Elicitor-mediated enhancement of secondary metabolite accumulation and biological activity in Microchirita involucrata in vitro root cultures

Elicitor-mediated enhancement of secondary metabolite accumulation and biological activity in Microchirita involucrata in vitro root cultures | 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 Elicitor-mediated enhancement of secondary metabolite accumulation and biological activity in Microchirita involucrata in vitro root cultures Apinun Limmongkon, Wipaporn Chuaymaung, Pathitta Sasiri, Butsakon Nisaipham, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8636341/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Mar, 2026 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted 4 You are reading this latest preprint version Abstract Microchirita involucrata (family Gesneriaceae) has recently been reassigned to the genus Microchirita ; however, its biological properties and potential applications remain unexplored in Thailand. Although the species is valued for its compact growth habit and attractive floral morphology, its phytochemical potential has received little attention. This study established an in vitro culture platform for M. involucrata , identified explants with high secondary metabolite potential, optimized metabolite accumulation through elicitation, and evaluated the biological activities of elicited root cultures. Thin-layer chromatography (TLC) screening revealed that root tissues contained the highest number of antioxidant-active bands; therefore, in vitro –derived root segments were selected for elicitation using methyl jasmonate (MeJA), chitosan (CHT), and β-cyclodextrin (CD), applied individually and in combination (MeJA + CD and CHT + CD). CD alone and combined treatments significantly enhanced antioxidant activity. Root tissue extracts exhibited higher antioxidant capacity, whereas culture medium extracts showed markedly stronger lipoxygenase (LOX) inhibitory and antibacterial activities. Culture media derived from CD and combined elicitor treatments displayed the highest LOX inhibition, with values ranging from 72.04% to 94.35%. Among all treatments, the MeJA + CD–elicited culture medium extract demonstrated the strongest antibacterial activity, with minimum inhibitory and bactericidal concentrations of 0.78 mg/mL against Staphylococcus aureus . Against Escherichia coli , the corresponding MIC and MBC values were 1.56 and 3.13 mg/mL, respectively. Overall, these findings provide new insights into elicitor-induced secondary metabolism in M. involucrata in vitro root cultures, supporting its relevance as a source of biologically active metabolites. Microchirita involucrata in vitro culture elicitation antioxidant antibacterial lipoxygenase inhibition Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Microchirita involucrata var. capitis (Craib) C. Puglisi is a member of the family Gesneriaceae. The genus Microchirita is distributed across central and southern China, India, Laos, Vietnam, Myanmar, Malaysia, and Thailand, where species predominantly inhabit limestone ecosystems characterized by environmental stress conditions that are often associated with unique metabolic profiles. Globally, approximately 48 species of Microchirita have been documented, with about 37 species reported in Thailand (Middleton et al. 2023 ). Despite its broad geographic distribution and ecological specialization, M. involucrata remains a largely underexplored species with respect to its biological properties, metabolic potential, and biotechnological utilization. There is limited information on its phytochemical composition, metabolite biosynthesis, or suitability for controlled production systems. M. involucrata was formerly placed in the genera Chirita and Didymocarpus before its reclassification into the genus Microchirita (Middleton and Puglisi 2017 ). Several species within the genus Didymocarpus , the former taxonomic placement of M. involucrata , have been traditionally used in folk medicine for the treatment of a wide range of ailments. Reported ethnomedicinal applications include the management of kidney stones, fever, inflammation, chronic asthma, influenza, diarrhea, wound healing, and anticancer purposes (Nanjala et al. 2022 ). Phytochemical investigations in genera of the family Gesneriaceae closely related to Microchirita have revealed the presence of several notable bioactive compounds. The studies of Chirita dryas extracts identified three phenylethanoid glucosides—Chiritoside A, B, and C (Damtoft and Jensen 1994 ). Similarly, dried leaf extracts of Didymocarpus tomentosa were found to contain 25 caryophyllene-rich essential oil constituents, which displayed cytotoxic activity against HeLa cancer cells (Gowda et al. 2012 ). A study investigating leaf, stem, and rhizome extracts of Sinningia bullata , a member of the family Gesneriaceae, reported that leaves extracted with 100% acetone yielded the highest total flavonoid content. These extracts exhibited strong antioxidant activity and demonstrated antibacterial effects against several pathogenic bacteria, including Escherichia coli, Staphylococcus aureus , and Pseudomonas aeruginosa . In addition, the extracts showed potent cytotoxic effects against B16F10 melanoma cells, completely inhibiting cancer cell migration, suppressing metastasis and colony formation, and inducing apoptosis as the primary mechanism of cytotoxicity (Chen et al. 2023 ). Plant tissue culture systems within the family Gesneriaceae have been successfully established in numerous genera, driven both by the commercial importance of these species as ornamental plants and the need for reliable propagation strategies. In addition to commercial applications, tissue culture has played a significant role in the conservation of rare or endangered taxa within the family. Notable examples include the successful in vitro culture of Chirita longgangensis (Tang et al. 2007 ), Chirita flavimaculata, C. eburnea , and C. speciosa (Nakano et al. 2009 ), as well as Ramonda serbica and R. nathaliae (Gashi et al. 2015 ). However, to date, no studies have reported the establishment of in vitro tissue culture systems for species within the genus Microchirita , including M. involucrata . Elicitor-based strategies are widely recognized for their ability to enhance secondary metabolite production in plants, with the underlying mechanisms varying according to elicitor type. Jasmonic acid (JA) and its derivative methyl jasmonate (MeJA) are well-established plant signaling molecules that regulate physiological processes and activate defense pathways in response to wounding and pathogen attack. MeJA has been successfully applied as an elicitor in in vitro root cultures of Astragalus aitosensis to enhance the production of cycloartane-type saponins, including astragalosides I, II, and IV—compounds known for their antitumor and immunomodulatory activities. Following MeJA elicitation, the yield of astragaloside I increased by approximately 68% compared with the control, and the production of astragalosides II and IV was also significantly enhanced (Enchev et al. 2024 ). Chitosan (CHT), a polysaccharide derived from crustacean shells and fungal cell walls, functions as a biotic elicitor by mimicking pathogen-associated molecular patterns (PAMPs), thereby triggering plant defense responses. Elicitation of Isatis tinctoria L. hairy root cultures with 150 mg/L chitosan for 36 hours resulted in a 7.08-fold increase in total flavonoid content—including rutin, quercetin, isorhamnetin, and isoliquiritigenin—compared with the control. This enhanced accumulation corresponded with the up regulation of key genes involved in the flavonoid biosynthetic pathway (Jiao et al. 2018 ). Cyclodextrins (CDs), cyclic oligosaccharides naturally produced by certain Bacillus species, serve as abiotic elicitors and possess the unique ability to form inclusion complexes with hydrophobic metabolites. This complexation not only promotes the secretion of secondary metabolites into the culture medium but also stabilizes them by preventing degradation and feedback inhibition. Treatment of Hordeum vulgare callus cultures with 5 ppm β-cyclodextrin has been shown to enhance the accumulation of phenolic and flavonoid compounds in both black and yellow barley varieties, thereby improving their antioxidant and anti-aging properties relevant to skincare applications (Arezoumand et al. 2025 ). The objectives of this study were to establish an in vitro cultivation system for M. involucrata using biotechnological approaches and to identify plant tissues with strong potential for producing bioactive secondary metabolites. The study further aimed to enhance metabolite production through the application of various elicitation strategies. In addition, the biological activities of the resulting extracts—including antioxidant, antibacterial, and lipoxygenase inhibition properties—were systematically evaluated. To date, no such investigation has been reported, and this study represents the first systematic evaluation of M. involucrata secondary metabolism under in vitro conditions. The findings provide new insights into the species’ biological and metabolic potential. 2. Materials and methods 2.1 Plant material Seeds of Microchirita involucrata var. capitis (Craib) C. Puglisi were collected from Phang Nga Province, Thailand, and subsequently cultivated under controlled greenhouse conditions at the Department of Biology, Naresuan University. Species authentication was performed by Assistant Professor Dr. Pranee Nangngam, an expert taxonomist of Department of Biology, Naresuan University. A voucher specimen was prepared and deposited at the Phitsanulok Naresuan University (PNU) herbarium (Voucher No. PNU05867). Mature plants were harvested, and various explants—including leaves, roots, capsules, stems, and whole plants—were carefully separated and prepared for subsequent extraction procedures. 2.2 In vitro plants Fully matured seeds of M. involucrata were surface sterilized using 5% (v/v) sodium hypochlorite (NaOCl) for 15 min, followed by three rinses with sterile distilled water to remove residual disinfectant. Sterilized seeds were aseptically transferred to culture on semi-solid Murashige & Skoog (MS) (Murashige and Skoog 1962 ) basal medium solidified with 7.5 g/L agar and supplemented with 30 g/L sucrose as a carbon source. In vitro plants were cultured for 4 weeks under controlled temperature conditions (25 ± 2°C) with a 16-hours photoperiod and a light intensity of 20 µmol m⁻² s⁻¹. In vitro plantlets were routinely subcultured onto fresh MS agar medium every 4 weeks to maintain healthy growth and to obtain sufficient biomass for subsequent extraction procedures. 2.3 Root Culture and Elicitor Treatments Root cultures were initiated by excising root segments from in vitro –grown M. involucrata plantlets. Approximately 4 g of fresh root tissue was transferred into 250-mL Erlenmeyer flasks containing 50 mL of half-strength MS liquid medium supplemented with 15 g/L sucrose. Elicitation was optimized by screening a series of concentrations for each elicitor— methyl jasmonate (MeJA) at 50, 100, 150, and 200 µM; chitosan (CHT) at 50, 100, 150, and 200 mg/L; and cyclodextrin (CD) at 2, 4, 6, and 8 mM —based on their effects on antioxidant activity. The concentrations that produced the highest antioxidant responses were selected and subsequently applied as individual and combined treatments, with comparisons made against the untreated control. Single elicitor treatments included 50 µM MeJA, CHT 100 mg/L, and CD 4 mM. Combination treatments consisted of 50 µM MeJA and 4 mM CD (MeJA + CD), and 100 mg/L CHT and 4 mM CD (CHT + CD). All cultures were exposed to the optimized elicitor concentrations on a rotary shaker (150 rpm) at 25 ± 2°C under dark conditions for 72 hours. Each elicitation treatment was performed using three biological replicates. 2.4 Root and culture medium extraction For natural M. involucrata plant material, leaves, roots, capsules, stems, and whole plants were dried in a hot-air oven at 60°C for 24 hours and subsequently ground into a fine powder. The powdered material was extracted by maceration with 95% ethanol at a ratio of 1:10 (w/v) for 24 hours, and the extraction process was performed in triplicate. The combined extracts were concentrated to dryness using a rotary evaporator. For root tissue cultures, following 72 hours of elicitation, root tissues were separated from the culture medium and subjected to extraction. The tissues were dried in a hot-air oven at 60°C for 24 hours and ground into a fine powder. The dried material was weighed and extracted with methanol by maceration at a plant-to-solvent ratio of 1:10 (w/v) for 24 hours, and the extraction was repeated three times. The filtrates were pooled and evaporated to dryness using a rotary evaporator. The culture medium was extracted separately using liquid-liquid extraction with ethyl acetate at a solvent-to-medium ratio of 1:1 (v/v). The extraction step was also performed three times and the combined ethyl acetate fractions were evaporated to dryness under reduced pressure at 40°C using a rotary evaporator. The resulting crude extracts were weighed and subsequently used for further analysis. 