To enhance the biosynthesis of SLs, Arnica montana and variety Arbo were elicited with jasmonic acid (JA) (Merck Millipore, Warsaw, Poland) and chitosan (Merck Millipore, Warsaw, Poland) at different concentration ranges. The plant cultures were maintained on MS medium solidified with agar, without plant hormones in a phytotron for 6 weeks without the addition of elicitors at 20 ± 2°C with a photoperiod of 16/8 h light/dark at a PPFD of 70 µmol m^-2 s^-1. After 6 weeks of culture, plants were transferred into a liquid MS medium with different concentrations of chitosan aqueous solutions and alcoholic solutions of JA. Sterile, filtered JA solution was prepared in 70% ethanol and diluted to final concentrations of 0.25, 0.50, 1.0, and 1.5 mg/L. Chitosan solution was prepared in sterile water to final concentrations of 50, 100, 150, 200, and 250 mg/L; pH was regulated prior to steam autoclaving and then added to the propagated plant cultures. The control in chitosan experiments was pure liquid MS medium. In the JA study, the medium supplemented with 70% ethanol in the same volume as in the case of JA was used. Cultures with elicitors were maintained in a phytotron for the next 7 days, extracted, and then the qualitative content of SL was analyzed by LC-QTOF-MS.

To investigate the effect of light wavelength on the biosynthesis of SLs, the propagated explants were exposed to four light spectra: yellow (570 nm), blue (465 nm), red (660 nm), and the full spectrum of light. The PPFD was set at the same level of 30 μmol m^-2 s^-1. The experiment was carried out in experimental cabinets using Philips fluorescent lamps emitting red light (‘TL’D 36W/15), blue light (‘TL’D 36W/18), yellow light (‘TL’D 36W/16), and full spectrum light (‘TL’D 36W/84). The air-conditioning was used to establish the same temperature in each cabinet with a separate conditioning system at 20 ± 2°C with a photoperiod of 16/8 h light/dark. The cultures were maintained in cabinets for 6 weeks.

Studies on the influence of PPFD on the biosynthesis of SLs were carried out, three-photon flux densities of 50, 100, and 150 μmol m^-2 s^-1 were used, and subsequently, qualitative analysis of SLs was carried out.

The SL concentrations in leaves of A. montana showed a marked increase, with the second and fifth leaves having double the amount of lactones found in the first leaf, and the third and fourth leaves displaying a threefold increase. The analysis shows that the level of lactones along the axis of the shoot is highest in the third and fourth leaves and the lowest in the first leaf. The distribution of lactones is diverse in the Arnica montana ‘Arbo’ ( Figure 2A ), where most lactones are found in the first leaf, with a gradual reduction in concentration in the leaves of the next examined nodes. In both taxa, the level of DHM changes the most significantly, with a decrease in variety and with a rise in the wild species leaves until the third node, whereas much smaller fluctuations were recorded in the case of tigloyldihydrohelenalin (DHT); however, overall, all derivatives follow the trends in the total SLs concentration.

Experiments were performed on a liquid medium that accelerates plant growth. After 9 weeks in the bioreactor of Arnica montana ‘Arbo’ ( Figure 2B ), the plant reached the fresh mass of 390.2 g, representing nearly a fivefold increase, with a moderate lactone content. The average SL component in the leaves was lower (40.25 mg/g DW) ( Table 1 ) than in the flasks culture (64.2 mg/g DW) ( Table 2 ), which may be the consequence of less transparency of the container walls, which reduced the light intensity by 20% and cut off wavelengths below 400 nm. It is also worth noting that the resulting clump is not built of leaves themselves; the analysis of the long, older shoots revealed the presence of lactones at 11.93 mg/g DW, whereas the apical shoots and short adventitious shoots contained 14.69 mg/g DW of these components ( Table 3 ). In contrast to aerial plant parts, the roots contained only small amounts of lactones (4.07 mg/g DW) ( Table 3 ). Additionally, access to light for plants growing in a compact clump is limited. The differences in the amount of lactones in various organs affect the efficiency of the in vitro bioreactor. In a 9-week cycle, it was estimated that leaves constitute 84% of the total mass, with shoots and roots 16%, which reduces the total productivity of the bioreactor. The efficiency of the bioreactor from Arnica montana ‘Arbo’ plants is 1319.40 mg/g DW of SLs calculated per leaf from one bioreactor’s 9-week cycle. This means that one cycle initiated from 80 g fresh weight of plants yields SLs equivalent to 156.97 g DW of field-grown leaves and 38.26 g DW of flowers. The underground parts of the plant - roots and rhizomes are a rich source of specific essential oils with thymol and its derivatives, which also possess therapeutic and pharmacological properties.

