capable of both inducing or increasing, as well as completely blocking, the biosynthesis of shikonin. The least studied effector is light. In 2002, it was shown that exposure to light has a negative effect on the biosynthesis of shikonin. The experiment was conducted as follows: the culture flask was covered with a commercially available colored cellophane sheet and irradiated with white light (10000 lx) from fluorescent lamps. The light intensities in the blue, red, and green cellophane-covered flasks were 600, 1000, and 900 lx, respectively. White and blue light were shown to have strongly negative effects, whereas red and green light slightly reduced shikonin accumulation (Yamamoto et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). However, despite the development of lighting technology and the possibility of regulating the intensity and spectral composition of light, studies on the effects of light on the biosynthesis of shikonin in cell cultures have not been carried out. However, such studies are of at least general interest, since in addition to shikonin, a number of other compounds are synthesized in cell cultures that accompany the shikonin biosynthetic pathway.\u003c/p\u003e\u003cp\u003eA detailed definition of the shikonin biosynthesis pathway allows for a more informed approach to the issue of its regulation. While free shikonin cannot be detected, it is present in living plant cells as esters of low-molecular-weight fatty acids such as acetate. Shikonin biosynthesis occurs through the phenylpropanoid and mevalonate pathways. The byproduct geranylhydroquinone is a precursor to both shikonin and shikonofurans. In addition to shikonin esters and shikonofurans, phenylpropanoid pathway products such as caffeic acid derivatives have been found in large quantities in \u003cem\u003eL. erythrorhizon\u003c/em\u003e cell cultures. Moreover, their biosynthesis competes with shikonin biosynthesis at the coumaroyl-CoA stage (Sun et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Thus, in addition to shikonin esters, \u003cem\u003eL. erythrorhizon\u003c/em\u003e cell cultures contain shikonofurans and CAD derivatives such as LAB, RA, and rabdosiin. The approach to the activation of shikonin biosynthesis must be based on differential regulation of the biosynthesis of the entire spectrum of compounds. Modern methods for modulating light exposure allow reproducible experiments on the influence of light factors on the secondary metabolism of plants to be conducted (Cavallaro and Rosario Muleo 2022). The effects of light exposure on the growth parameters of plant cell cultures are no less interesting. Thus, red light stimulates the growth of undifferentiated cultures without damaging secondary metabolism (Sobhani Najafabadi et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Veremeichik et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTherefore, in the present work, we investigated the effects of different types of light exposure on the growth and phytochemical content of long-term continuously cultivated calli of \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e. The Callus line BK-39 of \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e was obtained in 1993, while the first phytochemical analysis of this calli was performed in 2001 (Bulgakov et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). At that time, the 8-year-old \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e callus line contained 7 shikonin derivatives: acetylshikonin, propionylshikonin, isobutyrylshikonin, dimethyacrylshikonin, isovalerylshikonin, hydroxyisovalerylshikonin, and methylbutyrylshikonin. There are no published data about any other phytochemical compounds produced by the callus line BK-39 of \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e until 2005. In 2005, the 12-year-old callus line BK-39 of \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e produced approximately 1% DW of both rabdosiin and RA (Bulgakov et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). A more detailed analysis performed in 2021 (Shkryl et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) revealed the presence of caffeic acid derivatives (rabdosiin and RA), shikonofuran derivatives (shikonofuran D, E, and C), and shikonin derivatives (hydroxyisovalerylshikonin, acetylshikonin, isobutyrylshikonin, and isovalerylshikonin) in the 28-year-old callus line BK-39 of \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e. However, quantitative analysis of these compounds in the 28-year-old callus line BK-39 of \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e has not been performed.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Plant materials, growth conditions, and experimental design\u003c/h2\u003e\u003cp\u003eThe callus line BK-39 of \u003cem\u003eL. erythrorhizon\u003c/em\u003e was obtained previously, in 1993, from stem explants [ Bulgakov et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2001\u003c/span\u003e]. The modified W media (ammonium nitrate content was reduced to 400 mg/l) was supplemented with the following components (mg/l): nicotinic acid (0.5), thiamine-HCl (0.2), mesoinositol (100), peptone (100), pyridoxine-HCl (0.5), sucrose (25000), agar (6000), kinetin (2), indole-3-acetic acid (0.2), and CuSO4 (0.3). \u003cem\u003eL. erythrorhizon\u003c/em\u003e calli were continuously cultivated in the same solid medium in the dark at 24\u0026deg;C for more than 30 years and subcultured once every 30 days. The study employs specimens (strains) deposited in the Bioresource Collection of the Federal Scientific Centre of East Asia Terrestrial Biodiversity of the Far East Branch of the Russian Academy of Sciences (reg. number 2797657).\u003c/p\u003e\u003cp\u003eFor the experiments, after inoculation (2 g of inoculant per 50 mL of solid medium in Erlenmeyer flasks), the calli were immediately transferred under different light treatments for 30 days. As a control, we used calli growing in the dark. Thirty-day-old \u003cem\u003eL. erythrorhizon\u003c/em\u003e calli growing under different light conditions were photographed, harvested, weighed, and dried for chemical analysis. For a technical replication, 10 jars containing 2 grams of inoculant were exposed to each variant of LED light. Three separate experiments were conducted as biological replicates.\u003c/p\u003e\u003cp\u003eThe four-section growing chambers (100 \u0026times; 50 \u0026times; 50 cm) with light sources and a photoperiod (light/dark) of 16\u0026ndash;8 hours were designed and manufactured at the IACP FEB RAS. The light source matrices were made up of 24 three-watt LEDs (CHANZON, China) of various colours, creating one integrated light source as described previously (Veremeichik \u003cem\u003eet al\u003c/em\u003e., 2024). The reflective aluminum foil was used to diffuse light. The temperature (24\u0026deg;C) and air humidity (70%) were supported \u003cem\u003evia\u003c/em\u003e an FFB1212SH 12025 exhaust fan. The spectrum and intensities in each divided section were modulated according to the experimental design. In this study, in addition to warm white light (designated \u0026ldquo;W\u0026rdquo;), red (660 nm, designated \u0026ldquo;R\u0026rdquo;) and green (520 nm, designated \u0026ldquo;G\u0026rdquo;) monochromatic light as well as bichromatic red and blue (designated \u0026ldquo;RB\u0026rdquo;) monochromatic light were used. Each section of the chamber was equipped with LED lamps with different light intensities: 50, 100, or 300 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Each segment had 1\u0026ndash;10 light-emitting matrices, which produced the requisite level of photosynthetic photon flux density (PPFD). The intensity of the light in each portion of the chamber was adjusted by altering the supply current for each matrix. The spectra were measured with a PG200N spectrophotometer (UPRtek, Taiwan). Currents in the driver supply system were managed by a UT61A digital multimeter (Uni-T, China).