Biochemical profiling of callus and protocorm-like bodies during in vitro propagation of Dendrobium crepidatum lindl. and paxt

Introduction

When researchers at a Vietnamese ethnobotanical survey station opened a freshly collected Dendrobium crepidatum pseudobulb in 2019, the crude methanolic extract showed surprisingly strong free-radical scavenging stronger, in fact, than several commercially traded Dendrobium species [1] . That observation raised an obvious question: could the same bioactive profile be reproduced under laboratory conditions, where supply does not depend on dwindling wild stocks? Orchids of the genus Dendrobium have long occupied a dual role as ornamental plants and sources of traditional medicine across South and South-East Asia [2,3] . Alkaloids, phenanthrenes, bibenzyls, and polysaccharides isolated from various Dendrobium species display anti-inflammatory, antidiabetic, neuroprotective, and immunomodulatory effects in preclinical models [4,5] . D. crepidatum specifically is used in folk remedies for fever and gastrointestinal disorders in Bangladesh, Nepal, and parts of Indochina, but it remains one of the least biochemically characterised members of the genus [6] . Wild harvesting to meet medicinal and horticultural demand, combined with deforestation, has pushed D. crepidatum into threatened status across much of its range [7] . Micropropagation through callus and PLB routes offers a way to multiply plants without further pressure on natural populations [8] . However, tissue culture does not always preserve the metabolite profile that gives medicinal plants their therapeutic value. Growth regulators, light regime, carbon source, and passage number can each shift secondary metabolism in unpredictable directions [9,10] . For instance, elevated auxin concentrations sometimes suppress phenylpropanoid pathway enzymes, reducing phenolic output, while cytokinin-rich media may enhance alkaloid accumulation [11] . Unless the biochemical fidelity of cultured tissues is verified, there is a risk that mass-propagated plants will lack the compounds that made the species medicinally interesting in the first place. A handful of reports have examined phytochemistry in Dendrobium tissue cultures. Zhao et al. [12] tracked polysaccharide content in Dendrobium officinale PLBs and found levels 30-40% below those of greenhouse-grown stems. Conversely, Teixeira da Silva and Ng [13] reported that Dendrobium Nobile PLBs accumulated dendrobine at concentrations comparable to field plants when culture media included coconut water and activated charcoal. These contrasting outcomes highlight that results are species-and protocol-specific extrapolation from one Dendrobium to another is unreliable [14] . For D. crepidatum, Bhowmik et al. [8] recently established a reliable micropropagation protocol using nodal segments on MS medium, achieving high shoot multiplication rates. But that work did not extend to biochemical evaluation of the regenerated tissues. The present research fills that gap by quantifying total phenolics, flavonoids, alkaloids, proteins, carbohydrates, and antioxidant activity in callus, PLBs, and mother plant tissues of D. crepidatum propagated at Vietnam National University, Hanoi. By comparing metabolite levels across three developmental stages undifferentiated callus, organised PLBs, and mature field tissue we aimed to determine whether organogenesis restores the secondary metabolite signature and at which culture stage bioactive compound recovery is maximised. Such information is relevant both for conservation-oriented mass propagation and for any future effort to use in-vitro biomass as a raw material for pharmaceutical extraction [15,16] .

Extraction protocol

Callus, PLB, and mother plant samples were separately shade-dried at 40 °C for 72 h and ground to fine powder using a ceramic mortar. Powdered tissue (2 g) was extracted three times with 80% methanol (30 mL each) by magnetic stirring at room temperature for 24 h per cycle. The combined extracts were filtered through Whatman No. 1 paper and concentrated under reduced pressure at 40 °C using a rotary evaporator (Büchi R-210). The resulting crude methanolic extracts were stored at -20 °C in amber vials until analysis. Extraction yields were 11.3%, 14.7%, and 16.2% (w/w) for callus, PLBs, and mother plant tissue, respectively. A parallel aqueous extraction was performed for carbohydrate analysis: 1 g of powder was boiled in 50 mL distilled water for 30 min, cooled, and filtered. All extractions were carried out in triplicate within 48 h of drying to minimise compound degradation.