In summary, the bioreactor productivity of the applied type is 4025.02 mg SLs per 100 g of dry weight of leaves.

The experiments examined the influence of the light spectrum of different wavelengths on the production of SLs and the morphology of plants cultivated under these conditions. White, yellow, red, and blue light were used with the same PPFD intensity, i.e., expressed in micromoles per m^-2 s^-1. Preparing appropriate chambers for culture growth required cooling systems and ventilators of different power to obtain the same temperature of 20 ± 2°C and at PPFD of 30 µmol m^-2 s^-1. The spectral characteristics of the Philips fluorescent lamps, emitting different wavelengths of light, are shown in Figure 3 .

Among the wavelengths used, only red light induced the biosynthesis of SLs, both in the wild species and the variety. In this light spectrum, A. montana synthesizes three times more lactones after 2 months of culture, while an increase of over 60% is detected in ‘Arbo’ compared to controls. In wild species, dihydrohelenalin esters predominate, while helenalin derivatives are absent. In a variety, significantly more helenalin esters are synthesized in red light, mainly methacryloylhelenalin (HM) and tigloylhelenalin (HT). The lowest lactone synthesis occurs in the blue part of the spectrum. The morphogenetic influence of the light spectrum ( Figure 4 ) was manifested in the diverse development of leaves, the length of petioles, the intensity of rhizome growth, and the number and color of roots. Visually, plants cultivated under white ( Figure 4A ) and blue light ( Figure 4D ) contain the most chlorophyll, but they vary in the shape of the leaves, which are more elongated under white light than those exposed to blue light, and the formation of roots is also prolonged. Plants cultivated under red ( Figure 4C ) and yellow light ( Figure 4B ) contained less chlorophyll, but the leaves developed in both conditions had a completely different shape – in yellow light ( Figure 4B ), the blades were small and short, bent away from the stem. In the red part of the spectrum ( Figure 4C ), etiolated blades were juvenile, elongated, and erect. Intensive root development occurred on shoots growing under white light ( Figure 4A ), and the weakest under red light ( Figure 4C ). Yellow light ( Figure 4B ) promoted shoot branching the most, and white light ( Figure 4A ) the least of all applied conditions.

By analyzing the in vitro profiles of SLs in the studied Arnica taxa, it was proven that our native species A. montana, is characterized primarily by dihydrohelenalin and its derivatives (97.23%) ( Table 2 ). In comparison, in A. montana ‘Arbo’, these derivatives are slightly lower (80.5%), and, instead, with a larger fraction of helenalin and its esters ( Table 2 ).

Extraction and analyses performed using the LC-QTOF-MS on plant leaves cultivated on MS medium at PPFD of 50, 100, and 150 µmol m^-2 s^-1 show significant differences in their ability to biosynthesize SLs compared to the lowest used light intensity. In A. montana, it is 23.84 mg/g DW; in the Arbo variety, it is more than twice as high (59.5 mg/g DW) ( Table 2 ). It has been shown that the highest light level intensifies the biosynthesis of SLs by 24% in the wild species A. montana occurring in Lower Silesia and by 36.6% in the Arbo variety. Growing photon flux density in the Arnica montana ‘Arbo’ reduces the synthesis of helenalin derivatives, while it does not affect their synthesis in the A. montana wild species. In both taxa, unbound with carboxylic acid, dihydrohelenalin gradually rises with the increasing photon flux density, likely serving as a precursor to all its ester derivatives. As the light energy increases in A. montana ‘Arbo’, the content of helenalin esters declines while the analogous dihydrohelenalin esters rise. These findings indicate that light energy contributes to the conversion of helenalin esters into mostly the same dihydrohelenalin esters.