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Phenolic acid extraction and HPLC-DAD-ESI-MS/MS) conditions\u003c/h2\u003e\u003cp\u003e\u003cb\u003eChemicals\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAnalytical standard of rosmarinic acid was obtained from Aldrich (Germany). Analytical standard of rabdosiin with a purity of 98.5% was obtained previously from a callus culture of \u003cem\u003eEritrichium sericeum\u003c/em\u003e (Fedoreyev et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). A standard sample of shikonin with a purity of 98.5% was obtained previously from the roots of the plant \u003cem\u003eL. erythorhizon\u003c/em\u003e collected in Primorsky Krai (Fedoreev et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1979\u003c/span\u003e). All the eluents and extraction solutions were prepared with ultrapure water (Millipore, Bedford, MA, USA). All solvents were of analytical grade.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSample preparation for analytical chromatography\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePolyphenol extraction was performed according to a previously published protocol [Veremeichik \u003cem\u003eet al\u003c/em\u003e., 2024]. Briefly, ultrasonic extraction of dried and powdered callus material in 80% aqueous methanol was performed. The extracts were purified using a 0.45-\u0026micro;m membrane (Millipore, Bedford, MA, USA) before HPLC analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalytical chromatography and mass- spectrometry\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe polyphenol extracts were studied at the Instrumental Centre of Biotechnology and Gene Engineering of IBSS FEB RAS using an 1260 Infinity analytical HPLC system (Agilent Technologies, Santa Clara, California, USA) interfaced with an ion trap mass spectrometer (Bruker HCT ultra PTM Discovery System, Bruker Daltonik GmbH, Bremen, Germany). An analytical Zorbax C18 column (150 mm, 2.1 mm i.d., 3.5 \u0026micro;m, Agilent Technologies, USA) for polyphenol separation was applied at 40\u0026deg;C. The mobile phase consisted of a gradient elution of ultrapure water (A) and acetonitrile (B) with 0.1% formic acid added in both cases. The following linear gradient at a flow rate of 0.2 mL/min was used: 0 min, 5% B; 20 min, 30% B; and 30 min, 100% B. A photodiode array detector was employed in the range between 200 and 600 nm to obtain UV‒Vis spectra. Chromatograms for quantification were recorded at a wavelength of 325 nm. The MS instrument was operated in electrospray ionization (ESI) mode, and negative ions were detected. The following settings were used: the range of \u003cem\u003em/z\u003c/em\u003e detection was 100\u0026ndash;650, the drying gas (N\u003csub\u003e2\u003c/sub\u003e) flow rate was 8.0 L/min, the nebulizer gas (N\u003csub\u003e2\u003c/sub\u003e) pressure was 25 psi, the ion source potential was \u0026minus;\u0026thinsp;3.8 kV, and the drying gas temperature was 325\u0026deg;C. Tandem mass spectra were acquired in Auto-MS\u003csup\u003e2\u003c/sup\u003e mode (smart fragmentation) by increasing the collision energy. The fragmentation amplitude was set to 1 V.\u003c/p\u003e\u003cp\u003eThe productivity of the polyphenols was calculated as follows:\u003c/p\u003e\u003cp\u003eProductivity (mg/l)\u0026thinsp;=\u0026thinsp;Content \u0026times; DW,\u003c/p\u003e\u003cp\u003ewhere Content denotes the content of an individual compound (mg/g DW) and DW denotes the dry weight (g) of the callus biomass per liter of medium (g/l).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e2.3. Determination of shikonin ester content\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eThe content of shikonin esters in the cells was determined using a UV-1800 spectrophotometer (Shimadzu USA Manufacturing Inc., Oregon, USA). For extraction, 2.0 ml of ethanol was added to 50 mg of dried crushed calli, which were subsequently placed in an ultrasonic bath at 45\u0026deg;C for 10 min and left for 2 hours at room temperature. The resulting extract was filtered through a 0.45 \u0026micro;m syringe filter. The optical density of the extracts was measured at a wavelength of 526 nm. The content of shikonin derivatives in the sample was calculated using a calibration curve created from a solution of shikonin standard in ethanol (y\u0026thinsp;=\u0026thinsp;0.0199577x \u0026ndash; 0.0035186, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9994).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Statistical analysis\u003c/h2\u003e\u003cp\u003eThe STATISTICA software package (StatSoft, Inc., USA) was used for the statistical analysis. All values are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE). Student's t test was employed for the statistical assessment to compare two independent groups. Analysis of variance (ANOVA) was used, together with a multiple comparison approach, to compare several datasets. The cut-off point for statistical significance was fixed at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. Phytochemical composition of the 32-year-old callus line BK-39 of \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eIn the present work, we first analysed phytochemical compounds in the 32-year-old \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e callus line BK-39 and compared the obtained results with known data to determine the influence of continuous long-term cultivation on the productivity of the \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e callus line BK-39. The HPLC\u0026ndash;PDA-ESI-HR-MS/MS2 method was used to determine the phenolic compounds in the crude aqueous-metabololic extracts of \u003cem\u003eL. erythrorhizon\u003c/em\u003e cells. First, we ensured that all previously identified components [Shkryl \u003cem\u003eet al.\u003c/em\u003e,2021] were preserved in the studied cell culture. The chromatographic profile (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) of the control sample demonstrated the presence of several peaks divided into three groups. The two major peaks were identified as caffeic acid derivatives due to their full similarity with available standards: rabdosiin (\u003cstrong\u003e1\u003c/strong\u003e) and rosmarinic acid (\u003cstrong\u003e2\u003c/strong\u003e) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, a). We were also able to identify minor components \u003cem\u003evia\u003c/em\u003e identification carried out earlier [Shkryl \u003cem\u003eet al.