2.5 Antioxidant activity 2.5.1 ABTS antioxidant Assay The ABTS antioxidant assay was perform according to the method described by Re et al. ( 1999 ). The ABTS • ⁺ radical cation was generated by reacting 7 mM ABTS with 140 mM potassium persulfate (K₂S₂O₈) and allowing the mixture to stand in the dark for 16 hours. The resulting ABTS • ⁺ working solution was subsequently diluted to achieve an absorbance of 0.70 ± 0.02 at 734 nm. Antioxidant activity was assessed by adding 2 µL of the sample extract to 198 µL of the ABTS • ⁺ solution, followed by incubation at room temperature for 6 minutes. The absorbance was then measured at 734 nm. Trolox equivalent antioxidant capacity (TEAC) values were calculated from a Trolox standard calibration curve and expressed as TEAC per gram dry weight. 2.5.2 FRAP antioxidant assay The ferric reducing antioxidant power (FRAP) assay was carried out following the method of Benzie and Strain ( 1996 ) with minor modifications. The FRAP working reagent consisted of 300 mmol/L acetate buffer (pH 3.6), 10 mmol/L 2,4,6-tripyridyl-s-triazine (TPTZ) dissolved in 40 mmol/L HCl, and 20 mmol/L FeCl₃, mixed in a 10:1:1 proportion. For each reaction, 2 µL of sample extract was added to 198 µL of the freshly prepared FRAP reagent. The mixture was incubated at room temperature for 5 minutes, and the absorbance of the resulting Fe²⁺–TPTZ complex was measured at 593 nm. Antioxidant capacity was quantified using a standard curve prepared with ascorbic acid, and results were expressed as ascorbic acid equivalents (AAE) per gram dry weight. 2.6 Lipoxygenase Inhibition Assay The lipoxygenase (LOX) inhibition assay was performed by incubating the extract with a reaction mixture containing 10 mM phosphate buffer (pH 8.0) and 15-lipoxygenase enzyme (34,160 U/mL). After a 5-minutes pre-incubation at room temperature, the reaction was initiated by adding linoleic acid substrate (2,230 µM). Enzyme activity was monitored kinetically by measuring the increase in absorbance at 234 nm every 15 seconds for 3 minutes. The percentage of LOX inhibition was calculated by comparing the reaction rate (ΔA/min) of the sample with that of the solvent control using the following equation: 2.7 Thin layer chromatography (TLC) The compound profiles were screened using TLC. Crude extracts were spotted onto TLC silica gel 60 F₂₅₄ plates (Merck Millipore, Germany), and the plates were developed using a mobile phase of hexane: ethyl acetate (2.1:0.9, v/v). After development, the TLC chromatograms were visualized under UV light at 254 and 365 nm, followed by anisaldehyde staining with gentle heating. Antioxidant-active bands were further detected by spraying with DPPH solution. 2.8 Antimicrobial Activity Assay Antimicrobial activity was evaluated against two bacterial strains: Staphylococcus aureus TISTR 1466 (ATCC6538) and Escherichia coli TISTR 887 (ATCC 25922) which were obtained from Thailand institute of scientific and technological research culture collection (TISTR). 2.8.1 Disk Diffusion Assay Bacterial strains were cultured on Mueller–Hinton agar (MHA) plates and incubated at 37°C for 12–16 hours. A single colony from each bacterial species was suspended in sterile normal saline and adjusted to 0.5 McFarland standard. The bacterial suspension was swabbed onto the surface of MHA plates. The sterile paper disks (6-mm) were impregnated with the extract and placed on the bacterial plates, which were subsequently incubated at 37°C for 12–16 hours. The antimicrobial activity was assessed by measuring the diameter of the inhibition zone. 2.8.2 Determination of Minimum Inhibitory Concentration (MIC) Bacterial cultures were prepared in Mueller–Hinton broth (MHB) and adjusted to 0.5 McFarland standard. Extract samples were added to a 96-well plate and subjected to twofold serial dilution in MHB. All assays were performed in triplicate. The bacterial suspension was then added to each well, and the plate was incubated at 37°C for 12–16 hours. After incubation, resazurin solution was added to each well, followed by an additional 3-hours incubation at 37°C. Changes in resazurin color were monitored to determine bacterial viability. Wells remaining blue indicated the absence of bacterial growth, and the lowest concentration producing no color change was recorded as the MIC. Wells turning pink indicated bacterial growth. 2.8.3 Determination of Minimum Bactericidal Concentration (MBC) To determine the MBC, aliquots from wells showing no resazurin color change were dropped onto MHA plates. The plates were incubated at 37°C for 12–16 hours and examined for bacterial growth. The lowest concentration of extract that produced no visible bacterial colonies was recorded as the MBC. 2.9 Statistical analysis Statistical analysis was conducted using one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) post hoc test in IBM SPSS Statistics software (version 23). Data are presented as the mean ± standard deviation (SD) from three independent biological replicates, with significance defined at p < 0.05. 3. Results 3.1 Thin-layer chromatography ( TLC) profiling and antioxidant activity screening Natural M. involucrata plant materials collected from the controlled greenhouse, including flowers, roots, stems, capsules, leaves, and whole plants, were dried and individually extracted using 95% ethanol as demonstrated in Fig. 1 . Preliminary chemical profiling of the resulting extracts was performed by TLC. Each extract was applied to TLC plates, and chromatograms were visualized under UV illumination at 254 nm (Fig. 2 a) and 365 nm (Fig. 2 b). Distinct TLC banding patterns were observed among the different plant parts, with the root extract exhibiting a markedly unique profile compared to the other explants. Preliminary antioxidant screening was conducted by DPPH staining of the TLC plates (Fig. 2 c). The assay is based on the discoloration of the purple DPPH radical to yellow upon interaction with antioxidant constituents. Accordingly, bands displaying a purple-to-yellow transition after chromatographic separation were considered indicative of antioxidant activity. Among all tested plant parts, the root extract demonstrated the highest number and most intense of antioxidant-active bands, highlighting its potential as a rich source of bioactive compounds (Fig. 2 c). 3.2 Comparative TLC profiling of natural and in vitro –derived roots The preliminary findings indicated that the root extract was particularly promising, as it displayed the highest number of antioxidant-active bands. Accordingly, M. involucrata seeds were subjected to in vitro propagation under sterile laboratory conditions using tissue culture techniques. Notably, the in vitro culture system exhibited extensive root development compared to natural M. involucrata plant, as shown in Fig. 3 a-b. Root tissues obtained from the in vitro cultures were subsequently extracted and subjected to TLC analysis, which was compared with the TLC profile of roots from natural plants. The TLC fingerprints of both natural roots and in vitro –derived root cultures showed similar banding patterns when visualized under UV light at 254 nm (Fig. 3 c) and following DPPH staining (Fig. 3 d), indicating comparable phytochemical characteristics and antioxidant-active constituents. 3.3 Root characteristics and culture medium changes in response to elicitor treatments The optimal concentrations of methyl jasmonate (MeJA), cyclodextrin (CD), and chitosan (CHT) were selected based on preliminary screening experiments designed to identify the elicitation conditions that produced the highest antioxidant activity (Supplementary Table S1). The effects of the individual concentration ranges—MeJA at 50, 100, 150, and 200 µM; CD at 2, 4, 6, and 8 mM; and CHT at 50, 100, 150, and 200 mg/L—are presented in Supplementary Figure S1-S3. In this study, root elicitation was carried out using the optimal concentrations of individual elicitors, including 50 µM MeJA, 4 mM CD, and 100 mg/L CHT, as well as combined treatments consisting of 50 µM MeJA with 4 mM CD (MeJA + CD) and 100 mg/L CHT with 4 mM CD (CHT + CD). Root characteristics and culture medium of the control and elicitor-treated samples are presented in Fig. 4 . After 72 hours of elicitation, all culture medium exhibited noticeable color changes. The culture medium from the single CD treatment and the combined MeJA + CD treatment developed a pronounced bright yellow coloration, whereas the culture medium from the single CHT and the combined CHT + CD treatments turned dark brown. The coloration of the root tissues corresponded closely with the observed color changes in their respective culture medium. 3.4 TLC profiles of elicited root and culture medium extract Root tissues and culture medium from the control and elicitor-treated samples were separately extracted to obtain crude extracts. Crude extract yields from root tissues were relatively consistent across all treatments, including the control, ranging from 43 to 62 mg. In contrast, extract yields from the culture medium differed markedly among treatments, ranging from 2.3 to 13.7 mg. The single CD treatment and the combined elicitor treatments produced the highest yields, each generating approximately 13 mg of extract. Lower yields were obtained from the single MeJA and single CHT treatments. As expected, the control culture medium yielded only 1.9 mg of crude extract, the lowest amount among all samples. The TLC profiles of both the root tissue extracts and the corresponding culture medium extracts are shown in Fig. 5 . The TLC patterns of root tissue extracts were generally similar across all treatments, including the control (Fig. 5 a). In contrast, the TLC profiles of the culture medium extracts displayed clear variation between the control and each elicitor treatment (Fig. 5 b). The control medium exhibited fewer detectable bands compared to the elicited samples when visualized under UV 254 nm, UV 365 nm, and following anisaldehyde staining. Notably, more intense bands were observed in the combined elicitor treatments (MeJA + CD and CHT + CD) compared with the single-elicitor treatments (MeJA, CD, and CHT) (Fig. 5 b). Anisaldehyde staining further revealed elicitor-specific characteristic bands: a prominent purple band was observed in the CD-treated sample, while a notable brown band appeared in the MeJA-treated sample (Fig. 5 b, red arrows). These characteristic bands were absent in the CHT-treated sample. Interestingly, the combined MeJA + CD treatment exhibited both the prominent purple and brown bands, whereas the CHT + CD combination displayed only the prominent purple band (Fig. 5 b, red arrows). 3.5 Antioxidant activity of elicited root and culture medium extract The antioxidant activity of the root tissue and culture medium extracts, evaluated using the ABTS assay, demonstrated that root tissue extracts consistently exhibited higher antioxidant capacity than the corresponding culture medium extracts (Fig. 6 a and Supplementary Table S2). A similar trend in antioxidant response was observed across both extract types. Root tissue extracts showed activities ranging from 29.53 to 69.00 µmol Trolox/g dry weight, whereas culture medium extracts ranged from 0.40 to 7.20 µmol Trolox/g dry weight. In both datasets, the single MeJA and single CHT treatments yielded relatively low antioxidant activity. In contrast, treatment with single CD, as well as the combined CHT + CD and MeJA + CD treatments resulted in significantly higher antioxidant activity compared to the unelicited control. The FRAP assay revealed an antioxidant response pattern consistent with that observed in the ABTS assay. Root tissue extracts exhibited substantially higher antioxidant activity than the corresponding culture medium extracts (Fig. 6 b and Supplementary Table S2). The FRAP values of the tissue extracts ranged from 7.56 to 16.59 mg ascorbic acid equivalents (AAE)/g dry weight, whereas those of the culture medium extracts ranged from 0.11 to 1.75 mg AAE/g dry weight. In both extract types, the single MeJA treatment yielded the lowest antioxidant activity. In contrast, the single CD treatment and the combined MeJA + CD and CHT + CD treatments produced markedly higher AAE values, all of which were significantly greater than those of the control group. 