Due to the low concentration of helenalin and its derivatives in A. montana, only an increment in the synthesis of isobutyrylhelenalin (HIB), methacryloyldihydrohelenalin (DHM), isovaleryldihydrohelenalin (DHIV), and 2-methylbutyrylhelenalin (HMB) is visible under the growing light intensity, however, the same derivatives occur under these conditions in Arbo variety. Light intensity positively affects the similar profile distribution of SL derivatives, leading to an increased formation of dihydrohelenalin in both examined taxa. As the light fluence rate increases, the distribution profile of these SL derivatives becomes more uniform, further accelerating dihydrohelenalin production in both taxa. Notably, the Arbo variety contains more helenalin than compared to the wild species.

In biotechnology, JA is a known elicitor of the biosynthesis of secondary metabolites, consequently, the effect of this compound added to the agar medium in 4 doses (from 0.25 to 1.5 mg/L) on the SLs in leaves was determined. Preliminary experiments established that the greatest enhancement in the biosynthesis of these metabolites occurs after a short time, 7-day exposure of plants to this hormone. The results obtained after the application of JA indicate that the concentrations of 0.25 – 1.5 mg/L for A. montana ‘Arbo’ fit the Gauss curve with an optimum dose of 1 mg/L of nutrient solution, where the level of SLs in leaf tissues enlarges almost twofold compared to the control. In the case of the wild species, the best dose appears to be 0.25 mg/L, which brought about 34.5% more lactones than the leaves of control plants; the remaining concentrations were inferior.

In A. montana ‘Arbo’, compared to dihydrohelenalin an increase in the helenalin derivatives was also observed at all concentrations used. At the 0.25 mg/L dose, helenalin esters magnified fourfold, while the total dihydrohelenalin esters increased by 40%. JA induced the greatest formation of methacrylic and isovaleric derivatives of both helenalin and dihydrohelenalin. In the case of the wild species, such an effect of JA was not demonstrated, and the increased level of lactone correlates with the uniform synthesis of all derivatives of both groups of compounds.

JA, after a seven-day application in culture on MS agar medium, stimulates the biosynthesis of SLs, wild species respond already at a dose of 0.25 mg/L, and the variety requires a higher concentration of 1 mg/L. In A. montana ‘Arbo’, this growth regulator enhances biosynthesis by 1.5 times and changes the proportions between helenalin and dihydrohelenalin esters, promoting helenanolides biosynthesis. It can be assumed that the wild species originating from a site in southwestern Poland in the Izera Mountains is more sensitive to the JA used, and the optimal effect occurs at lower doses of this hormone.

Another well-known elicitor used in in vitro cultures is chitosan, a polysaccharide resulting from the deacetylation of chitin (poly-β-1,4-D-glucosamine). This compound was used in 5 doses (50–250 mg/L agar MS medium) for the Arnica species and variety. The tested plants responded with increased biosynthesis of SLs to the application of chitosan in the medium, and these effects were visible after 7 days of culture. The sensitivity of each taxon to the doses of chitosan varies. For the wild form of Arnica montana, the optimal concentration for lactone production is 50 mg/L, while for the Arbo variety, it is 100 mg/L. In the case of the wild species, the lactone content was elevated fivefold compared to the control on the medium without chitosan. From among all lactones, this polymer mainly stimulated the biosynthesis of dihydrohelenalin derivatives, i.e., DHM, isobutyryldihydrohelenalin (DHIB), DHT, and 2-methylbutyryldihydrohelenalin (DHMB), isovaleryldihydrohelenalin (DHIV). For the A. montana ‘Arbo’, the most effective concentration was 100 mg/L, where the contents of formed lactones amplified almost twice, and the ratio of helenalin esters to dihydrohelenalin changed from 1:5 to 1:2.5, and helenalin and dihydrohelenalin derivatives are synthesized evenly. At all doses of chitosan, except the highest, the total lactones level is higher than in the control, whereas in the wild species, it is equal to or lower than in the control. It is worth adding that both elicitors (JA and chitosan) induce rapid reactions; however, the remarkable effect diminishes with prolonged exposure. JA serves as an effective induction factor for the Arbo variety, while applying chitosan significantly increases the synthesis of SLs in A. montana, resulting in a fivefold higher concentration. The observed data suggest the effectiveness of chitosan, a factor imitating the attack of fungal pathogens and insects, and primarily highlights the different sensitivity of both taxa to the elicitors used.