\u003c/em\u003e,2021]. The next three peaks were assigned as shikonofurans: shikonofuran D (\u003cstrong\u003e3\u003c/strong\u003e), shikonofuran E (\u003cstrong\u003e4\u003c/strong\u003e) and shikonofuran C (\u003cstrong\u003e5\u003c/strong\u003e) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, a). Red naphthoquinone pigments (shikonin derivatives) were also detected: hydroxyisovalerylshikonin (\u003cstrong\u003e6\u003c/strong\u003e), acetylshikonin (\u003cstrong\u003e7\u003c/strong\u003e), isobutyrylshikonin (\u003cstrong\u003e8\u003c/strong\u003e) and isovalerylshikonin (\u003cstrong\u003e9\u003c/strong\u003e) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, b). All information about the determined phenolic compounds is summarized in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. The quantitative measurement of rabdosiin (\u003cstrong\u003e1\u003c/strong\u003e) was performed \u003cem\u003evia\u003c/em\u003e the external standard method with a research-grade standard sample of previously isolated rabdosiin. Rosmarinic acid (\u003cstrong\u003e2\u003c/strong\u003e) and shikonofurans (\u003cstrong\u003e3\u0026ndash;5\u003c/strong\u003e) were quantified on the basis of four-point regression curves built with the reference commercial standard of rosmarinic acid.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eList of phenolic compounds produced by \u003cem\u003eL. erythrorhizon\u003c/em\u003e calli grown under control dark conditions.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePeak number\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCompound\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRt, min\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eUV max, nm\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eESI-MS, [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e, \u003cem\u003em/z\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eContent, % DW\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRabdosiin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e253, 285, 346\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e717\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRosmarinic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e287, 327\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e359\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.138\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eShikonofuran D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e25.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e269, 323\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e343\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eShikonofuran E\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e26.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e271, 328\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e355\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eShikonofuran C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e26.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e270, 325\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e357\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHydroxyisovalerylshikonin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e27.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e272, 518\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e387\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAcetylshikonin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e274, 516\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e329\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIsobutyrylshikonin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e273, 517\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e357\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.037\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIsovalerylshikonin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e33.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e273, 517\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e371\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.024\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eThe results of the quantitative analysis (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) revealed that the contents of the major compounds, rabdosiin and RA, did not change beginning in 2005 during continuous long-term cultivation and reached 1% DW. However, the content of shikonin derivatives was significantly lower than that in 2001. Only four of the seven compounds were detected in the 32-year-old callus line BK-39 of \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e. Interestingly, these four shikonin derivatives were predominant in the 8-year-old callus line BK-39 of \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e. While minor compounds were completely absent, the compositions of these four major shikonin derivatives changed. In 2001, the major compounds were isobutyrylshikonin, acetylshikonin, isovalerylshikonin, and hydroxyisovalerylshikonin (up to 38, 27, 12, and 9% of shikonin derivatives, respectively). In 2025, the major compounds were isobutyrylshikonin, isovalerylshikonin, and hydroxyisovalerylshikonin (48, 35, and 12% of shikonin derivatives, respectively), while the content of acetylshikonin was reduced to 4% of that of shikonin derivatives. Unfortunately, we cannot assess changes in the content of shikonofurans since there are no earlier data. The total content of shikonofurans in the 32-year-old callus line BK-39 was not greater than 0.06% DW.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. The growth of \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli cultivated under different light treatments\u003c/h2\u003e\n \u003cp\u003ePreviously, we showed that different LED treatments can improve the productivity of long-term cultivated calli (Veremeichik et al., \u003cspan class=\"CitationRef\"\u003e2024a\u003c/span\u003e). On the basis of previous studies, we can also conclude that the most appropriate intensities of LED light exposure are 100 and 300 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. In 2002, light exposure was shown to block shikonin biosynthesis (Yamamoto et al., \u003cspan class=\"CitationRef\"\u003e2002\u003c/span\u003e). However, despite significant advances in photobiology, similar studies using finely tuned lighting have not been carried out. In the present study, we investigated the phytochemical content of the 32-year-old callus line BK-39 of \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e grown for 30 days under monochromatic red, green, bichromatic red and blue LED light at intensities of 100 and 300 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e: R100 and R300; G100 and G300; RB100 and RB300. Dark conditions (D) were used as positive controls; and warm white LED light with an intensity of 100 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (W100) was used as a negative control.\u003c/p\u003e\n \u003cp\u003eFirst, we investigated the effects of different light conditions on the growth of \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e callus culture. \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli were cultivated once under different light condition for 30 days. In our work, we investigated the effects of two main variables of LED lighting on the productivity of \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e cell cultures. These are the spectral composition and the illumination intensity, expressed in PPFD. The four-section chambers are equipped with adjustable lamps. The LEDs are located on one board and combined into one matrix (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, a). Each board is powered by a current driver of the DS-EUM-075S105DG type, with which the level of irradiation can be adjusted by changing the current for the LEDs. Warm white (W) and monochromatic, red (R) and green (R), and bichromatic red and blue (RB) lighting options (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, b-e) with different characteristics were used (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, f). The light intensities chosen for the experiments were 100 and 300 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Calli grown in the dark were used as a control.