3.6 Lipoxygenase inhibition assay of elicited root and culture medium extract Root tissue and culture medium extracts were evaluated for lipoxygenase (LOX) inhibitory activity using linoleic acid as the substrate for the 15-LOX enzyme reaction. Crude extracts were tested at a uniform concentration of 0.5 mg/mL, and the percentage inhibition was calculated following extract addition to the assay system. As shown in Fig. 7 , all root tissue extracts exhibited less than 50% LOX inhibition. Interestingly, the culture medium extracts displayed a markedly different inhibition pattern. Minimal LOX inhibition (3–5%) was observed in the control and in the single MeJA- and CHT-treated samples. In contrast, substantially higher inhibition was detected in the culture medium of samples treated with single CD and with the combined CHT + CD and MeJA + CD treatments, yielding inhibition percentages of 72.04 ± 6.71%, 82.05 ± 8.41%, and 94.35 ± 4.41%, respectively (Supplementary Table S3). These findings indicate that elicitation with single CD or the combined CHT + CD and MeJA + CD treatments promote the release of LOX-inhibitory bioactive compounds into the culture medium. 3.7 Antibacterial assay of elicited root and culture medium extract Antibacterial activity was evaluated using the disk diffusion assay. A total of 1 mg of crude extract from both root tissues and culture medium was applied to sterile disks and tested against the Gram-positive bacterium S. aureus and the Gram-negative bacterium E. coli . The diameters of the inhibition zones were measured, and the 6-mm paper disk diameter was subtracted to determine the antibacterial potency. As shown in Fig. 8 and Table 1 , the root tissue extracts produced relatively small inhibition zones compared to the corresponding culture medium extracts for both S. aureus and E. coli . Enhanced antibacterial activity was observed in the culture medium of elicited samples, with inhibition zones of 8–12 mm for S. aureus and 7.33–13.67 mm for E. coli , compared with the untreated control. Notably, the combination treatment MeJA + CD produced the largest inhibition zones for both S. aureus (12.00 ± 2.06 mm) and E. coli (13.67 ± 0.58 mm), surpassing all single-elicitor treatments. Table 1 Antibacterial activity of Microchirita involucrata root tissue and culture medium crude extracts against Staphylococcus aureus and Escherichia coli following elicitation with individual elicitors (CD, MeJA, and CHT) and combined treatments (CHT + CD and MeJA + CD), compared with the unelicited control. Disk diffusion results are reported as the diameter of the clear zone minus the 6-mm disk diameter. Inhibition zone values are presented as mean ± SD (n = 3). MIC denotes the minimum inhibitory concentration, and MBC denotes the minimum bactericidal concentration. ND indicates not detected. Sample type Elicitors S. aureus E. Coli Inhibition zone (mm) MIC (mg/mL) MBC (mg/mL) Inhibition zone (mm) MIC (mg/mL) MBC (mg/mL) Tissue Control 7.35 ± 1.26 ND ND 4.25 ± 1.23 ND ND 4 mM CD 4.33 ± 2.15 ND ND 4.64 ± 0.64 ND ND 50 µM MeJA 4.25 ± 1.32 ND ND ND ND ND 100 mg/L CHT 5.58 ± 1.62 ND ND 2.65 ± 0.40 ND ND 100 mg/L CHT + 4 mM CD 2.32 ± 0.46 ND ND 1.80 ± 0.65 ND ND 50 µM MeJA + 4 mM CD 7.50 ± 1.52 ND ND 5.54 ± 1.48 ND ND Medium Control 7.00 ± 1.54 6.25 > 25 6.00 ± 1.32 6.25 > 25 4 mM CD 11.00 ± 1.08 3.13 12.5 10.33 ± 2.08 3.13 6.25 50 µM MeJA 8.00 ± 3.05 3.13 > 25 9.33 ± 1.15 6.25 > 25 100 mg/L CHT 10.67 ± 0.62 3.13 > 25 9.67 ± 1.53 6.25 > 25 100 mg/L CHT + 4 mM CD 8.33 ± 0.68 3.13 12.5 7.33 ± 2.31 3.13 12.5 50 µM MeJA + 4 mM CD 12.00 ± 2.06 0.78 0.78 13.67 ± 0.58 1.56 3.13 Given the higher antibacterial activity observed in the culture medium extracts, only these samples were further evaluated using the broth microdilution method to determine the minimum inhibitory concentration (MIC), followed by the minimum bactericidal concentration (MBC). As shown in Table 1 , elicitor-treated samples exhibited lower MIC values against S. aureus compared with the untreated control. Notably, the combined MeJA + CD treatment exhibited the greatest antibacterial potency, presenting the lowest MIC (0.78 mg/mL) and MBC (0.78 mg/mL) among all treatments. For E. coli , the single CD treatment as well as the combined CHT + CD and MeJA + CD treatments produced lower MIC and MBC values than the control. Consistent with the results for S. aureus , the MeJA + CD combination showed the strongest activity against E. coli , with MIC and MBC values of 1.56 mg/mL and 3.13 mg/mL, respectively, representing the lowest among all tested samples. 4. Discussion Microchirita involucrata var. capitis (Craib) C. Puglisi was reclassified into the genus Microchirita along with species formerly placed in the genera Chirita and Didymocarpus . To date, no bioactivity studies have been reported specifically for M. involucrata . However, numerous studies on species within its former genera have documented diverse biological activities, including antioxidant, antimicrobial, and anti-inflammatory properties (Kindo et al. 2014 ; Nanjala et al. 2022 ). Traditionally, plants from these groups have been widely used as herbal remedies for various conditions such as allergic skin disorders, inflammation, fever, diabetes, and several other ailments (Bhongade et al. 2021 ). In this study, we examined the TLC profiles of ethanol extracts prepared from different parts of M. involucrata , including leaves, flowers, roots, capsules, and stems. The results demonstrated that the root extract displayed the most distinct TLC fingerprint, clearly differentiating it from the other plant parts. Notably, the root extract also exhibited the most intense antioxidant-active bands following DPPH staining, indicating a higher abundance of antioxidant constituents. Roots have frequently been highlighted as important reservoirs of bioactive phytochemicals. For example, qualitative and quantitative analyses of Calotropis gigantea and C. procera roots revealed that flavonoids, alkaloids, tannins, and phenolic compounds are the predominant constituents, contributing to their extensive use in traditional medicine (Mishra et al. 2018 ). Similarly, the roots of Marsdenia macrantha , traditionally used to treat mouth infections, constipation, and urinary retention, were found to contain abundant flavonoids and alkaloids with demonstrated antibacterial activity against foodborne pathogens (Shikwambi et al. 2021 ). As underground organs continually exposed to soilborne pathogens and environmental stressors, roots often accumulate a wide variety of defensive phytochemicals synthesized to protect the plant. Consequently, root-based defense mechanisms play a crucial role in plant survival against the diverse array of belowground attackers (Touw and van Dam 2025 ). The initial screening indicated that the root extract was the most promising sample, exhibiting the highest number of antioxidant-active bands. To generate sufficient root biomass for further analysis, an in vitro tissue culture system for M. involucrata was established—representing the first report of sterile tissue culture propagation for this species within the genus Microchirita . The in vitro cultures produced extensive root formation, and the TLC profiles of the in vitro –derived roots closely resembled those of roots collected from natural plants. These findings demonstrate that in vitro root cultures of M. involucrata are capable of producing phytochemical patterns comparable to naturally grown roots. These findings are consistent with previous reports demonstrating that adventitious root cultures can produce secondary metabolite profiles comparable to those of naturally grown roots (Khanam et al. 2022 ; Reis et al. 2011 ). The application of biotechnology to root tissue culture is therefore crucial for the rapid and large-scale production of root biomass under controlled laboratory conditions. The production of secondary metabolites in root cultures can be substantially enhanced through the application of elicitors that simulate biotic or abiotic stress, thereby triggering the plant’s defensive metabolic pathways. A wide range of elicitors has been utilized depending on species-specific responses, including methyl jasmonate, chitosan, salicylic acid, and certain heavy metals (Rahmat and Kang 2019 ). Appropriate selection of elicitors and optimization of elicitation conditions can markedly increase the accumulation and yield of secondary metabolites in root tissue cultures. In this study, root elicitation of M. involucrata was performed by excising root segments from in vitro –grown plantlets and cultivating them in liquid medium supplemented with various concentrations of single elicitors, including MeJA, CHT, and CD. Optimal concentrations for each elicitor were selected based on antioxidant activity screening of both root tissue and culture medium extracts in comparison with the unelicited control. Because CD treatment produced high antioxidant activity in both tissue and medium extracts, CD was subsequently combined with MeJA or CHT to evaluate the effects of the combined treatments (MeJA + CD and CHT + CD). TLC fingerprinting revealed that the root tissue extracts displayed similar banding patterns across all treatments and the control, whereas the culture medium extracts showed clear and distinct variations among treatments (Fig. 5 ). More intense and diverse TLC patterns were observed in the combined elicitor treatments (MeJA + CD and CHT + CD) compared with the single-elicitor treatments. Notably, anisaldehyde staining revealed elicitor-specific characteristic bands, indicating that each elicitor induced the synthesis and secretion of particular metabolites into the culture medium (Fig. 5 b). These metabolite-specific patterns corresponded with the visible color changes in the culture medium following elicitation. Overall, while root tissue extracts maintained similar profiles across treatments, the culture medium exhibited distinct TLC patterns reflective of the different elicitation conditions. The application of elicitor-based strategies can enhance the synthesis of secondary metabolites during in vitro culture. In addition to enhancing the synthesis and accumulation of secondary metabolites, elicitor application in the culture medium has also been reported to stimulate the efflux of intracellular metabolites (Wang et al. 2025 ). This promotes the release of specific compounds into the culture medium, which can be observed as distinct differences in TLC profiles following elicitor treatment. Furthermore, several studies have demonstrated that the application of combined elicitors can elicit synergistic responses, resulting in greater secondary metabolite accumulation compared with single-elicitor treatments. This enhanced production is generally attributed to elicitor-induced signaling crosstalk, which leads to stronger activation and upregulation of the relevant biosynthetic pathways. Such synergistic action was observed in adventitious roots of Primula veris , where the combined application of L-proline and jasmonic acid resulted in significant increases in phenolic acid, glycoside, and saponin accumulation. These findings highlight the importance of strategic elicitor selection and that appropriate elicitation can substantially enhance secondary metabolite production of the in vitro culture systems (Sarropoulou et al. 2023 ). Treatment with single CD, as well as the combined CHT + CD and MeJA + CD elicitors, resulted in significantly higher antioxidant activity compared with the unelicited control. Antioxidant evaluation further revealed that root tissue extracts consistently exhibited greater antioxidant capacity than the corresponding culture medium extracts. In contrast, the LOX inhibition assay—reflecting in vitro anti-inflammatory potential—and the antibacterial assay showed markedly higher activity in the culture medium extracts than in the tissue extracts. This divergence is likely attributable to differential metabolite localization and elicitor-induced metabolite secretion. Antioxidant compounds are typically retained within plant tissues, where they provide intracellular protection against oxidative stress, thereby contributing to the higher antioxidant activity observed in tissue extracts (Shen et al. 2022 ). Conversely, elicitors can selectively promote the efflux of particular secondary metabolites into the culture medium. Cyclodextrin, in particular, enhances the secretion and stabilization of hydrophobic defense-related metabolites by forming inclusion complexes, leading to their extracellular accumulation (Farhadi et al. 2020 ). These secreted metabolites, therefore, contribute more strongly to LOX inhibitory and antibacterial activities in the culture medium. Such differential metabolite distribution suggests that elicitation not only stimulates biosynthesis but also regulates metabolite compartmentalization, producing distinct biological activity profiles between tissues and their culture medium (Massalha et al. 2017 ). Similar phenomena have been reported in Arabidopsis, where the volatile homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) functions as a defensive metabolite against the root rot pathogen Pythium irregulare . DMNT, released as a breakdown product of arabidiol, was shown to reduce oospore germination during early infection stages (Sohrabi et al. 2017 ). Given the diverse bioactivities observed in the culture medium extracts beyond antioxidant activity, these findings suggest promising potential for further exploration of the bioactive compounds produced and secreted by M. involucrata in response to elicitor treatment. Future studies should focus on the isolation and structural characterization of the metabolites responsible for the observed biological activities, enabling direct links between specific secondary metabolites and their functional roles. 