Light affects the quality and efficiency of in vitro culture through spectrum, irradiance, and exposure length. It has been shown that an increase in photon flux density correlates with higher SL content in leaf tissues ( Table 2 ) The radiation sources used in our study in vitro were fluorescent lamps emitting white light or broadband spectra of blue, yellow, and red. Experiments conducted at the light intensities (PPFD) of 50, 100, and 150 µmol m^-2 s^-1 with a 16-hour photoperiod demonstrated that higher photon flux densities correlate with increased SL content in leaf tissues ( Table 2 ). Usually, light intensities typically used in in vitro culture range from 20 to 30 µmol m² s^-1, resulting in low SL content. Light has a formative effect on the development of Arnica plants by promoting the enlargement of the leaf blade and enhancing the formation of secretory structures on the leaf surface where lactones should be synthesized.

The positive impact of light on the biosynthesis of lactones in sunflower seedlings was demonstrated by Spring et al. (1986), who found that as light intensity increased to 100 W m² (approximately 267.36 µmol m^-2 s^-1), the concentration of SLs (niveusin C and 15-hydroxy-3-dehydrodesoxyfruticin) rose six-fold. However, further increments in fluency rates over 100 W m² did not lead to additional accumulation of lactones. The light-induced production of 15-hydroxy-3-dehydrodesoxyfruticin occurred after a short lag phase of 40 to 60 min, while the level of niveusin C remained unchanged for the next 12 h of exposure (Spring et al., 1986). Similar findings are reported by (Scavo and Mauromicale, 2020) regarding the phytotoxic lactone cynaropicrin, which synthesis grows under 60% shading in field conditions. Additionally, the SL content in lettuce rises after the plants are covered with foils that block ultraviolet (UV) radiation (Laurenčíková et al., 2023). Previous reports on Arnica montana ‘Arbo’ by Spitaler et al. (2006) show that the total contents of SLs and flavonoids were not positively correlated with the altitude of the growth site. Phenolics protect plants from UV radiation, and increased altitude enhances the synthesis of caffeic acid derivatives and specific ortho-hydroxylated flavonoids (Spitaler et al., 2006). These findings indicate that intense sunlight exposure may reduce the synthesis of lactones while enhancing the production of compounds that protect plants from strong solar radiation. Similar observations were made by Stefanache et al. (2014) in their study of Romanian A. montana, comparing the phenolic content in wild populations to that cultivated in vitro. Their findings revealed that samples from in vitro cultures had a low phenolic acid content and high SL levels, whereas wild-growing populations exhibited the opposite trends (Stefanache et al., 2014). Ecologically, SLs manifest antibacterial and antifungal properties and inhibit the biosynthesis of nucleic acids in invading pathogens, which is crucial for protecting susceptible young tissues under low light exposure (Drogosz and Janecka, 2018). Additionally, lactones can interact with plant hormones to promote elongational growth toward the light, as some lactones have been found in shoots at concentrations of 11–14 mg/g DW and in roots at 4 mg/g DW. High light intensities affect plant structure, morphology, and physiological changes associated with tissue maturity. Under full irradiance, specific synthesized metabolites can partially perform the functions of SLs.