\u003c/p\u003e\n \u003cp\u003eWe analysed the effects of warm white light, red, green and a combination of red and blue light with intensities of 100 and 300 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Compared with the dark-grown control, all the light treatments had no negative effect on the growth of the \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e callus culture. However, warm white, green and a combination of red and blue light resulted in the loss of color in the culture. While \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli grown in the dark are rich in crimson color, the calli grown under light conditions are devoid of color. However, the calli grown under red light remained colored (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. Phytochemical contents in \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli cultivated under different light treatments\u003c/h2\u003e\n \u003cp\u003eFirst, we were interested in how different LED light treatments affect the contents of shikonin derivatives. As shown in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, all the light treatments completely blocked the biosynthesis of the shikonin derivatives in \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli, despite red light of both intensities (100 and 300 \u0026micro;mol m \u003csup\u003e2\u003c/sup\u003es \u003csup\u003e1\u003c/sup\u003e). Red light treatment led to a 20-fold reduction in the contents of the major compounds isobutyrylshikonin and isovalerylshikonin. Biosynthesis of minor compounds (hydroxyisovalerylshikonin and acetylshikonin) as well as the biosynthesis of shikonofurans were blocked in red light-treated \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eHPLC analysis of shikonin derivatives in \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli cultivated for 30 days under various light treatments: D, darkness; warm white, monochromatic, and bichromatic sources designated W, R, G, and RB, respectively, with intensities of 100 and 300 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"8\"\u003e\n \u003cp\u003eLight treatment, \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eW100\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eR100\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eR300\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eG100\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eG300\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRB100\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRB300\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"9\"\u003e\n \u003cp\u003eShikonins, mg/g DW\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHydroxyisovalerylshikonin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAcetylshikonin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.028\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIsobutyrylshikonin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.366*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.193\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.235\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIsovalerylshikonin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.236*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.225\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.215\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"9\"\u003e\n \u003cp\u003eShikonofurans, mg/g DW\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eShikonofuran D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.028\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eShikonofuran E\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eShikonofuran C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.028\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"9\"\u003eND, not detected.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"9\"\u003eThe mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean is used to show the data from three separate studies with ten biological replicates.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"9\"\u003e* above the error indicates statistically significant differences (ANOVA, p ˂0.05).\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eWe analysed the effects of different light treatments on the contents of the major polyphenolic compounds, rabdosiin and RA. Warm white light with an intensity of 100 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e did not affect the biosynthesis or growth of either caffeic acid derivative compared with the control dark-grown \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Interestingly, whole green and red‒blue light treatments with an intensity of 100 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e led to a 1.5- and 2-fold increase in the rabdosiin content, respectively, whereas red light treatment resulted in an almost twofold rabdosiin content compared with that of the control dark-grown \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, a). Increasing the light treatment intensity to 300 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e did not have positive effects on the rabdosiin content. Considering the impact of light treatments on the growth of \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli, the productivity of rabdosiin in G100 and RB100 light-treated \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli was greater than 1.5 times greater than that in the control dark-grown \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, a). At that time, no light treatment had any positive effect on the biosynthesis or productivity of RA (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, b).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3.4. The growth and phytochemical content of\u003c/strong\u003e \u003cstrong\u003eL. eryrthrorhizon\u003c/strong\u003e \u003cstrong\u003ecalli cultivated under low-intensity red and blue light treatments\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eSince exposure to red light did not block shikonin biosynthesis in \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli, we next tested the effect of red light at a reduced intensity of up to 50 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (R50). In addition, we studied the effects of supplementation with 1/5 (R40B10) or half (R25B25) blue light on the overall intensity of 50 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. We showed that all three light treatments had a strongly positive effect on callus growth, with an increase of more than 15% (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, a). However, when the calli were exposed to pure red light, the color of the culture was no less intense than that of the dark-grown control. When blue light was supplemented, the color of the calli visually became less saturated, which indicates a decrease in the accumulation of shikonin (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, b).