5. Conclusion This study establishes, for the first time, a controlled in vitro root culture and elicitation system for Microchirita involucrata and provides new insights into its secondary metabolic capacity. Root tissues were identified as the most metabolically responsive explant, exhibiting pronounced antioxidant-associated chemical profiles. Targeted elicitation using cyclodextrin (CD), either alone or in combination with methyl jasmonate (MeJA) or chitosan (CHT), significantly enhanced secondary metabolite accumulation and associated biological activities. Distinct tissue- and compartment-specific responses were observed, with root extracts showing higher antioxidant capacity, whereas culture medium extracts displayed markedly stronger lipoxygenase-inhibitory and antibacterial activities. These findings indicate that elicitation not only activates intracellular secondary metabolic pathways but also promotes the extracellular secretion of metabolites into the culture medium. The establishment of a reproducible in vitro root culture platform, together with elicitor-driven metabolic modulation, provides a robust experimental framework for future studies focused on metabolite isolation, structural elucidation, and the investigation of regulatory mechanisms underlying secondary metabolite biosynthesis and secretion in M. involucrata . Collectively, this work expands current understanding of elicitor-mediated secondary metabolism in in vitro root systems and highlights M. involucrata as a valuable model for studying metabolite regulation and secretion dynamics in Gesneriaceae species. Declarations The authors have no competing interests to declare that are relevant to the content of this article. Ethics declaration Not applicable Author contribution statement Apinun Limmongkon : Conceptualization, Supervision, Visualization, Data curation, Writing – original draft. Wipaporn Chuaymaung : Methodology, Formal analysis, Validation. Pathitta Sasiri : Methodology, Formal analysis, Validation. Butsakon Nisaipham : Methodology, Formal analysis, Validation. Sirianong Khongwet : Methodology, Formal analysis, Validation. Onrut Sapatee : Visualization, Investigation. Thanakorn Wongsa : Conceptualization, Visualization. Anupan Kongbangkerd : Conceptualization, Resources, Writing – review and editing. Wannapa Khanthit : Methodology, Formal analysis. Sirinan Temwong : Methodology, Formal analysis. Arpassara Maliprom : Methodology, Formal analysis. Wuthinant Piriyanupong : Methodology, Formal analysis. Data availability statement The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Funding sources This work was supported by Naresuan University (NU) and the National Science, Research and Innovation Fund (NSRF) [grant number R2569B087]. References Arezoumand E, Bagheri K, Mazloum S, Noh GM, Hamishehkar H, Kosari-Nasab M, Kim KH (2025) β-Cyclodextrin as an elicitor of polyphenolic contents of barley (Hurdeum vulgare) callus with antioxidant and anti-aging properties on human skin fibroblast cells (HFF2). 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Front Microbiol 16:1558567. 10.3389/fmicb.2025.1558567 Supplementary Files Supplementarydata.pdf Cite Share Download PDF Status: Published Journal Publication published 30 Mar, 2026 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted Reviewers agreed at journal 22 Jan, 2026 Reviewers invited by journal 22 Jan, 2026 Editor assigned by journal 22 Jan, 2026 First submitted to journal 20 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-8636341","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":578773059,"identity":"bbe0bcb8-205e-40e8-a883-9c40e6df157d","order_by":0,"name":"Apinun 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University","correspondingAuthor":false,"prefix":"","firstName":"Wuthinant","middleName":"","lastName":"Piriyanupong","suffix":""}],"badges":[],"createdAt":"2026-01-19 08:31:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8636341/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8636341/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11240-026-03437-8","type":"published","date":"2026-03-30T15:59:56+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":101345556,"identity":"bbf75cf4-f7e7-40cd-9090-ab5cec6063bd","added_by":"auto","created_at":"2026-01-28 17:03:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4217960,"visible":true,"origin":"","legend":"\u003cp\u003eNatural \u003cem\u003eM. involucrata\u003c/em\u003e plant materials. (a) Plant parts including flowers, roots, stems, capsules, leaves, and whole plants; (b) dried plant materials; and (c) solvent extracts obtained following maceration with 95% ethanol.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8636341/v1/237711e095f06762172ba987.png"},{"id":101345555,"identity":"6422eb7a-a5d6-4ebd-b62d-e1f29edaa952","added_by":"auto","created_at":"2026-01-28 17:03:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1788732,"visible":true,"origin":"","legend":"\u003cp\u003eChemical profiling of natural \u003cem\u003eM. involucrata\u003c/em\u003eplant parts—including flowers, roots, stems, capsules, leaves, and whole plants—using TLC. Chromatograms were visualized under (a) UV at 254 nm, (b) UV at 365 nm, and (c) DPPH staining for antioxidant screening.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8636341/v1/11e9f87e326a8079775932a0.png"},{"id":101345564,"identity":"e81f9c53-2707-4b01-ab84-e79c0dcedb52","added_by":"auto","created_at":"2026-01-28 17:03:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7944021,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Natural \u003cem\u003eM. involucrata\u003c/em\u003eplant; (b) \u003cem\u003ein vitro\u003c/em\u003e–cultured \u003cem\u003eM. involucrata\u003c/em\u003e plant; and TLC fingerprints comparing natural roots and \u003cem\u003ein vitro\u003c/em\u003e–derived root cultures visualized under (c) UV 254 nm and (d) DPPH staining for antioxidant screening.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8636341/v1/c87a9af59c205bb333a2c08a.png"},{"id":101345560,"identity":"3c2cb3b8-8b08-4bee-8826-0f923c3621cd","added_by":"auto","created_at":"2026-01-28 17:03:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4943642,"visible":true,"origin":"","legend":"\u003cp\u003eRoot morphology and culture medium appearance of control and elicitor-\u003cem\u003etreated M. involucrata\u003c/em\u003e root cultures at Day 0 and Day 3. Following elicitation, the culture medium was separated from the root tissues for subsequent extraction. Scale bar = 2 cm.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8636341/v1/f182d509cce09de449da1221.png"},{"id":101345561,"identity":"c99f64dc-d40c-4764-aa8f-4ef0a7016bcf","added_by":"auto","created_at":"2026-01-28 17:03:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4026398,"visible":true,"origin":"","legend":"\u003cp\u003eTLC profiles of (a) root tissue extracts and (b) the corresponding culture medium extracts from \u003cem\u003eM. involucrate\u003c/em\u003eroot cultures. Samples include the control, CD-treated, MeJA-treated, CHT-treated, MeJA + CD–treated, and CHT + CD–treated groups. TLC plates were visualized under UV 254 nm, UV 365 nm, and anisaldehyde staining.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8636341/v1/fc2bc78ecacd056890cd6a67.png"},{"id":101345563,"identity":"807b82b6-ed3a-4b49-b98d-5bec4fa200ed","added_by":"auto","created_at":"2026-01-28 17:03:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":809240,"visible":true,"origin":"","legend":"\u003cp\u003eAntioxidant activity of \u003cem\u003eM. involucrata\u003c/em\u003e root cultures elicited with individual elicitors (CD, MeJA, and CHT) and combined treatments (CHT + CD and MeJA + CD), compared with the unelicited control. Antioxidant capacity was assessed using (a) the ABTS assay and (b) the FRAP assay. Green bars represent root tissue extracts, and yellow bars represent culture medium extracts. Different lowercase letters indicate statistically significant differences (p \u0026lt; 0.05) among root tissue extracts, whereas different uppercase letters indicate statistically significant differences (p \u0026lt; 0.05) among culture medium extracts.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8636341/v1/178b4f3bed1109c784b153a9.png"},{"id":101397835,"identity":"6a563df6-bbb6-4814-81a7-a7fdda22031f","added_by":"auto","created_at":"2026-01-29 09:37:29","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1239224,"visible":true,"origin":"","legend":"\u003cp\u003eLipoxygenase inhibition activity of \u003cem\u003eM. involucrata\u003c/em\u003e root cultures elicited with individual elicitors (CD, MeJA, and CHT) and combined treatments (CHT + CD and MeJA + CD), compared with the unelicited control. Blue bars represent root tissue extracts, and orange bars represent culture medium extracts. Different lowercase letters indicate statistically significant differences (p \u0026lt; 0.05) among root tissue extracts, while different uppercase letters indicate statistically significant differences (p \u0026lt; 0.05) among culture medium extracts.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8636341/v1/cc2cbd19978e4902fe205ad4.png"},{"id":101345562,"identity":"d7a5d5b3-7578-44fc-9106-816ec53c005c","added_by":"auto","created_at":"2026-01-28 17:03:36","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":9083026,"visible":true,"origin":"","legend":"\u003cp\u003eAntibacterial activity of \u003cem\u003eM. involucrata\u003c/em\u003eroot tissue and culture medium crude extracts against a) \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and b) \u003cem\u003eEscherichia coli\u003c/em\u003e following elicitation with individual elicitors (CD, MeJA, and CHT) and combined treatments (CHT + CD and MeJA + CD), compared with the unelicited control. DMSO was used as a negative control. The paper disk diameter was 6 mm.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-8636341/v1/50fe2d20f18f9a59e15976a7.png"},{"id":106344286,"identity":"95bdd40f-d0f5-4770-a20b-b585121d04b9","added_by":"auto","created_at":"2026-04-07 16:13:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":40297584,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8636341/v1/a774fd5f-f80c-403b-83cb-630fcc2bfde0.pdf"},{"id":101345558,"identity":"a1d97870-3411-460b-86c2-a8885797b2bf","added_by":"auto","created_at":"2026-01-28 17:03:36","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":895329,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8636341/v1/574b83643e8dc4918cdcb239.pdf"}],"financialInterests":"","formattedTitle":"Elicitor-mediated enhancement of secondary metabolite accumulation and biological activity in Microchirita involucrata in vitro root cultures","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cem\u003eMicrochirita involucrata var. capitis\u003c/em\u003e (Craib) C. Puglisi is a member of the family Gesneriaceae. The genus \u003cem\u003eMicrochirita\u003c/em\u003e is distributed across central and southern China, India, Laos, Vietnam, Myanmar, Malaysia, and Thailand, where species predominantly inhabit limestone ecosystems characterized by environmental stress conditions that are often associated with unique metabolic profiles. Globally, approximately 48 species of \u003cem\u003eMicrochirita\u003c/em\u003e have been documented, with about 37 species reported in Thailand (Middleton et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Despite its broad geographic distribution and ecological specialization, \u003cem\u003eM. involucrata\u003c/em\u003e remains a largely underexplored species with respect to its biological properties, metabolic potential, and biotechnological utilization. There is limited information on its phytochemical composition, metabolite biosynthesis, or suitability for controlled production systems.