Material and methods

Material

Healthy D. crepidatum plants bearing mature pseudobulbs were collected from a private orchid nursery in Hoang Mai district, Hanoi (20.98° N, 105.85° E) in May 2022. Voucher specimens were authenticated at the Botany Herbarium of Vietnam National University (accession VNU-HN-2022-0437). Nodal segments (1.5-2.0 cm) excised from secondyear pseudobulbs served as explants. All chemicals Murashige and Skoog basal salt mixture, 2,4-D, BAP, naphthaleneacetic acid (NAA), Folin-Ciocalteu reagent, gallic acid, quercetin, aluminium chloride, DPPH, ascorbic acid, bovine serum albumin, Bradford reagent, and anthrone were of analytical grade and sourced from Sigma-Aldrich and HiMedia. Glassware was autoclaved at 121 °C for 20 min prior to use. A UV-Vis spectrophotometer (Shimadzu UV-1800) was used for all colorimetric assays.

Methods

Explants were surface-sterilised with 70% ethanol for 60 s, followed by 0.1% mercuric chloride for 8 min, and rinsed five times with sterile distilled water. For callus induction, sterilised segments were inoculated onto MS medium supplemented with 1.5 mg/L 2, 4-D, 30 g/L sucrose, and 7 g/L agar (pH 5.8). Cultures were maintained at 25±2 °C under a 16/8 h photoperiod (2,000 lux cool-white fluorescent light). Callus initiation was scored at weekly intervals; established callus was subcultured every four weeks. For PLB differentiation, six-week-old callus masses (approximately 0.5 g) were transferred to MS medium containing 2.0 mg/L BAP and 0.5 mg/L NAA. PLB emergence was recorded from week eight, and fully developed PLBs were harvested at week twelve for biochemical analysis. Total phenolic content was determined using the Folin-Ciocalteu method with gallic acid as standard (0-200 µg/mL calibration curve, R² = 0.997). Total flavonoid content was measured by the aluminium chloride colorimetric assay with quercetin as standard. Alkaloid content was estimated using the bromocresol green method. Protein was quantified by the Bradford assay with bovine serum albumin standards. Total soluble carbohydrates were measured by the anthronesulphuric acid method with glucose as standard. DPPH radical scavenging activity was assessed at five concentrations (25, 50, 100, 200, 400 µg/mL) with ascorbic acid as positive control; percentage inhibition was calculated as [(Abs_control-Abs_sample) / Abs_control] × 100. All assays were performed in triplicate; results are expressed as mean±standard deviation. One-way analysis of variance (ANOVA) followed by Tukey's HSD test (p < 0.05) was used to compare tissue types, computed in SPSS v26. The research was conducted between June 2022 and March 2023 at the Plant Biochemistry Laboratory, Vietnam National University, Hanoi.

Quality control

Instrument calibration was verified at the start of each analytical run. The UV-Vis spectrophotometer was zeroed against a methanol blank, and calibration curves were freshly prepared for each assay session using certified reference standards. Spike recovery tests were performed by adding known amounts of gallic acid and quercetin to representative callus extracts; recoveries ranged from 96.2% to 102.8%, confirming acceptable accuracy. Coefficient of variation across triplicate measurements did not exceed 4.7% for any analyte. Reagent blanks were run alongside every batch to monitor background interference. All glassware used for the DPPH assay was wrapped in aluminium foil to prevent photodegradation of the radical solution.

Results

Callus initiation was observed on 87% of explants within 28 days on 1.5 mg/L 2, 4-D medium, and PLBs differentiated on 74% of transferred callus masses by week twelve on BAP-supplemented medium. Biochemical quantification revealed a consistent gradient: metabolite concentrations were lowest in undifferentiated callus, intermediate in PLBs, and highest in mother plant tissue, a pattern that held across all six parameters tested. The radar overlay shows that PLBs (medium pink line) tracked the mother plant profile (light pink) more closely than callus (dark pink) on every axis, with the largest gap between callus and the other two tissue types appearing in total phenolics and antioxidant activity.

Comprehensive interpretation

The biochemical gradient from callus through PLBs to mother plant tissue was consistent across every metabolite class measured. PLBs recovered between 77% and 91% of the mother plant's metabolite concentrations depending on the parameter, while callus reached only 50-72%. The doseresponse curve for DPPH scavenging confirmed that antioxidant capacity tracked total phenolic and flavonoid content, as expected from the well-documented correlation between phenylpropanoid metabolites and radicalquenching ability. Ascorbic acid remained the strongest scavenger at every concentration, but the gap between PLB extract and the positive control narrowed at higher doses at 400 µg/mL, PLBs reached 84% of ascorbic acid's activity. These patterns suggest that organogenesis progressively reactivates secondary metabolic pathways suppressed during dedifferentiation, and that PLB-stage biomass may offer a practical harvest point for bioactive compound recovery.