\u003c/p\u003e\n \u003cp\u003eSurprisingly, R50 treatment led to a significant increase in shikonin biosynthesis. As shown by HPLC analysis, the contents of hydroxyisovalerylshikonin and isovalerylshikonin were increased approximately 2- and 1.4-fold, respectively, compared with those in the control dark-grown \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The contents of isobutyrylshikonin and acetylshikonin did not change. At that time, the content of shikonofuranes was reduced approximately 2-fold in R50-treated \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli compared with the control dark-grown \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Supplementation of red light with blue light led to a dramatic decrease in the shikonin derivative content and total blockade of shikonofurane biosynthesis. Interestingly, none of these light treatments led to significant changes in the rabdosiin content; however, R50 treatment led to a significant decrease in the RA content (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, a). However, considering the impact of these light treatments on growth, the productivity of both rabdosiin and RA was more than 20% greater in R40B10-treated \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli than in control dark-grown R40B10-treated calli (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, b).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eHPLC analysis of shikonin derivatives in \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli cultivated for 30 days under various light treatments: D, darkness; monochromatic, and bichromatic sources designated R and RB, respectively, with intensities of 50 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eLight treatment, \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eR50\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eR25B25\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eR40B10\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eShikonins, mg/g DW\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHydroxyisovalerylshikonin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.021\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.637\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAcetylshikonin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.028\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.035\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIsobutyrylshikonin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.366\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.371\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.098\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIsovalerylshikonin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.236\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.457\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.136\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eShikonofurans, mg/g DW\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eShikonofuran D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.028\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eShikonofuran E\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.024\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eShikonofuran C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.028\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003eND, not detected.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003eThe mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean is used to show the data from three separate studies with ten biological replicates.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003eThe different letters above the error indicate statistically significant differences (ANOVA, p ˂0.05).\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cstrong\u003e3.5. The impact of inductors and low-intensity red light treatment on the productivity of shikonin in the\u003c/strong\u003e \u003cstrong\u003eL. eryrthrorhizon\u003c/strong\u003e \u003cstrong\u003ecalli\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eCopper ions are the most effective inducers of shikonin biosynthesis in the callus cultures of \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e (Sun et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). In the present study, \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli were stably grown on solid media supplemented with copper glycerate (0.3 mg/l). We investigated the combined effects of low-intensity red LED light and increased concentrations of inducers on shikonin productivity in \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli. \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli were grown for one passage (30 days) in the control dark conditions (D) and under low-intensity (50 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) red LED light (R50). For cultivation, solid medium supplemented with 0.3 copper glycerate was used, and the concentrations were increased to 1.2 and 2.4 (mg/L).\u003c/p\u003e\n \u003cp\u003eIncreasing the copper glycerate concentration to 1.2 mg/L did not suppress growth and increased the content and production of shikonin esters by 20% when the samples were grown in the dark (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). When grown in the dark, increasing the copper glycerate concentration to 2.4 mg/L had an inhibitory effect on growth (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e, a). However, the content of shikonin esters did not increase compared with that in the 1.2 cultivar (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e, b). Moreover, the production of shikonin increased by 20% with the addition of 1.2 and insignificantly with the addition of 2.4 (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e, c). When \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli were grown under red light with an intensity of 50 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, we did not find a negative effect of increasing the concentration of copper glycerate on the growth of the culture. Increasing the concentration of copper glycerate to 1.2 mg/L and growing \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli under red light with an intensity of 50 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e allowed us to increase the productivity of the culture by more than 2 times (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIt is believed that with long-term perennial cultivation, the ability of calli not only to regenerate but also to produce secondary metabolites decreases (Liu et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In the present work, we analysed the growth and biosynthetic characteristics of the \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e callus line BK-39 obtained in 1993 after 32 years of cultivation (more than 380 passages). Thus, the growth of the callus line BK-39 was approximately 19 g/L DW in 2001, after 8 years of continuous cultivation. As we showed in the present work, after 32 years of continuous cultivation, the growth of the \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e callus line BK-39 was approximately 5 g/L DW. We suggest that long-term cultivation may be the reason for this significant (approximately fourfold) decrease in growth. The effects of over a decade of continuous cultivation on the growth of a callus culture are poorly understood. In a recent study, we demonstrated that long-term cultured calli did not experience a decline in growth when their secondary metabolite biosynthesis decreased (Veremeichik et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e; Veremeichik \u003cem\u003eet al\u003c/em\u003e., 2025). We assumed that the youthful culture was characterized by explosive growth, which eventually levelled off to a comfortable level.