\u003c/p\u003e \u003cp\u003e \u003cem\u003eM. involucrata\u003c/em\u003e was formerly placed in the genera \u003cem\u003eChirita\u003c/em\u003e and \u003cem\u003eDidymocarpus\u003c/em\u003e before its reclassification into the genus \u003cem\u003eMicrochirita\u003c/em\u003e (Middleton and Puglisi \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Several species within the genus \u003cem\u003eDidymocarpus\u003c/em\u003e, the former taxonomic placement of \u003cem\u003eM. involucrata\u003c/em\u003e, have been traditionally used in folk medicine for the treatment of a wide range of ailments. Reported ethnomedicinal applications include the management of kidney stones, fever, inflammation, chronic asthma, influenza, diarrhea, wound healing, and anticancer purposes (Nanjala et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Phytochemical investigations in genera of the family Gesneriaceae closely related to \u003cem\u003eMicrochirita\u003c/em\u003e have revealed the presence of several notable bioactive compounds. The studies of \u003cem\u003eChirita dryas\u003c/em\u003e extracts identified three phenylethanoid glucosides\u0026mdash;Chiritoside A, B, and C (Damtoft and Jensen \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Similarly, dried leaf extracts of \u003cem\u003eDidymocarpus tomentosa\u003c/em\u003e were found to contain 25 caryophyllene-rich essential oil constituents, which displayed cytotoxic activity against HeLa cancer cells (Gowda et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). A study investigating leaf, stem, and rhizome extracts of \u003cem\u003eSinningia bullata\u003c/em\u003e, a member of the family Gesneriaceae, reported that leaves extracted with 100% acetone yielded the highest total flavonoid content. These extracts exhibited strong antioxidant activity and demonstrated antibacterial effects against several pathogenic bacteria, including \u003cem\u003eEscherichia coli, Staphylococcus aureus\u003c/em\u003e, and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e. In addition, the extracts showed potent cytotoxic effects against B16F10 melanoma cells, completely inhibiting cancer cell migration, suppressing metastasis and colony formation, and inducing apoptosis as the primary mechanism of cytotoxicity (Chen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePlant tissue culture systems within the family Gesneriaceae have been successfully established in numerous genera, driven both by the commercial importance of these species as ornamental plants and the need for reliable propagation strategies. In addition to commercial applications, tissue culture has played a significant role in the conservation of rare or endangered taxa within the family. Notable examples include the successful \u003cem\u003ein vitro\u003c/em\u003e culture of \u003cem\u003eChirita longgangensis\u003c/em\u003e (Tang et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), \u003cem\u003eChirita flavimaculata, C. eburnea\u003c/em\u003e, and \u003cem\u003eC. speciosa\u003c/em\u003e (Nakano et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), as well as \u003cem\u003eRamonda serbica\u003c/em\u003e and \u003cem\u003eR. nathaliae\u003c/em\u003e (Gashi et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). However, to date, no studies have reported the establishment of \u003cem\u003ein vitro\u003c/em\u003e tissue culture systems for species within the genus \u003cem\u003eMicrochirita\u003c/em\u003e, including \u003cem\u003eM. involucrata\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e Elicitor-based strategies are widely recognized for their ability to enhance secondary metabolite production in plants, with the underlying mechanisms varying according to elicitor type. Jasmonic acid (JA) and its derivative methyl jasmonate (MeJA) are well-established plant signaling molecules that regulate physiological processes and activate defense pathways in response to wounding and pathogen attack. MeJA has been successfully applied as an elicitor in \u003cem\u003ein vitro\u003c/em\u003e root cultures of \u003cem\u003eAstragalus aitosensis\u003c/em\u003e to enhance the production of cycloartane-type saponins, including astragalosides I, II, and IV\u0026mdash;compounds known for their antitumor and immunomodulatory activities. Following MeJA elicitation, the yield of astragaloside I increased by approximately 68% compared with the control, and the production of astragalosides II and IV was also significantly enhanced (Enchev et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Chitosan (CHT), a polysaccharide derived from crustacean shells and fungal cell walls, functions as a biotic elicitor by mimicking pathogen-associated molecular patterns (PAMPs), thereby triggering plant defense responses. Elicitation of \u003cem\u003eIsatis tinctoria\u003c/em\u003e L. hairy root cultures with 150 mg/L chitosan for 36 hours resulted in a 7.08-fold increase in total flavonoid content\u0026mdash;including rutin, quercetin, isorhamnetin, and isoliquiritigenin\u0026mdash;compared with the control. This enhanced accumulation corresponded with the up regulation of key genes involved in the flavonoid biosynthetic pathway (Jiao et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Cyclodextrins (CDs), cyclic oligosaccharides naturally produced by certain Bacillus species, serve as abiotic elicitors and possess the unique ability to form inclusion complexes with hydrophobic metabolites. This complexation not only promotes the secretion of secondary metabolites into the culture medium but also stabilizes them by preventing degradation and feedback inhibition. Treatment of \u003cem\u003eHordeum vulgare\u003c/em\u003e callus cultures with 5 ppm β-cyclodextrin has been shown to enhance the accumulation of phenolic and flavonoid compounds in both black and yellow barley varieties, thereby improving their antioxidant and anti-aging properties relevant to skincare applications (Arezoumand et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe objectives of this study were to establish an \u003cem\u003ein vitro\u003c/em\u003e cultivation system for \u003cem\u003eM. involucrata\u003c/em\u003e using biotechnological approaches and to identify plant tissues with strong potential for producing bioactive secondary metabolites. The study further aimed to enhance metabolite production through the application of various elicitation strategies. In addition, the biological activities of the resulting extracts\u0026mdash;including antioxidant, antibacterial, and lipoxygenase inhibition properties\u0026mdash;were systematically evaluated. To date, no such investigation has been reported, and this study represents the first systematic evaluation of \u003cem\u003eM. involucrata\u003c/em\u003e secondary metabolism under \u003cem\u003ein vitro\u003c/em\u003e conditions. The findings provide new insights into the species\u0026rsquo; biological and metabolic potential.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Plant material\u003c/h2\u003e \u003cp\u003eSeeds of \u003cem\u003eMicrochirita involucrata\u003c/em\u003e var. \u003cem\u003ecapitis\u003c/em\u003e (Craib) C. Puglisi were collected from Phang Nga Province, Thailand, and subsequently cultivated under controlled greenhouse conditions at the Department of Biology, Naresuan University. Species authentication was performed by Assistant Professor Dr. Pranee Nangngam, an expert taxonomist of Department of Biology, Naresuan University. A voucher specimen was prepared and deposited at the Phitsanulok Naresuan University (PNU) herbarium (Voucher No. PNU05867). Mature plants were harvested, and various explants\u0026mdash;including leaves, roots, capsules, stems, and whole plants\u0026mdash;were carefully separated and prepared for subsequent extraction procedures.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 \u003cem\u003eIn vitro\u003c/em\u003e plants\u003c/h2\u003e \u003cp\u003eFully matured seeds of \u003cem\u003eM. involucrata\u003c/em\u003e were surface sterilized using 5% (v/v) sodium hypochlorite (NaOCl) for 15 min, followed by three rinses with sterile distilled water to remove residual disinfectant. Sterilized seeds were aseptically transferred to culture on semi-solid Murashige \u0026amp; Skoog (MS) (Murashige and Skoog \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1962\u003c/span\u003e) basal medium solidified with 7.5 g/L agar and supplemented with 30 g/L sucrose as a carbon source. \u003cem\u003eIn vitro\u003c/em\u003e plants were cultured for 4 weeks under controlled temperature conditions (25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) with a 16-hours photoperiod and a light intensity of 20 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1;. \u003cem\u003eIn vitro\u003c/em\u003e plantlets were routinely subcultured onto fresh MS agar medium every 4 weeks to maintain healthy growth and to obtain sufficient biomass for subsequent extraction procedures.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Root Culture and Elicitor Treatments\u003c/h2\u003e \u003cp\u003eRoot cultures were initiated by excising root segments from \u003cem\u003ein vitro\u003c/em\u003e\u0026ndash;grown \u003cem\u003eM. involucrata\u003c/em\u003e plantlets. Approximately 4 g of fresh root tissue was transferred into 250-mL Erlenmeyer flasks containing 50 mL of half-strength MS liquid medium supplemented with 15 g/L sucrose. Elicitation was optimized by screening a series of concentrations for each elicitor\u0026mdash; methyl jasmonate (MeJA) at 50, 100, 150, and 200 \u0026micro;M; chitosan (CHT) at 50, 100, 150, and 200 mg/L; and cyclodextrin (CD) at 2, 4, 6, and 8 mM \u0026mdash;based on their effects on antioxidant activity. The concentrations that produced the highest antioxidant responses were selected and subsequently applied as individual and combined treatments, with comparisons made against the untreated control. Single elicitor treatments included 50 \u0026micro;M MeJA, CHT 100 mg/L, and CD 4 mM. Combination treatments consisted of 50 \u0026micro;M MeJA and 4 mM CD (MeJA\u0026thinsp;+\u0026thinsp;CD), and 100 mg/L CHT and 4 mM CD (CHT\u0026thinsp;+\u0026thinsp;CD). All cultures were exposed to the optimized elicitor concentrations on a rotary shaker (150 rpm) at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C under dark conditions for 72 hours. Each elicitation treatment was performed using three biological replicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Root and culture medium extraction\u003c/h2\u003e \u003cp\u003eFor natural \u003cem\u003eM. involucrata\u003c/em\u003e plant material, leaves, roots, capsules, stems, and whole plants were dried in a hot-air oven at 60\u0026deg;C for 24 hours and subsequently ground into a fine powder. The powdered material was extracted by maceration with 95% ethanol at a ratio of 1:10 (w/v) for 24 hours, and the extraction process was performed in triplicate. The combined extracts were concentrated to dryness using a rotary evaporator.\u003c/p\u003e \u003cp\u003eFor root tissue cultures, following 72 hours of elicitation, root tissues were separated from the culture medium and subjected to extraction. The tissues were dried in a hot-air oven at 60\u0026deg;C for 24 hours and ground into a fine powder. The dried material was weighed and extracted with methanol by maceration at a plant-to-solvent ratio of 1:10 (w/v) for 24 hours, and the extraction was repeated three times. The filtrates were pooled and evaporated to dryness using a rotary evaporator.\u003c/p\u003e \u003cp\u003eThe culture medium was extracted separately using liquid-liquid extraction with ethyl acetate at a solvent-to-medium ratio of 1:1 (v/v). The extraction step was also performed three times and the combined ethyl acetate fractions were evaporated to dryness under reduced pressure at 40\u0026deg;C using a rotary evaporator. The resulting crude extracts were weighed and subsequently used for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Antioxidant activity\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1 ABTS antioxidant Assay\u003c/h2\u003e \u003cp\u003eThe ABTS antioxidant assay was perform according to the method described by Re et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). The ABTS\u003csup\u003e\u0026bull;\u003c/sup\u003e⁺ radical cation was generated by reacting 7 mM ABTS with 140 mM potassium persulfate (K₂S₂O₈) and allowing the mixture to stand in the dark for 16 hours. The resulting ABTS\u003csup\u003e\u0026bull;\u003c/sup\u003e⁺ working solution was subsequently diluted to achieve an absorbance of 0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 at 734 nm. Antioxidant activity was assessed by adding 2 \u0026micro;L of the sample extract to 198 \u0026micro;L of the ABTS\u003csup\u003e\u0026bull;\u003c/sup\u003e⁺ solution, followed by incubation at room temperature for 6 minutes. The absorbance was then measured at 734 nm. Trolox equivalent antioxidant capacity (TEAC) values were calculated from a Trolox standard calibration curve and expressed as TEAC per gram dry weight.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003e2.5.2 FRAP antioxidant assay\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe ferric reducing antioxidant power (FRAP) assay was carried out following the method of Benzie and Strain (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) with minor modifications. The FRAP working reagent consisted of 300 mmol/L acetate buffer (pH 3.6), 10 mmol/L 2,4,6-tripyridyl-s-triazine (TPTZ) dissolved in 40 mmol/L HCl, and 20 mmol/L FeCl₃, mixed in a 10:1:1 proportion. For each reaction, 2 \u0026micro;L of sample extract was added to 198 \u0026micro;L of the freshly prepared FRAP reagent. The mixture was incubated at room temperature for 5 minutes, and the absorbance of the resulting Fe\u0026sup2;⁺\u0026ndash;TPTZ complex was measured at 593 nm. Antioxidant capacity was quantified using a standard curve prepared with ascorbic acid, and results were expressed as ascorbic acid equivalents (AAE) per gram dry weight.