It might be asked why the lactone content in field conditions is lower than in the in vitro culture, despite the fact that the dry mass of leaves growing in the field is half to three quarters higher, the lactone content is several times lower. We suspect several reasons for this phenomenon: strong insolation promoting rapid maturation and accumulation of phenolic compounds, including thymol, whose biosynthesis occurs from the same precursors (acetate/mevalonic pathway; unpublished data); the action of atmospheric factors: wind, which can damage the structure, and rain, which can aid leakage partially water-soluble lactones from the trichomes on the leaf surface. In summary, our findings and those from various authors indicate a duality in the plant protection system of A. montana under different light conditions. Specifically, low light intensity promotes the active synthesis of lactones, while high light intensity, particularly with short wavelengths, encourages the production of phenols and ortho-hydroxylated flavonoids. Additionally, SLs, as a group of substances related to strigolactones, can influence plant growth under varying light conditions, as demonstrated by (Spring et al., 1986).

Both the quantity and quality of light influence photosynthesis and plant morphogenesis, reflecting the multifaceted impact of this factor on the receptor system, including phytochrome, cryptochrome, phototropins, zeaxanthins, and others. Analysis of different light spectra shows a modification in plant morphology ( Figure 4) . Plants exposed to blue light were shorter than the control group but exhibited a similarly intense green color. In contrast, plants grown under yellow light were more branched, had smaller leaf blades, and showed greater etiolation. Those grown under the red part of the spectrum were the most etiolated, displaying elongated, stretched shoots and narrow, long leaf blades.

In both the wild species and the cultivar, only the red part of the spectrum initiated the rise in SL synthesis by over 63%, leading to an increase in helenalin derivatives in the Arnica montana ‘Arbo’ and a threefold increase in dihydrohelenalin esters in A. montana species compared to control under the white light of the same photon flux density. A similar effect of red light on sunflowers was evidenced by Spring et al. (1986), furthermore, the action of red light was partially altered by a subsequent far-red pulse. The total synthesis of SLs was also associated with red light in holy basil (Chutimanukul et al., 2022). These findings suggest that SL synthesis is controlled by phytochrome, with the red light-induced form, Pfr, dominating over Pr. This dominance triggers the biosynthesis of SLs, especially in low light conditions (PPFD 30-40 µmol m^-2 s^-1).

Phytochromes are built from two polypeptide regions: the photo-sensing domain on the amino-terminal end and the carboxy-terminal module involved in the dimerization of phytochrome polypeptide of histidine kinase-related domain chains. The carboxy-terminal ends in directed nuclear localization and exhibits serine/threonine kinase activity, facilitating the degradation of the transcription factor–phytochrome interaction factor 3 (PIF3), which activates photosynthetic genes in the dark (Sharrock, 2008). The interaction between the C-terminal module and the degradation of the transcription factor causes photobody biogenesis, which generates the regulated phytochrome-mediated signaling and physiological outputs (Matsushita et al., 2003). Consistent with these findings and the model of red light causing photomorphogenesis, Ahn et al. (2022) proposed that phytochrome B (phyB) Pfr is required for the endoplasmic reticulum (ER) stress response via induction of expression of ER luminal protein chaperones and unfolded protein response (UPR) genes in the nucleus signaling. In stress reaction, reactive oxygen species (ROS) are accumulated in Arabidopsis cells (Ahn et al., 2022). It may be hypothesized that they are responsible for incorporating an oxygen atom in a lactone ring formation, like during the synthesis of leucodin in chicory by cytochrome P450. Furthermore, most cytochrome P450 is also localized in the ER, leading to blue light-reversible inhibition of enzyme activity (de Kraker et al., 2002). Our results support the generally presented statement that red light enhances the accumulation of terpenoids, whereas blue light inhibits terpenoid biosynthesis (Kuo et al., 2015).