\u003c/p\u003e\u003cp\u003eIn this study, we also examined phytochemicals in the 32-year-old \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e callus line BK-39. To determine the impact of continuous long-term cultivation on the production of the \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e callus line BK-39, we compared the results with existing data. The chromatographic profile of the \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e callus line BK-39 demonstrated the presence of two major peaks, rabdosiin and RA, and minor components such as shikonofurans and shikonin derivatives. The Callus line BK-39 of \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e was obtained in 1993, while the first phytochemical analysis of this callus was performed in 2001 (Bulgakov et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). In 2005, the 12-year-old callus line BK-39 of \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e produced approximately 1% DW of both rabdosiin and RA (Bulgakov et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Quantitative analysis revealed that the contents of the major compounds, rabdosiin and RA, did not change beginning in 2005 during continuous long-term cultivation and reached 1% DW. However, the content of shikonin derivatives was significantly lower than that in 2001. Only four of the seven compounds were detected in the 32-year-old callus line BK-39 of \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eWe previously demonstrated that various LED treatments can increase the productivity of \u003cem\u003eMertensia maritima\u003c/em\u003e calli cultivated for a long period of time (Veremeichik et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e). We may infer from earlier research that the ideal range for LED light exposure intensity is between 100 and 300 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Shikonin production was demonstrated to be blocked by light exposure in 2002 (Yamamoto et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). However, comparable investigations with precisely calibrated lighting have not been conducted, despite notable advancements in photobiology. First, we compared the phytochemical content of a 32-year-old \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e callus line grown for 30 days under monochromatic red and green light and bichromatic red and blue LED light treatment to that of calli grown under warm white light as a negative control and dark conditions as a positive control. Compared with that of the dark-grown control, the growth of the \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e callus culture was unaffected by any of the light treatments. However, the color of the culture was lost as a result of warm white, green, and a mix of red and blue light. The calli of \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e that are grown in the dark have a deep crimson hue, but those that are grown in light have no colour at all. The color of the calli that were exposed to red light, however, was maintained. Therefore, regardless of intensity, red light has the strongest growth-stimulating effect. Growth is not adversely affected by the impacts of green light. This pattern is typical of cell cultures in general. The information gathered for this study is consistent with earlier findings for plants and callus cultivation. Accordingly, cultivation of \u003cem\u003eHypericum perforatum\u003c/em\u003e callus cultures under red light and dark conditions resulted in much greater biomass accumulation, whereas cultivation of the cultures under blue light had the opposite effect (Najafabadi et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Notably, cardoon seedlings grow 60\u0026ndash;100% faster under red light than they do in a greenhouse, whereas blue light inhibits their growth (Rabara et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). According to our earlier findings, blue light at an intensity of 100 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e completely inhibited the growth of \u003cem\u003eM. maritima\u003c/em\u003e, whereas red and green light had no detrimental effects on cell culture growth (Veremeichik et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFirst, we wanted to determine how the content of shikonin derivatives was affected by various LED light treatments. Despite red light, all light treatments completely prevented the production of shikonin compounds in \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli. The amount of the main shikonin derivatives was reduced by 20 times when the samples were exposed to red light at intensities of 100 and 300 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. We examined how the various light treatments affected the levels of rabdosiin and RA, two important polyphenolic chemicals. Compared with that of the control dark-grown \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli, the production of both caffeic acid derivatives was unaffected by warm white light. Intriguingly, the rabdosiin content increased 1.5- and 2-fold in response to the green and red\u0026ndash;blue light treatments, but it decreased nearly twofold-fold in response to the red light treatment compared with that in the control dark-grown \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli. Raising the light treatment intensities to 300 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e did not have the same beneficial effect on the amount of rabdosiin. Compared with that in the control dark-grown \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli, the productivity of rabdosiin in the G100 and RB100 light-treated \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli was more than 1.5 times greater, considering the effects of the light treatments on growth. None of the light treatments had any beneficial effects on RA biosynthesis or output.\u003c/p\u003e\u003cp\u003eIt was previously demonstrated that blue light suppressed the formation of shikonin while increasing the level of RA (Gaisser and Heide, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). These findings imply that Lithospermum cells have two routes, one that leads to the biosynthesis of polyphenols and the other to the creation of shikonin, both of which share an early biosynthetic sequence. Importantly, the pattern of RA accumulation in the aerial and underground sections of the whole plant differed; that is, shikonin was found only in the underground tissues, whereas RA was essentially undetectable in the root tissues. Nevertheless, it is unknown whether light regulates the biosynthesis of these caffeic acid oligomers in the same way that it governs the biosynthesis of shikonin (Yamamoto et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). However, effective approaches for the regulation of RA biosynthesis include the use of RA, which is one of the most frequently occurring caffeic acid esters in the plant kingdom in addition to chlorogenic acid. RA has numerous biological and pharmacological activities (Petersen \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In plants, RA is believed to serve as a preformed defence compound against pathogens and herbivores (Petersen and Simmonds, 2003). Moreover, caffeic acid esters can act as UV protectants (Cle\u0026acute; \u003cem\u003eet al.