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Lipoxygenase Inhibition Assay\u003c/h2\u003e \u003cp\u003e The lipoxygenase (LOX) inhibition assay was performed by incubating the extract with a reaction mixture containing 10 mM phosphate buffer (pH 8.0) and 15-lipoxygenase enzyme (34,160 U/mL). After a 5-minutes pre-incubation at room temperature, the reaction was initiated by adding linoleic acid substrate (2,230 \u0026micro;M). Enzyme activity was monitored kinetically by measuring the increase in absorbance at 234 nm every 15 seconds for 3 minutes. The percentage of LOX inhibition was calculated by comparing the reaction rate (ΔA/min) of the sample with that of the solvent control using the following equation:\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Thin layer chromatography (TLC)\u003c/h2\u003e \u003cp\u003eThe compound profiles were screened using TLC. Crude extracts were spotted onto TLC silica gel 60 F₂₅₄ plates (Merck Millipore, Germany), and the plates were developed using a mobile phase of hexane: ethyl acetate (2.1:0.9, v/v). After development, the TLC chromatograms were visualized under UV light at 254 and 365 nm, followed by anisaldehyde staining with gentle heating. Antioxidant-active bands were further detected by spraying with DPPH solution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Antimicrobial Activity Assay\u003c/h2\u003e \u003cp\u003eAntimicrobial activity was evaluated against two bacterial strains: \u003cem\u003eStaphylococcus aureus\u003c/em\u003e TISTR 1466 (ATCC6538) and \u003cem\u003eEscherichia coli\u003c/em\u003e TISTR 887 (ATCC 25922) which were obtained from Thailand institute of scientific and technological research culture collection (TISTR).\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.8.1 Disk Diffusion Assay\u003c/h2\u003e \u003cp\u003eBacterial strains were cultured on Mueller\u0026ndash;Hinton agar (MHA) plates and incubated at 37\u0026deg;C for 12\u0026ndash;16 hours. A single colony from each bacterial species was suspended in sterile normal saline and adjusted to 0.5 McFarland standard. The bacterial suspension was swabbed onto the surface of MHA plates. The sterile paper disks (6-mm) were impregnated with the extract and placed on the bacterial plates, which were subsequently incubated at 37\u0026deg;C for 12\u0026ndash;16 hours. The antimicrobial activity was assessed by measuring the diameter of the inhibition zone.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.8.2 Determination of Minimum Inhibitory Concentration (MIC)\u003c/h2\u003e \u003cp\u003eBacterial cultures were prepared in Mueller\u0026ndash;Hinton broth (MHB) and adjusted to 0.5 McFarland standard. Extract samples were added to a 96-well plate and subjected to twofold serial dilution in MHB. All assays were performed in triplicate. The bacterial suspension was then added to each well, and the plate was incubated at 37\u0026deg;C for 12\u0026ndash;16 hours. After incubation, resazurin solution was added to each well, followed by an additional 3-hours incubation at 37\u0026deg;C. Changes in resazurin color were monitored to determine bacterial viability. Wells remaining blue indicated the absence of bacterial growth, and the lowest concentration producing no color change was recorded as the MIC. Wells turning pink indicated bacterial growth.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.8.3 Determination of Minimum Bactericidal Concentration (MBC)\u003c/h2\u003e \u003cp\u003eTo determine the MBC, aliquots from wells showing no resazurin color change were dropped onto MHA plates. The plates were incubated at 37\u0026deg;C for 12\u0026ndash;16 hours and examined for bacterial growth. The lowest concentration of extract that produced no visible bacterial colonies was recorded as the MBC.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was conducted using one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) post hoc test in IBM SPSS Statistics software (version 23). Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) from three independent biological replicates, with significance defined at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.1 Thin-layer chromatography\u003c/b\u003e (\u003cb\u003eTLC) profiling and antioxidant activity screening\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eNatural \u003cem\u003eM. involucrata\u003c/em\u003e plant materials collected from the controlled greenhouse, including flowers, roots, stems, capsules, leaves, and whole plants, were dried and individually extracted using 95% ethanol as demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Preliminary chemical profiling of the resulting extracts was performed by TLC. Each extract was applied to TLC plates, and chromatograms were visualized under UV illumination at 254 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) and 365 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Distinct TLC banding patterns were observed among the different plant parts, with the root extract exhibiting a markedly unique profile compared to the other explants. Preliminary antioxidant screening was conducted by DPPH staining of the TLC plates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). The assay is based on the discoloration of the purple DPPH radical to yellow upon interaction with antioxidant constituents. Accordingly, bands displaying a purple-to-yellow transition after chromatographic separation were considered indicative of antioxidant activity. Among all tested plant parts, the root extract demonstrated the highest number and most intense of antioxidant-active bands, highlighting its potential as a rich source of bioactive compounds (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Comparative TLC profiling of natural and \u003cem\u003ein vitro\u003c/em\u003e\u0026ndash;derived roots\u003c/h2\u003e \u003cp\u003eThe preliminary findings indicated that the root extract was particularly promising, as it displayed the highest number of antioxidant-active bands. Accordingly, \u003cem\u003eM. involucrata\u003c/em\u003e seeds were subjected to \u003cem\u003ein vitro\u003c/em\u003e propagation under sterile laboratory conditions using tissue culture techniques. Notably, the \u003cem\u003ein vitro\u003c/em\u003e culture system exhibited extensive root development compared to natural \u003cem\u003eM. involucrata\u003c/em\u003e plant, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-b. Root tissues obtained from the \u003cem\u003ein vitro\u003c/em\u003e cultures were subsequently extracted and subjected to TLC analysis, which was compared with the TLC profile of roots from natural plants. The TLC fingerprints of both natural roots and \u003cem\u003ein vitro\u003c/em\u003e\u0026ndash;derived root cultures showed similar banding patterns when visualized under UV light at 254 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec) and following DPPH staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), indicating comparable phytochemical characteristics and antioxidant-active constituents.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Root characteristics and culture medium changes in response to elicitor treatments\u003c/h2\u003e \u003cp\u003eThe optimal concentrations of methyl jasmonate (MeJA), cyclodextrin (CD), and chitosan (CHT) were selected based on preliminary screening experiments designed to identify the elicitation conditions that produced the highest antioxidant activity (Supplementary Table S1). The effects of the individual concentration ranges\u0026mdash;MeJA at 50, 100, 150, and 200 \u0026micro;M; CD at 2, 4, 6, and 8 mM; and CHT at 50, 100, 150, and 200 mg/L\u0026mdash;are presented in Supplementary Figure S1-S3. In this study, root elicitation was carried out using the optimal concentrations of individual elicitors, including 50 \u0026micro;M MeJA, 4 mM CD, and 100 mg/L CHT, as well as combined treatments consisting of 50 \u0026micro;M MeJA with 4 mM CD (MeJA\u0026thinsp;+\u0026thinsp;CD) and 100 mg/L CHT with 4 mM CD (CHT\u0026thinsp;+\u0026thinsp;CD).\u003c/p\u003e \u003cp\u003eRoot characteristics and culture medium of the control and elicitor-treated samples are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. After 72 hours of elicitation, all culture medium exhibited noticeable color changes. The culture medium from the single CD treatment and the combined MeJA\u0026thinsp;+\u0026thinsp;CD treatment developed a pronounced bright yellow coloration, whereas the culture medium from the single CHT and the combined CHT\u0026thinsp;+\u0026thinsp;CD treatments turned dark brown. The coloration of the root tissues corresponded closely with the observed color changes in their respective culture medium.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.4 TLC profiles of elicited root and culture medium extract\u003c/h2\u003e \u003cp\u003eRoot tissues and culture medium from the control and elicitor-treated samples were separately extracted to obtain crude extracts. Crude extract yields from root tissues were relatively consistent across all treatments, including the control, ranging from 43 to 62 mg. In contrast, extract yields from the culture medium differed markedly among treatments, ranging from 2.3 to 13.7 mg. The single CD treatment and the combined elicitor treatments produced the highest yields, each generating approximately 13 mg of extract. Lower yields were obtained from the single MeJA and single CHT treatments. As expected, the control culture medium yielded only 1.9 mg of crude extract, the lowest amount among all samples.\u003c/p\u003e \u003cp\u003eThe TLC profiles of both the root tissue extracts and the corresponding culture medium extracts are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The TLC patterns of root tissue extracts were generally similar across all treatments, including the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). In contrast, the TLC profiles of the culture medium extracts displayed clear variation between the control and each elicitor treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). The control medium exhibited fewer detectable bands compared to the elicited samples when visualized under UV 254 nm, UV 365 nm, and following anisaldehyde staining. Notably, more intense bands were observed in the combined elicitor treatments (MeJA\u0026thinsp;+\u0026thinsp;CD and CHT\u0026thinsp;+\u0026thinsp;CD) compared with the single-elicitor treatments (MeJA, CD, and CHT) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnisaldehyde staining further revealed elicitor-specific characteristic bands: a prominent purple band was observed in the CD-treated sample, while a notable brown band appeared in the MeJA-treated sample (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, red arrows). These characteristic bands were absent in the CHT-treated sample. Interestingly, the combined MeJA\u0026thinsp;+\u0026thinsp;CD treatment exhibited both the prominent purple and brown bands, whereas the CHT\u0026thinsp;+\u0026thinsp;CD combination displayed only the prominent purple band (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, red arrows).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Antioxidant activity of elicited root and culture medium extract\u003c/h2\u003e \u003cp\u003eThe antioxidant activity of the root tissue and culture medium extracts, evaluated using the ABTS assay, demonstrated that root tissue extracts consistently exhibited higher antioxidant capacity than the corresponding culture medium extracts (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea and Supplementary Table S2). A similar trend in antioxidant response was observed across both extract types. Root tissue extracts showed activities ranging from 29.53 to 69.00 \u0026micro;mol Trolox/g dry weight, whereas culture medium extracts ranged from 0.40 to 7.20 \u0026micro;mol Trolox/g dry weight. In both datasets, the single MeJA and single CHT treatments yielded relatively low antioxidant activity. In contrast, treatment with single CD, as well as the combined CHT\u0026thinsp;+\u0026thinsp;CD and MeJA\u0026thinsp;+\u0026thinsp;CD treatments resulted in significantly higher antioxidant activity compared to the unelicited control.