Phytochromes, particularly phytochrome A (phyA) and phytochrome B (phyB), are pivotal in mediating plant responses to light, influencing various developmental processes, including seed germination, stem elongation, and flowering. These responses are intricately linked to gibberellin (GA) biosynthesis and signaling pathways (Blázquez and Weigel, 1999). In Arabidopsis, red light exposure induces the expression of GA biosynthetic genes, including GA4 and GA4H, which encode GA 3β-hydroxylases responsible for the final steps in GA biosynthesis. Notably, GA4H expression is regulated explicitly by (phyB), whereas GA4 expression is influenced by other phytochromes, indicating a complex regulation of GA biosynthesis by light quality (Yamaguchi et al., 1998). Phytochromes, particularly phyB, can modulate plant responses to GAs by influencing GA sensitivity. For instance, in Arabidopsis, phyB mutants exhibit enhanced hypocotyl elongation in response to exogenous GAs, despite having wild-type levels of endogenous GAs. This indicates that phyB may regulate GA-dependent responses by altering GA sensitivity rather than biosynthesis. Phytochromes interact with other phytohormones, such as brassinosteroids (BRs), to regulate GA biosynthesis and signaling. BRs can enhance the transcriptional activity of phytochrome-interacting factors (PIFs), which modulate the expression of GA biosynthetic genes like GA3ox and GA20ox. This crosstalk ensures coordinated regulation of growth processes like cell elongation (Castro-Camba et al., 2022). The interplay between phytochrome signaling and GA biosynthesis is crucial for plants to adapt to changing light conditions. By modulating the expression of key enzymes like CPS and KS, phytochromes integrate light signals with hormonal pathways to regulate growth and development processes. Understanding these mechanisms provides insights into how plants coordinate internal and external signals to optimize their developmental strategies.

Red light converts phytochromes from their inactive Pr to active Pfr form. Pfr accumulates in the nucleus and modulates gene expression by interacting with PIFs, PHYTOCHROME INTERACTING FACTORS, leading to their degradation and releasing the suppression of downstream transcription factors involved in secondary metabolism (Legris et al., 2019). Pfr activation upregulates MYB (PAP1), which promotes anthocyanin biosynthesis by activating CHS, DFR, and ANS genes (Zhang et al., 2023). WRKY33 controls the biosynthesis of camalexin and other phytoalexins (Chen et al., 2022). The bHLH TFs work in concert with MYBs to regulate flavonoid pathway genes.

Both JA and chitosan induced a rapid response in Arnica plants, resulting in increased synthesis of SLs. The reaction in A. montana was stronger after chitosan treatment than JA. In media containing chitosan, the level of lactones expanded fourfold in the wild species and doubled in the Arbo variety. The wild species A. montana was more sensitive to the doses used; the lowest dose of chitosan was 50 mg/L, which caused the maximum response, while the optimum dose for Arnica montana ‘Arbo’ was 100 mg/L. Chitosan, a fungal cell wall polysaccharide, is recognized by plants as a microbial factor, triggering defense reactions and stimulating systemic immunity. Iula et al. (2022) studied broad reprogramming induced by molecular elicitors using metabolome analysis of tomatoes as a model plant. The response of tomato plants to chitosan was specific; secondary metabolites of the glucosinolate type, strong defense compounds characteristic to the Brassicaceae family, accumulated in the leaves, while isoprenoid derivatives decreased. These results demonstrate that different plant families and species have specific secondary metabolites, and the same molecular elicitor can trigger diverse reactions and initiate broad changes in phytohormone levels (Iula et al., 2022).

Our examination shows that reactions to the administration of chitosan in the medium were rapid, observable within 7 days of culture, and gradually disappeared after 2 weeks. This study also shows a change in the profile of SL derivatives associated with a threefold higher content of helenalin esters compared to the control. In the Arbo variety, higher amounts of HT, HM, HIB, and HA were noted, while A. montana showed a slight increase in helenanolides, but a significant rise in dihydrohelenalin derivatives (DHM, DHT, and DHMB/DHIV).

Previous studies revealed that chitosan induces acquired resistance in plants to various pathogens by the production of proteins, enzymes, and secondary metabolites. It enhances the levels and activities of glucanase, chitinase, phenylalanine ammonia-lyase, polyphenol oxidase, superoxide dismutase, and catalase, promoting the synthesis of phytoalexins and the formation of suberin, lignin, and phenols (Hidangmayum et al., 2019).Chitosan itself also has a destructive effect on fungal and bacterial pathogens. In its low molecular mass water-soluble (LMWS) form, chitosan shows an affinity for plasmalemma lipids; its specificity in the microbial systems is associated with a positive charge, which promotes interaction with anionic components of the plasma membrane (Park et al., 2008). Using the technique developed by Park et al. (2008), which employs fluorescein isothiocyanate (FITC)-labeled chitosan derivatives (Geisberger et al., 2013), showed that labeled molecules attach to a microbial wall and intracellular ultrastructure in tested species. It seems interesting to consider whether the interaction with oil bodies inherent in cells capable of synthesizing phytoalexins triggers their biosynthesis.