\u003c/em\u003e, 2008). Numerous pharmacological and biological activities, such as anti-inflammatory, antioxidative, antidiabetes, antivirus, antitumour, neuroprotective, and hepatoprotection effects, of RA and related compounds have been described (Guan et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Antiviral activity was shown for extracts from \u003cem\u003eMelissa officinalis\u003c/em\u003e against \u003cem\u003eHerpes simplex\u003c/em\u003e infections (Astani et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Owing to their high productivity, plant cell cultures are a potential source of RA. Thus, \u003cem\u003eSalvia officinalis\u003c/em\u003e suspension culture produce approximately 36% (Hippolyte et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). The regulation of rhabdosin biosynthesis is of no less interest. The RA dimer rabdosiin belongs to the lignans, constituting an abundant class of phenylpropanoids and having a number of medically important biological activities, such as antitumor, antimitotic, and antiviral properties (Umezawa, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Rabdosiin was detected in \u003cem\u003eRabdosia japonica\u003c/em\u003e (Lamiaceae) (Agata et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1988\u003c/span\u003e)d \u003cem\u003eerythrorhizon\u003c/em\u003e (Boraginaceae) (Yamamoto et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Recently, it was shown that rabdosiin has anticancer (Flegkas et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), antiallergic (Ito et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), and nefroprotective activities (Inyushkina et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSince exposure to red light did not block shikonin biosynthesis in \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli, we tested the effect of red light at a reduced intensity of up to 50 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. In addition, we studied the effect of supplementation with 1/5 and half blue light on the overall intensity of 50 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. These light treatments had a strongly positive effect on callus growth, with an increase of more than 15%. R50 treatment led to a significant increase in shikonin biosynthesis, whereas blue light supplementation led to a decrease in the accumulation of shikonin and total blockade of shikonin biosynthesis. Interestingly, none of these light treatments led to significant changes in the rabdosiin content; however, the R50 treatment led to a significant decrease in the RA content. However, considering the impact of these light treatments on growth, the productivity of both rabdosiin and RA was more than 20% greater in R40B10-treated \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli than in the dark-grown control. Copper ions are the most effective inducers of shikonin biosynthesis in the callus cultures of \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e (Sun et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). We investigated the combined effects of low-intensity red LED light and increased concentrations of inducers on shikonin productivity in \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli. Increasing the copper glycerate concentration to 1.2 mg/L did not suppress growth and increased the content and production of shikonin esters by 20% when the samples were grown in the dark. When the mixture was grown in the dark, the production of shikonin increased by 20% with the addition of 1.2, but the increase was not significant with the addition of 2.4. When \u003cem\u003eL. eryrthrorhizon\u003c/em\u003e calli were grown under R50, we did not find a negative effect of increasing the concentration of copper glycerate on the growth of the culture. Increasing the concentration of copper glycerate to 1.2 mg/l allowed us to increase the productivity of the culture by more than 2 times.\u003c/p\u003e\u003cp\u003eIn summary, we propose the following scheme for the light-dependent differential regulation of the biosynthesis of shikonin and CADs in \u003cem\u003eL. erythrorhizon\u003c/em\u003e calli (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Shikonin and shikonofurans are byproducts of geranylhydroquinone. Two key precursors of geranylhydroquinone, geranyl diphosphate (GPP), are derived \u003cem\u003evia\u003c/em\u003e the mevalonate pathway, and \u003cem\u003ep\u003c/em\u003e-hydroxybenzoic acid (PHB) is derived \u003cem\u003evia\u003c/em\u003e the phenylpropanoid pathway. Biosynthesis of the aromatic intermediate PHB derived from coumaroyl-CoA, which is a key precursor for RA biosynthesis derived from tyrosine. The main derivatives of RA in \u003cem\u003eL. erythrorhizon\u003c/em\u003e are lithospermic acid B (LAB) and rabdosiin (Sun et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). As we showed in the present work, monochromatic green and bichromatic red and blue LED light treatments shifted biosynthesis from the coumaroyl-CoA stage to the RA stage, whereas low-intensity red light treatments, in contrast, shifted biosynthesis to the shikonin stage. Moreover, low-intensity red light treatment shifted the biosynthesis to the side of shikonin in the geranylhydroquinone stage.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eResearch on light sources and environmental factors that can increase the sustainability and profitability of PFALs has become increasingly important in recent years (Orsini et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this study, the impact of artificial monochromatic and bichromatic LED light at wide ranges of intensities (50, 100, and 300 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) on the growth and biosynthesis of caffeic acid derivatives as well as shikonin and shikonofurans in long-term cultivated \u003cem\u003eL. erythrorhizon\u003c/em\u003e callus cultures was investigated for the first time. In general, the following conclusions can be drawn: i) In long-term cultivated \u003cem\u003eL. erythrorhizon\u003c/em\u003e callus cultures, the content of shikonin decreased after more than 30 years of cultivation, whereas the content of CADs did not significantly change. ii) Red light has the greatest growth-stimulating effect regardless of intensity. It can also be assumed, on the basis of literary data, that this pattern is characteristic of cell cultures in general. iii) The most effective treatment for CAD productivity (both RA and rabdosiin) is red/blue and green light treatment. iv) The most effective way to produce shikonin in long-term cultivated \u003cem\u003eL. erythrorhizon\u003c/em\u003e calli is to use red light with an intensity of 50 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and increase the copper glycerate concentration. v) It may also be assumed that the blue and green components of white light have a negative effect on shikonin biosynthesis because of the light-dependent shift in the biosynthesis of CADs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThe analyses described in this work were performed \u003cem\u003evia\u003c/em\u003e equipment from the Instrumental Centre for Biotechnology and Gene Engineering at the Federal Scientific Centre of East Asia Terrestrial Biodiversity of the Far East Branch of the Russian Academy of Sciences within the state assignment of the Ministry of Science and Higher Education of the Russian Federation (0207-2024-0022) \u003cem\u003evia\u003c/em\u003e lightning equipment from the Institute of Automation and Control Processes, Far Eastern Branch of the Russian Academy of Sciences within the state assignment of the Ministry of Science and Higher Education of the Russian Federation (FWFW-2024-0004). The spectrophotometric analysis was performed within the state assignment of the G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Science (agreement No. 075-03-2025-231).