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe FRAP assay revealed an antioxidant response pattern consistent with that observed in the ABTS assay. Root tissue extracts exhibited substantially higher antioxidant activity than the corresponding culture medium extracts (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb and Supplementary Table S2). The FRAP values of the tissue extracts ranged from 7.56 to 16.59 mg ascorbic acid equivalents (AAE)/g dry weight, whereas those of the culture medium extracts ranged from 0.11 to 1.75 mg AAE/g dry weight. In both extract types, the single MeJA treatment yielded the lowest antioxidant activity. In contrast, the single CD treatment and the combined MeJA\u0026thinsp;+\u0026thinsp;CD and CHT\u0026thinsp;+\u0026thinsp;CD treatments produced markedly higher AAE values, all of which were significantly greater than those of the control group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Lipoxygenase inhibition assay of elicited root and culture medium extract\u003c/h2\u003e \u003cp\u003eRoot tissue and culture medium extracts were evaluated for lipoxygenase (LOX) inhibitory activity using linoleic acid as the substrate for the 15-LOX enzyme reaction. Crude extracts were tested at a uniform concentration of 0.5 mg/mL, and the percentage inhibition was calculated following extract addition to the assay system. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, all root tissue extracts exhibited less than 50% LOX inhibition. Interestingly, the culture medium extracts displayed a markedly different inhibition pattern. Minimal LOX inhibition (3\u0026ndash;5%) was observed in the control and in the single MeJA- and CHT-treated samples. In contrast, substantially higher inhibition was detected in the culture medium of samples treated with single CD and with the combined CHT\u0026thinsp;+\u0026thinsp;CD and MeJA\u0026thinsp;+\u0026thinsp;CD treatments, yielding inhibition percentages of 72.04\u0026thinsp;\u0026plusmn;\u0026thinsp;6.71%, 82.05\u0026thinsp;\u0026plusmn;\u0026thinsp;8.41%, and 94.35\u0026thinsp;\u0026plusmn;\u0026thinsp;4.41%, respectively (Supplementary Table S3). These findings indicate that elicitation with single CD or the combined CHT\u0026thinsp;+\u0026thinsp;CD and MeJA\u0026thinsp;+\u0026thinsp;CD treatments promote the release of LOX-inhibitory bioactive compounds into the culture medium.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Antibacterial assay of elicited root and culture medium extract\u003c/h2\u003e \u003cp\u003eAntibacterial activity was evaluated using the disk diffusion assay. A total of 1 mg of crude extract from both root tissues and culture medium was applied to sterile disks and tested against the Gram-positive bacterium \u003cem\u003eS. aureus\u003c/em\u003e and the Gram-negative bacterium \u003cem\u003eE. coli\u003c/em\u003e. The diameters of the inhibition zones were measured, and the 6-mm paper disk diameter was subtracted to determine the antibacterial potency. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the root tissue extracts produced relatively small inhibition zones compared to the corresponding culture medium extracts for both \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e. Enhanced antibacterial activity was observed in the culture medium of elicited samples, with inhibition zones of 8\u0026ndash;12 mm for \u003cem\u003eS. aureus\u003c/em\u003e and 7.33\u0026ndash;13.67 mm for \u003cem\u003eE. coli\u003c/em\u003e, compared with the untreated control. Notably, the combination treatment MeJA\u0026thinsp;+\u0026thinsp;CD produced the largest inhibition zones for both \u003cem\u003eS. aureus\u003c/em\u003e (12.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.06 mm) and \u003cem\u003eE. coli\u003c/em\u003e (13.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 mm), surpassing all single-elicitor treatments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntibacterial activity of \u003cem\u003eMicrochirita involucrata\u003c/em\u003e root tissue and culture medium crude extracts against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e following elicitation with individual elicitors (CD, MeJA, and CHT) and combined treatments (CHT\u0026thinsp;+\u0026thinsp;CD and MeJA\u0026thinsp;+\u0026thinsp;CD), compared with the unelicited control. Disk diffusion results are reported as the diameter of the clear zone minus the 6-mm disk diameter. Inhibition zone values are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;3). MIC denotes the minimum inhibitory concentration, and MBC denotes the minimum bactericidal concentration. ND indicates not detected.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSample type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eElicitors\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003e\u003cem\u003eE. Coli\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eInhibition zone\u003c/p\u003e \u003cp\u003e(mm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIC\u003c/p\u003e \u003cp\u003e(mg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMBC\u003c/p\u003e \u003cp\u003e(mg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eInhibition zone\u003c/p\u003e \u003cp\u003e(mm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMIC\u003c/p\u003e \u003cp\u003e(mg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMBC\u003c/p\u003e \u003cp\u003e(mg/mL)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eTissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 mM CD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50 \u0026micro;M MeJA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100 mg/L CHT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.58\u0026thinsp;\u0026plusmn;\u0026thinsp;1.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100 mg/L CHT\u0026thinsp;+\u0026thinsp;4 mM CD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50 \u0026micro;M MeJA\u0026thinsp;+\u0026thinsp;4 mM CD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.54\u0026thinsp;\u0026plusmn;\u0026thinsp;1.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eMedium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 mM CD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50 \u0026micro;M MeJA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.00\u0026thinsp;\u0026plusmn;\u0026thinsp;3.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100 mg/L CHT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100 mg/L CHT\u0026thinsp;+\u0026thinsp;4 mM CD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e12.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50 \u0026micro;M MeJA\u0026thinsp;+\u0026thinsp;4 mM CD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eGiven the higher antibacterial activity observed in the culture medium extracts, only these samples were further evaluated using the broth microdilution method to determine the minimum inhibitory concentration (MIC), followed by the minimum bactericidal concentration (MBC). As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, elicitor-treated samples exhibited lower MIC values against \u003cem\u003eS. aureus\u003c/em\u003e compared with the untreated control. Notably, the combined MeJA\u0026thinsp;+\u0026thinsp;CD treatment exhibited the greatest antibacterial potency, presenting the lowest MIC (0.78 mg/mL) and MBC (0.78 mg/mL) among all treatments. For \u003cem\u003eE. coli\u003c/em\u003e, the single CD treatment as well as the combined CHT\u0026thinsp;+\u0026thinsp;CD and MeJA\u0026thinsp;+\u0026thinsp;CD treatments produced lower MIC and MBC values than the control. Consistent with the results for \u003cem\u003eS. aureus\u003c/em\u003e, the MeJA\u0026thinsp;+\u0026thinsp;CD combination showed the strongest activity against \u003cem\u003eE. coli\u003c/em\u003e, with MIC and MBC values of 1.56 mg/mL and 3.13 mg/mL, respectively, representing the lowest among all tested samples.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e \u003cem\u003eMicrochirita involucrata\u003c/em\u003e var. \u003cem\u003ecapitis\u003c/em\u003e (Craib) C. Puglisi was reclassified into the genus \u003cem\u003eMicrochirita\u003c/em\u003e along with species formerly placed in the genera \u003cem\u003eChirita\u003c/em\u003e and \u003cem\u003eDidymocarpus\u003c/em\u003e. To date, no bioactivity studies have been reported specifically for \u003cem\u003eM. involucrata\u003c/em\u003e. However, numerous studies on species within its former genera have documented diverse biological activities, including antioxidant, antimicrobial, and anti-inflammatory properties (Kindo et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Nanjala et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Traditionally, plants from these groups have been widely used as herbal remedies for various conditions such as allergic skin disorders, inflammation, fever, diabetes, and several other ailments (Bhongade et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In this study, we examined the TLC profiles of ethanol extracts prepared from different parts of \u003cem\u003eM. involucrata\u003c/em\u003e, including leaves, flowers, roots, capsules, and stems. The results demonstrated that the root extract displayed the most distinct TLC fingerprint, clearly differentiating it from the other plant parts. Notably, the root extract also exhibited the most intense antioxidant-active bands following DPPH staining, indicating a higher abundance of antioxidant constituents. Roots have frequently been highlighted as important reservoirs of bioactive phytochemicals. For example, qualitative and quantitative analyses of \u003cem\u003eCalotropis gigantea\u003c/em\u003e and \u003cem\u003eC. procera\u003c/em\u003e roots revealed that flavonoids, alkaloids, tannins, and phenolic compounds are the predominant constituents, contributing to their extensive use in traditional medicine (Mishra et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Similarly, the roots of \u003cem\u003eMarsdenia macrantha\u003c/em\u003e, traditionally used to treat mouth infections, constipation, and urinary retention, were found to contain abundant flavonoids and alkaloids with demonstrated antibacterial activity against foodborne pathogens (Shikwambi et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). As underground organs continually exposed to soilborne pathogens and environmental stressors, roots often accumulate a wide variety of defensive phytochemicals synthesized to protect the plant. Consequently, root-based defense mechanisms play a crucial role in plant survival against the diverse array of belowground attackers (Touw and van Dam \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe initial screening indicated that the root extract was the most promising sample, exhibiting the highest number of antioxidant-active bands. To generate sufficient root biomass for further analysis, an \u003cem\u003ein vitro\u003c/em\u003e tissue culture system for \u003cem\u003eM. involucrata\u003c/em\u003e was established\u0026mdash;representing the first report of sterile tissue culture propagation for this species within the genus \u003cem\u003eMicrochirita\u003c/em\u003e. The \u003cem\u003ein vitro\u003c/em\u003e cultures produced extensive root formation, and the TLC profiles of the \u003cem\u003ein vitro\u003c/em\u003e\u0026ndash;derived roots closely resembled those of roots collected from natural plants. These findings demonstrate that \u003cem\u003ein vitro\u003c/em\u003e root cultures of \u003cem\u003eM. involucrata\u003c/em\u003e are capable of producing phytochemical patterns comparable to naturally grown roots. These findings are consistent with previous reports demonstrating that adventitious root cultures can produce secondary metabolite profiles comparable to those of naturally grown roots (Khanam et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Reis et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The application of biotechnology to root tissue culture is therefore crucial for the rapid and large-scale production of root biomass under controlled laboratory conditions.