JA exhibited effects similar to those of chitosan, but the enhancement of biosynthesis in the wild species was weaker, and the only effective dose was the lowest (0.2 mg/L). After applying both compounds, no significant changes in the SL profile were observed. However, the increase in SLs synthesis in the Arbo variety was greater than with chitosan, occurring across a wider range of doses.

JA is part of the signal transduction system that regulates the growth and response of plants to abiotic (cold, drought, salinity, metals, light) and biotic stresses. It operates within a complex signaling network involving phytohormones, integrating regulatory transcription factors and their associated genes, including those responsible for defense reactions. At a quantity of 10–⁴ M, it induces the synthesis of mimilactone A, the main phytoalexin in rice suspension culture (Nojiri et al., 1993). In-ground cultivation administering 1 mM methyl jasmonate (MeJA) with 0.025 mg/L of zinc increased menthol content in essential oil from Mentha piperita, affecting both its quality and quantity (Mehdizadeh et al., 2024). Wang et al. (2020) believe that JA does not play an independent role in the regulatory network, but acts by integrating with transcription factors of related genes while acting synergistically and antagonistically with abscisic acid, ethylene, and salicylic acid (Wang et al., 2020). The red light modulates, creating a synergistic regulatory effect on secondary metabolites, promoting JA accumulation or signaling. Pfr suppresses JAZ repressor, releasing MYC2, a key transcription factor for secondary metabolism. The red light also interacts with ABA and may enhance flavonoid accumulation during drought stress mediated by MYB/WRKY activation (Zvi et al., 2012; Johnson et al., 2023).

The receptor for JAs is the CORONATINE INSENSITIVE 1 (COI1) protein, which associates with Skp1-like, cullin 1, and ring-box protein to form the ubiquitin ligase SCF^COI1 complex, participating in the degradation of negative regulators of JA signaling (Yan et al., 2009). The negative regulator of JA of the transcriptional network is the JASMONATE ZIM DOMAIN protein (JAZ). JAZ protein at the NT domain interacts with DELLA protein, inhibiting JA signaling. The C-terminus of the JAZ protein regulates function and binding to COI1 and MYC2/3/4 for transduction of downstream signaling. In the absence of JA, JAZ binds TF (MYC/MYB/WD40 and others), inhibiting genes that respond to JA. The presence of JA promotes recognition of JAZ by ubiquitin ligase. It directs this protein to degradation in the SCF^COI1 complex, which releases TFs inhibiting or activating expression of JA-responsive genes (Zhao et al., 2024). In our experiments, the effect of the application of JA disappears after 10 days, which indicates rapid synthesis of new JAZ proteins after signal transduction (Zhao et al., 2024). Additionally, Yan et al. (2022) showed that NbJAZ3 was highly expressed in trichomes, and lines with overexpression of NbJAZ3 trichome density significantly decreased with lower levels of expression of NbWo, NbCycB2, and NbMIXTA. Lines of NbJAZ3 RNAi slightly increased trichome density with higher expression of NbWo. The authors’ findings give a novel JA-mediated glandular trichome development model consisting of NbJAZ3-NbWo-NbCycB2 axis.