\u003c/p\u003e\n\u003cp\u003eCRediT author statement\u003c/p\u003e\n\u003cp\u003eG.N.\u0026nbsp;Veremeichik: Conceptualization, Data curation, Project administration, Supervision, Validation, Visualization, Writing \u0026ndash; original draft.\u003c/p\u003e\n\u003cp\u003eG.N.\u0026nbsp;Veremeichik; V.P. Grigorchuk; S.A. Silantieva; E.P. Subbotin; E.V. Brodovskaya, G.K. Tchernoded; O.A. Tikhonova; S.A. Fedoreyev; N.P. Mishchenko; E. A. Vasileva; A.A. Khopta;\u0026nbsp;S.O. Kozhanov: Investigation, Methodology, Formal Analysis.\u003c/p\u003e\n\u003cp\u003eV.P. Bulgakov; Y.N. Kulchin;\u0026nbsp;Siberev A.S.: Resources, Funding acquisition.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis research was funded by a grant from the Ministry of Science and Higher Education of the Russian Federation for large scientific projects in priority areas of scientific and technological development (subsidy identifier 075-15-2024-540).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData\u003c/strong\u003e \u003cstrong\u003eavailability\u003c/strong\u003e The datasets generated during and/or analysed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode\u003c/strong\u003e \u003cstrong\u003eavailability\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics\u003c/strong\u003e \u003cstrong\u003eapproval\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent\u003c/strong\u003e \u003cstrong\u003eto\u003c/strong\u003e \u003cstrong\u003eparticipate\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent\u003c/strong\u003e \u003cstrong\u003efor\u003c/strong\u003e \u003cstrong\u003epublication\u003c/strong\u003e All the authors whose names appeared on the submission approved the version to be published and agreed to be accountable for all aspects of the work in ensuring that the questions related to the accuracy of integrity of any part of the work were appropriately investigated and resolved.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict\u003c/strong\u003e \u003cstrong\u003eof\u003c/strong\u003e \u003cstrong\u003einterest\u003c/strong\u003e The authors declare that they have no competing interests\u003cins cite=\"mailto:Rubriq\" datetime=\"2025-06-26T14:24\"\u003e.\u003c/ins\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAgata I, Hatano T, Nishibe S, Okuda T (1988) Rabdosiin, a new rosmarinic acid dimer with a lignan skeleton, from \u003cem\u003eRabdosia japonica\u003c/em\u003e. Chem Pharm Bull 36:3223\u0026ndash;3225. https://doi.org/10.1248/cpb.36.3223\u003c/li\u003e\n \u003cli\u003eAstani A, Reichling J, Schnitzler P (2012) \u003cem\u003eMelissa officinalis\u003c/em\u003e extract inhibits attachment of herpes simplex virus in vitro. Chemother 58:70\u0026ndash;77. https://doi.org/10.1159/000335590\u003c/li\u003e\n \u003cli\u003eBulgakov VP, Kozyrenko MM, Fedoreyev SA, Mischenko NP, Denisenko VA, Zvereva LV, Pokushalova TV, Zhuravlev YuN (2001) Shikonin production by p-fluorophenylalanine resistant cells of \u003cem\u003eLithospermum erythrorhizon\u003c/em\u003e. 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Plant Biotechnol. 34:131\u0026ndash;142. https://doi.org/10.5511/plantbiotechnology.17.0823a\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Artificial light, rosmarinic acid, long-term cultured calli, Lithospermum erythrorhizon Siebold \u0026 Zucc, shikonin derivatives, rabdosiin","lastPublishedDoi":"10.21203/rs.3.rs-7030114/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7030114/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCurrently, global challenges require the search for alternative sources of phytochemicals used by Mankind throughout its history. Plant cell culture technology can provide efficient and sustainable sources of phytochemicals with reduced energy and carbon footprints. The first patented industrial process of cultivating calluses as sources of phytochemicals was based on the calli of \u003cem\u003eLithospermum erythrorhizon\u003c/em\u003e Siebold \u0026amp; Zucc (Boraginaceae). The high pharmacological activity of \u003cem\u003eL.\u0026nbsp;eryrthrorhizon\u003c/em\u003e is due to the content of the naphthoquinone shikonin. Despite the development of LED lighting technology, in-depth studies of the effects of light on the biosynthesis of shikonin in cell cultures have not been carried out. In the present work, the impact of artificial monochromatic and bichromatic LED light at wide ranges of intensities (50, 100, and 300 µmol m\u003csup\u003e-2\u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e) on the growth and biosynthesis of caffeic acid derivatives (CADs) as well as shikonin and shikonofurans in long-term continuously cultivated \u003cem\u003eL.\u0026nbsp;erythrorhizon\u003c/em\u003e calli was investigated for the first time. Red light has the greatest growth-stimulating effect regardless of intensity. The most effective treatment for CADs productivity is red/blue and green light treatment. The most effective way to produce shikonin in long-term cultivated \u003cem\u003eL.\u0026nbsp;erythrorhizon\u003c/em\u003e calli is to use red light with an intensity of 50 µmol m\u003csup\u003e-2\u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e and increase the inductor concentration. It may also be assumed that the blue and green components of white light have a negative effect on shikonin biosynthesis because of the light-dependent shift in the biosynthesis of CADs.\u003c/p\u003e","manuscriptTitle":"Low-intensity monochromatic red LED light can improve shikonin productivity in long-term cultivated Lithospermum erythrorhizon Sieb. et Zucc. calli","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-09 10:44:30","doi":"10.21203/rs.3.rs-7030114/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-07-07T11:59:52+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-07T09:51:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-03T14:55:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2025-07-02T10:03:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0cf0c830-3bc6-46fc-b09b-379d06e7bb6f","owner":[],"postedDate":"July 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-10T15:59:24+00:00","versionOfRecord":{"articleIdentity":"rs-7030114","link":"https://doi.org/10.1007/s11240-025-03280-3","journal":{"identity":"plant-cell-tissue-and-organ-culture-pctoc","isVorOnly":false,"title":"Plant Cell, Tissue and Organ Culture (PCTOC)"},"publishedOn":"2025-11-05 15:57:07","publishedOnDateReadable":"November 5th, 2025"},"versionCreatedAt":"2025-07-09 10:44:30","video":"","vorDoi":"10.1007/s11240-025-03280-3","vorDoiUrl":"https://doi.org/10.1007/s11240-025-03280-3","workflowStages":[]},"version":"v1","identity":"rs-7030114","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7030114","identity":"rs-7030114","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}