Discussion

Taken as a whole, the data show that D. crepidatum tissues cultured in vitro retain a measurable but reduced pool of secondary metabolites relative to field-grown plants, and that the degree of reduction depends on the developmental state of the culture. PLBs approximate mother plant chemistry far more closely than undifferentiated callus, a pattern that holds for phenolics, flavonoids, alkaloids, and antioxidant capacity alike.

This finding aligns with reports from other Dendrobium species. Zhao et al. [12] observed that polysaccharide content in D. officinale PLBs reached about 65% of greenhouse stem levels, a proportion similar to the phenolic recovery (86%) seen here for D. crepidatum PLBs. Teixeira da Silva and Ng [13] likewise reported that D. nobile PLBs accumulated dendrobine at concentrations approaching those of mature stems when coconut water was added to the medium. The agreement across species suggests a shared mechanism: as cells reorganise into meristematic centres during PLB formation, gene networks governing specialised metabolism particularly the phenylpropanoid and terpenoid indole alkaloid pathways are progressively reactivated [10,14]. On the other hand, the callus-stage metabolite levels recorded here were lower than those reported by Kaur and Bhutani [9] for Dendrobium fimbriatum callus, where total phenolics exceeded 6 mg GAE/g DW at a similar 2,4-D concentration. One possible explanation is genotype sensitivity: D. crepidatum may channel more carbon toward primary growth under auxin stimulation, leaving fewer precursors for the shikimate pathway. Another factor could be light intensity our cultures received 2,000 lux, whereas Kaur and Bhutani used 3,000 lux, and phenylalanine ammonia-lyase activity is known to be light-responsive [11] . From a broader standpoint, the results have two practical bearings. First, for conservation-driven mass propagation as recently described by Bhowmik et al. [8] it is reassuring that PLBs carry a biochemical profile close to wild-type tissue;

plants regenerated from PLBs are therefore likely to retain medicinal value once established in the field. Second, if the goal shifts toward using in-vitro biomass itself as a pharmaceutical feedstock, the data suggest harvesting at the PLB stage rather than the callus stage, because PLBs yield roughly 50% more phenolics and flavonoids per gram of dry tissue, with correspondingly stronger antioxidant activity. Optimisation of culture conditions increased light, elicitor treatments, or precursor feeding could potentially close the remaining gap between PLBs and mother plant tissue [15,16] .

Instrumentation and analytical equipment

All spectrophotometric measurements were carried out on a Shimadzu UV-1800 double-beam UV-Vis spectrophotometer fitted with 1 cm matched quartz cuvettes. Wavelengths used were 765 nm (Folin-Ciocalteu phenolics), 415 nm (aluminium chloride flavonoids), 470 nm (bromocresol green alkaloids), 595 nm (Bradford protein), 630 nm (anthrone carbohydrates), and 517 nm (DPPH scavenging). A Büchi R-210 rotary evaporator with a V-700 vacuum pump was used for solvent concentration. Analytical balances (Mettler Toledo ME204E, readability 0.1 mg) were employed for all weighing operations. Autoclaving was performed in a Hirayama HVE-50 vertical autoclave. The laminar flow cabinet (Esco Airstream) was UV-sterilised for 30 min before each culture session.

Conclusion

The main contribution of this research is a side-by-side biochemical comparison of callus, PLBs, and mother plant tissues of D. crepidatum a data set that was previously unavailable for this threatened medicinal orchid. Across six analytes, PLBs consistently recovered 77-91% of the mother plant's metabolite concentrations, while callus tissues retained only 50-72%. DPPH scavenging assays reinforced this hierarchy, with PLB extracts reaching 78.9% inhibition at 400 µg/mL compared to 67.3% for callus. These figures carry significance on two fronts. For conservation programmes that rely on micropropagation to produce planting stock, the nearwild-type chemistry of PLBs provides confidence that regenerated plants will retain their medicinal properties once reintroduced. For pharmaceutical research, PLB-stage biomass represents a more productive starting material than callus for bioactive compound extraction, offering higher yields per unit of culture effort.

Several questions remain open. Whether the metabolite recovery seen in PLBs persists through subsequent subculture passages has not been tested here and warrants longitudinal tracking. The influence of elicitor treatments methyl jasmonate, chitosan, or salicylic acid on pushing PLB metabolite levels closer to or beyond mother plant values is another promising avenue. And molecular confirmation, using techniques such as RAPD or ISSR markers, would verify that the genetic integrity underpinning these biochemical profiles remains intact through the culture process.

Acknowledgement

Acknowledgements Contributions not qualifying for authorshipThe authors acknowledge Ms. Le Thi Mai and Mr. Pham Quang Huy for technical help with spectrophotometric analyses and culture maintenance.

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