Arnica montana is protected in many countries, and for medicinal purposes, plant cultivation is introduced. Plantations of this species occupy a small area in Germany, France, and the Netherlands. In Poland, attempts were made to cultivate Arnica montana, but difficulties were encountered in establishing and maintaining cultivation (Kozłowski et al., 1999) (Sugier, 2007). achieved the first successes in the experiments by applying different elements of agricultural technology. On the investigational fields, the wild species grown from seedlings gave high yields of raw material in the third (97.5 g/m²) and fourth (99.7 g/m²) year of cultivation, while plants from shoot cuttings showed significantly lower harvests and the maximum flower yield was obtained earlier, in the third year of vegetation. Taking into account the variability of yield depending on the method of establishing the plantation in subsequent years, an average of 85.1 g of dry flower mass per square meter was annually obtained. The content of lactones in flowers ranges between 8–12 mg/g DW, which gives 600-1021.2 mg of lactones per m² of cultivation. In the bioreactor, from the initial plant mass of 80 g FW, after 9 weeks, we obtain 13.82 g/g DW of SLs. The growing plant clump contains 84% leaves, with a weight of 32.78 g DW. The Arbo variety in the bioreactor contains, on average, 40.25 mg/g DW lactones, giving 1319.40 mg lactones at 100 µmol m^-2 s^-1 lighting, which is equivalent to the flower yield from approximately 1 m² of ground cultivation. This quantity was obtained after one 9-week cycle in a bioreactor and not after several years of cultivation.

The content of lactones in leaves collected from a cold frame in the Botanical Garden in Wroclaw was estimated at 0.85 mg/g DW for the species and 8.40 mg/g DW for the variety (Parafiniuk et al., 2023). The species also contains significantly smaller amounts of lactones in the in vitro culture, which shifts into a lower reproduction coefficient. This feature is associated with faster maturation of rhizomes and roots, manifested by darkening and hardening of these tissues, which indicates a higher content of phenolic compounds. Analyzing the level of lactones in the wild species and the variety, it can be concluded that lactones are responsible for greater plant vigor. In the optimal environment of in vitro culture, this manifests itself in a reduced micropropagation rate, while in field conditions, it causes a low reproduction coefficient and a lack of plant expansion. In contrast, the variety quickly colonizes all available space. It can be argued that this is determined by the ratio of lactones to phenolic compounds in the internal tissues of both taxa. This places SLs in the category of compounds acting in a hormone-like manner, influencing cytokinin metabolism or influencing the GA and ABA ratio, which maintains significant plant juvenility and generates strong expansion in field cultivation.

The propagation of whole plants in the bioreactor is derived from observations of Malarz et al. (1993), who noted the influence of the root system on the level of SL biosynthesis and on our knowledge resulting from anatomical structure examination of the internal and external secretory system of Arnica montana (Kromer et al., 2016). The lipid distribution and fatty acid composition of triglycerides contained in the oil bodies of various organs were studied. The previously unknown connection of the axial internal secretory system of all organs into one coherent whole affects the availability of triglycerides contained in oil bodies as a substrate for the synthesis of essential oils, including SLs. In addition to the axial system of secretory channels, the plant has a system of discontinuous channels in the primary cortex and idioblasts that supply intermediates for the final synthesis of compounds stored in places critical for the health and ecological safety of the plant. For SLs, bitter substances, herbivore repellents, and perhaps also allelopathic compounds, the final site of biosynthesis is glandular hairs on the surface of leaves. The explanation presented above prompted us to use whole plant cultures in experiments with great success. The currently developed bioreactor cultivation method allows for a 7-fold increase in fresh mass increments within one month, which is serious competition in relation to extremely difficult field cultivation.

The extract from plants cultivated in vitro is devoid of even traces of pesticides, fungicides, or herbicides and is additionally sterile, which is currently required by the European Union. Production in bioreactors also does not pollute the environment, which is why, in our opinion, it is the best future solution. Our research can be applied to eliminate the harvesting of Arnica montana, a legally protected endangered plant, from its natural habitats, which also possesses significant beneficial medicinal properties. Therefore, production in bioreactors is the most suitable solution to protect endangered A. montana plants and produce sterile extracts for various applications.

Various techniques are employed to obtain secondary metabolites, utilizing methods such as cell suspension cultures, callus cultures, transformed organs, and whole plants. Typically, these cultures require the addition of phytohormones and involve several phases, including the selection of highly productive lines, the multiplication of plant material, and the production stage. The method we propose is relatively simple and cost-effective, not requiring hormone supplementation. Furthermore, when using tetraploid varieties of A. montana bred in the Plant Tissue Culture Laboratory and registered with COBORU (Research Center for Cultivar Testing), the efficiency of lactone biosynthesis can be significantly enhanced.