\u003c/p\u003e \u003cp\u003eThe production of secondary metabolites in root cultures can be substantially enhanced through the application of elicitors that simulate biotic or abiotic stress, thereby triggering the plant\u0026rsquo;s defensive metabolic pathways. A wide range of elicitors has been utilized depending on species-specific responses, including methyl jasmonate, chitosan, salicylic acid, and certain heavy metals (Rahmat and Kang \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Appropriate selection of elicitors and optimization of elicitation conditions can markedly increase the accumulation and yield of secondary metabolites in root tissue cultures. In this study, root elicitation of \u003cem\u003eM. involucrata\u003c/em\u003e was performed by excising root segments from \u003cem\u003ein vitro\u003c/em\u003e\u0026ndash;grown plantlets and cultivating them in liquid medium supplemented with various concentrations of single elicitors, including MeJA, CHT, and CD. Optimal concentrations for each elicitor were selected based on antioxidant activity screening of both root tissue and culture medium extracts in comparison with the unelicited control. Because CD treatment produced high antioxidant activity in both tissue and medium extracts, CD was subsequently combined with MeJA or CHT to evaluate the effects of the combined treatments (MeJA\u0026thinsp;+\u0026thinsp;CD and CHT\u0026thinsp;+\u0026thinsp;CD). TLC fingerprinting revealed that the root tissue extracts displayed similar banding patterns across all treatments and the control, whereas the culture medium extracts showed clear and distinct variations among treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). More intense and diverse TLC patterns were observed in the combined elicitor treatments (MeJA\u0026thinsp;+\u0026thinsp;CD and CHT\u0026thinsp;+\u0026thinsp;CD) compared with the single-elicitor treatments. Notably, anisaldehyde staining revealed elicitor-specific characteristic bands, indicating that each elicitor induced the synthesis and secretion of particular metabolites into the culture medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). These metabolite-specific patterns corresponded with the visible color changes in the culture medium following elicitation. Overall, while root tissue extracts maintained similar profiles across treatments, the culture medium exhibited distinct TLC patterns reflective of the different elicitation conditions. The application of elicitor-based strategies can enhance the synthesis of secondary metabolites during \u003cem\u003ein vitro\u003c/em\u003e culture. In addition to enhancing the synthesis and accumulation of secondary metabolites, elicitor application in the culture medium has also been reported to stimulate the efflux of intracellular metabolites (Wang et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This promotes the release of specific compounds into the culture medium, which can be observed as distinct differences in TLC profiles following elicitor treatment. Furthermore, several studies have demonstrated that the application of combined elicitors can elicit synergistic responses, resulting in greater secondary metabolite accumulation compared with single-elicitor treatments. This enhanced production is generally attributed to elicitor-induced signaling crosstalk, which leads to stronger activation and upregulation of the relevant biosynthetic pathways. Such synergistic action was observed in adventitious roots of \u003cem\u003ePrimula veris\u003c/em\u003e, where the combined application of L-proline and jasmonic acid resulted in significant increases in phenolic acid, glycoside, and saponin accumulation. These findings highlight the importance of strategic elicitor selection and that appropriate elicitation can substantially enhance secondary metabolite production of the \u003cem\u003ein vitro\u003c/em\u003e culture systems (Sarropoulou et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTreatment with single CD, as well as the combined CHT\u0026thinsp;+\u0026thinsp;CD and MeJA\u0026thinsp;+\u0026thinsp;CD elicitors, resulted in significantly higher antioxidant activity compared with the unelicited control. Antioxidant evaluation further revealed that root tissue extracts consistently exhibited greater antioxidant capacity than the corresponding culture medium extracts. In contrast, the LOX inhibition assay\u0026mdash;reflecting \u003cem\u003ein vitro\u003c/em\u003e anti-inflammatory potential\u0026mdash;and the antibacterial assay showed markedly higher activity in the culture medium extracts than in the tissue extracts. This divergence is likely attributable to differential metabolite localization and elicitor-induced metabolite secretion. Antioxidant compounds are typically retained within plant tissues, where they provide intracellular protection against oxidative stress, thereby contributing to the higher antioxidant activity observed in tissue extracts (Shen et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Conversely, elicitors can selectively promote the efflux of particular secondary metabolites into the culture medium. Cyclodextrin, in particular, enhances the secretion and stabilization of hydrophobic defense-related metabolites by forming inclusion complexes, leading to their extracellular accumulation (Farhadi et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These secreted metabolites, therefore, contribute more strongly to LOX inhibitory and antibacterial activities in the culture medium. Such differential metabolite distribution suggests that elicitation not only stimulates biosynthesis but also regulates metabolite compartmentalization, producing distinct biological activity profiles between tissues and their culture medium (Massalha et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Similar phenomena have been reported in Arabidopsis, where the volatile homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) functions as a defensive metabolite against the root rot pathogen \u003cem\u003ePythium irregulare\u003c/em\u003e. DMNT, released as a breakdown product of arabidiol, was shown to reduce oospore germination during early infection stages (Sohrabi et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGiven the diverse bioactivities observed in the culture medium extracts beyond antioxidant activity, these findings suggest promising potential for further exploration of the bioactive compounds produced and secreted by \u003cem\u003eM. involucrata\u003c/em\u003e in response to elicitor treatment. Future studies should focus on the isolation and structural characterization of the metabolites responsible for the observed biological activities, enabling direct links between specific secondary metabolites and their functional roles.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study establishes, for the first time, a controlled \u003cem\u003ein vitro\u003c/em\u003e root culture and elicitation system for \u003cem\u003eMicrochirita involucrata\u003c/em\u003e and provides new insights into its secondary metabolic capacity. Root tissues were identified as the most metabolically responsive explant, exhibiting pronounced antioxidant-associated chemical profiles. Targeted elicitation using cyclodextrin (CD), either alone or in combination with methyl jasmonate (MeJA) or chitosan (CHT), significantly enhanced secondary metabolite accumulation and associated biological activities. Distinct tissue- and compartment-specific responses were observed, with root extracts showing higher antioxidant capacity, whereas culture medium extracts displayed markedly stronger lipoxygenase-inhibitory and antibacterial activities. These findings indicate that elicitation not only activates intracellular secondary metabolic pathways but also promotes the extracellular secretion of metabolites into the culture medium. The establishment of a reproducible \u003cem\u003ein vitro\u003c/em\u003e root culture platform, together with elicitor-driven metabolic modulation, provides a robust experimental framework for future studies focused on metabolite isolation, structural elucidation, and the investigation of regulatory mechanisms underlying secondary metabolite biosynthesis and secretion in \u003cem\u003eM. involucrata\u003c/em\u003e. Collectively, this work expands current understanding of elicitor-mediated secondary metabolism in \u003cem\u003ein vitro\u003c/em\u003e root systems and highlights \u003cem\u003eM. involucrata\u003c/em\u003e as a valuable model for studying metabolite regulation and secretion dynamics in Gesneriaceae species.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declaration\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eApinun Limmongkon\u003c/strong\u003e: Conceptualization, Supervision, Visualization, Data curation, Writing – original draft. \u003cstrong\u003eWipaporn Chuaymaung\u003c/strong\u003e: Methodology, Formal analysis, Validation. \u003cstrong\u003ePathitta Sasiri\u003c/strong\u003e: Methodology, Formal analysis, Validation. \u003cstrong\u003eButsakon Nisaipham\u003c/strong\u003e: Methodology, Formal analysis, Validation. \u003cstrong\u003eSirianong Khongwet\u003c/strong\u003e: Methodology, Formal analysis, Validation.\u0026nbsp;\u003cstrong\u003eOnrut Sapatee\u003c/strong\u003e: Visualization, Investigation. \u003cstrong\u003eThanakorn Wongsa\u003c/strong\u003e: Conceptualization, Visualization. \u003cstrong\u003eAnupan Kongbangkerd\u003c/strong\u003e: \u0026nbsp; Conceptualization, Resources, Writing – review and editing. \u003cstrong\u003eWannapa Khanthit\u003c/strong\u003e: Methodology, Formal analysis. \u003cstrong\u003eSirinan Temwong\u003c/strong\u003e: Methodology, Formal analysis. \u003cstrong\u003eArpassara Maliprom\u003c/strong\u003e: Methodology, Formal analysis. \u003cstrong\u003eWuthinant Piriyanupong\u003c/strong\u003e: Methodology, Formal analysis. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Naresuan University (NU) and the National Science, Research and Innovation Fund (NSRF) [grant number R2569B087].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eArezoumand E, Bagheri K, Mazloum S, Noh GM, Hamishehkar H, Kosari-Nasab M, Kim KH (2025) β-Cyclodextrin as an elicitor of polyphenolic contents of barley (Hurdeum vulgare) callus with antioxidant and anti-aging properties on human skin fibroblast cells (HFF2). 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Front Microbiol 16:1558567. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fmicb.2025.1558567\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2025.1558567\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\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":"Microchirita involucrata, in vitro culture, elicitation, antioxidant, antibacterial, lipoxygenase inhibition","lastPublishedDoi":"10.21203/rs.3.rs-8636341/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8636341/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eMicrochirita involucrata\u003c/em\u003e (family Gesneriaceae) has recently been reassigned to the genus \u003cem\u003eMicrochirita\u003c/em\u003e; however, its biological properties and potential applications remain unexplored in Thailand. Although the species is valued for its compact growth habit and attractive floral morphology, its phytochemical potential has received little attention. This study established an \u003cem\u003ein vitro\u003c/em\u003e culture platform for \u003cem\u003eM. involucrata\u003c/em\u003e, identified explants with high secondary metabolite potential, optimized metabolite accumulation through elicitation, and evaluated the biological activities of elicited root cultures. Thin-layer chromatography (TLC) screening revealed that root tissues contained the highest number of antioxidant-active bands; therefore, \u003cem\u003ein vitro\u003c/em\u003e\u0026ndash;derived root segments were selected for elicitation using methyl jasmonate (MeJA), chitosan (CHT), and β-cyclodextrin (CD), applied individually and in combination (MeJA\u0026thinsp;+\u0026thinsp;CD and CHT\u0026thinsp;+\u0026thinsp;CD). CD alone and combined treatments significantly enhanced antioxidant activity. Root tissue extracts exhibited higher antioxidant capacity, whereas culture medium extracts showed markedly stronger lipoxygenase (LOX) inhibitory and antibacterial activities. Culture media derived from CD and combined elicitor treatments displayed the highest LOX inhibition, with values ranging from 72.04% to 94.35%. Among all treatments, the MeJA\u0026thinsp;+\u0026thinsp;CD\u0026ndash;elicited culture medium extract demonstrated the strongest antibacterial activity, with minimum inhibitory and bactericidal concentrations of 0.78 mg/mL against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. Against \u003cem\u003eEscherichia coli\u003c/em\u003e, the corresponding MIC and MBC values were 1.56 and 3.13 mg/mL, respectively. Overall, these findings provide new insights into elicitor-induced secondary metabolism in \u003cem\u003eM. involucrata in vitro\u003c/em\u003e root cultures, supporting its relevance as a source of biologically active metabolites.\u003c/p\u003e","manuscriptTitle":"Elicitor-mediated enhancement of secondary metabolite accumulation and biological activity in Microchirita involucrata in vitro root cultures","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-28 17:03:31","doi":"10.21203/rs.3.rs-8636341/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-01-22T22:32:15+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-22T13:10:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-22T05:54:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2026-01-20T07:10:17+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":"e6293758-d74f-422e-8407-be24a0fc6a98","owner":[],"postedDate":"January 28th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-07T16:07:09+00:00","versionOfRecord":{"articleIdentity":"rs-8636341","link":"https://doi.org/10.1007/s11240-026-03437-8","journal":{"identity":"plant-cell-tissue-and-organ-culture-pctoc","isVorOnly":false,"title":"Plant Cell, Tissue and Organ Culture (PCTOC)"},"publishedOn":"2026-03-30 15:59:56","publishedOnDateReadable":"March 30th, 2026"},"versionCreatedAt":"2026-01-28 17:03:31","video":"","vorDoi":"10.1007/s11240-026-03437-8","vorDoiUrl":"https://doi.org/10.1007/s11240-026-03437-8","workflowStages":[]},"version":"v1","identity":"rs-8636341","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8636341","identity":"rs-8636341","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}