Abstract Cytokinins promote growth and secondary metabolism in aromatic herbs, but optimal dosing for Vietnamese balm remains unresolved. This study evaluated foliar 6-benzylaminopurine (6-BA; 0–25 ppm) on leaf development, physiology, and essential oil (EO) in Elsholtzia ciliata. Leaf traits, chlorophyll, soluble sugars, and antioxidant enzymes (POD, SOD) were measured; EO yield and composition were determined by GC–MS. Analyses included ANOVA, correlations, and PCA. All 6-BA doses enhanced the growth of leaves; 15 ppm was consistently the optimal dose. At 15 ppm, chlorophyll reached 2.54 mg/g FW and EO content rose to 3.27% (4.61-fold vs 0.71% control), accompanied by higher sugars and POD/SOD activities. GC–MS identified 26 constituents (95.88% of oil) at 15 ppm, with enrichment of oxygenated monoterpenes (neral+geranial 32.44%) and sesquiterpenes, notably (Z)-β-farnesene (21.43%). Correlations linked BA dose with leaf number (r=0.73), chlorophyll (r = 0.63), POD (r = 0.71), and SOD (r = 0.58); leaf size covaried with biomass, while EO showed weak direct correlation (r = 0.16). PCA (PC1 74.0%; PC2 8.7%) separated treatments along the BA gradient, with 15 ppm aligning with chlorophyll, sugar, and antioxidant vectors. Collectively, 15 ppm 6-BA maximized vegetative performance, physiological vigor, and EO yield/quality in E. ciliata, providing a practical regimen and a basis for mechanistic and agronomic optimization. Findings guide efficient, scalable horticultural management.

Keywords: Antioxidant enzymes, Chlorophyll, Elsholtzia ciliata, Essential oils, Principal component analysis

Citation:  Tho, L.T.L., Dung, T.T.P., and Khang, L.T.P. 2026. Impact of boiling, ultrasonic, and microwave–ultrasonic assisted extraction on phenoli Influence of 6-Benzylaminopurine on leaf parameters, biochemical characteristics, essential oil yeilds and composition in Vietnamese balm - Elsholtzia ciliata (Thunb.) Hyland. Natural and Life Sciences Communications. 25(2): e2026036.

Graphical Abstract:

INTRODUCTION

Elsholtzia ciliata (Thunb.) Hyland (E. cilitata) is a member of the Lamiaceae family and is native to Asia, where it is widely distributed in Asian countries (Luu et al., 2023). E. ciliata thrives in areas with abundant sunlight, typically in mountainous regions and along riverbanks (Guo et al., 2012). E. ciliata has warm and spicy properties, making it a valuable component of traditional Chinese medicine. It has been shown to be effective in treating common ailments such as colds, allergies, dermatitis, bleeding, limb constriction, high fever, hemorrhoids, measles, and hemostasis (Liu et al., 2007). Additionally, E. ciliata essential oil (Ec EOs) is known for its antibacterial, anti-inflammatory, antioxidant, antiviral, and potential cancer-fighting properties (Mahmoud and Croteau, 2002; Luu et al., 2023). As a result, there is a growing demand for Vietnamese balm as a raw material, primarily for leaf collection, which is the primary source of essential oil extraction (She et al., 2009; Luu et al., 2023; Tho et al., 2024).

Cytokinin is a type of plant growth regulator that stimulates cell division in both shoots and roots. The accumulation of cytokinins in specific plant parts and tissues is age-dependent (Taiz and Zeiger, 2002). Cytokinins have a significant impact on cell growth and differentiation. They also influence the development of axillary buds, apical dominance, leaf senescence, and the aging process, while preventing leaf drop and yellowing by suppressing enzyme activity related to tissue aging (Bharati et al., 2023). Furthermore, cytokinins play a key role in controlling apical dominance by interacting with auxin to promote accessory shoot formation (Baskaran and Jayabalan, 2008). In terms of essential oil accumulation, cytokinins promote the process by boosting growth parameters. This includes increasing number of leaves, expanding leaf area, enhancing gland cell formation, and increasing the density of secretory cells, both in field conditions and during in vitro treatment (Baskaran and Jayabalan, 2008). El-keltawi and Croteau (1987) attribute this effect to cytokinins' influence on metabolism, which regulates the growth of mature and immature tissues, thereby stimulating the production of monoterpenes (El-keltawi and Croteau, 1987).

Among the cytokinin group, 6-Benzylaminopurine (6-BA) is a particularly potent agent. Incorporating BA into plant life cycles can induce changes in plants' physiological and morphological characteristics. It helps plants better adapt to environmental conditions by improving growth and yield (Puripunyavanich et al., 2021; Gulzar et al., 2024). Currently, the cultivation of leafy plants, especially E. ciliata, is often practised in a rudimentary manner, with farming households frequently using high doses of plant growth regulators to expedite growth and early harvesting (Tho et al., 2024). Controlled use of BA content not only promotes healthy plant development but also ensures the accumulation of essential oils. Given the existing research landscape and the explanations provided, this study aims to investigate the impact of the plant growth regulator 6-BA on leaf parameters and essential oil content in E. ciliata.

MATERIALS AND METHODS

Study area

The research was investigating the influence of BA on E. ciliata’s growth was carried out at the nursery garden in Can Giuoc, Long An province (10°36 '21.4" N 106°36'49.4" E). Leaf parameters were conducted at the Teaching Methods - Plant Physiology laboratory - at Ho Chi Minh City University of Education. The essential oil extraction process and GC/MS analysis were carried out at the Research Institute of Biotechnology and Environment - Nong Lam University, Ho Chi Minh City.

Chemicals

Alkane standard solutions C₅ - C₂₄ and 6-BA were purchased from Sigma–Aldrich Co. (Steineheim, Germany). Ethanol and anhydrous sodium sulphate were bought from Merck Co. (Darmstadt, Germany).

Plant material and field site description

A total of 2,000 E. ciliata seeds were obtained from Huong Nong Seed Company, Ho Chi Minh City, Vietnam. Five seeds were sown in each plastic pot (20 cm in diameter and 35 cm in height). After four weeks, the seedlings were thinned and transplanted to a nursery in Long An province (10°36'21.4"N 106°36'49.4"E). The soil characteristics were as follows: 1.3% coarse sand, 1.0% fine sand, 52.9% lightness rate, 2.06% organic matter content, and a pH of 5.16 with a conductivity of 188.00 µS/cm.

Each experiment was conducted in triplicate. Within each plot, E. ciliata were arranged in six evenly spaced rows, each 0.4 m apart. To ensure adequate moisture, plants were watered daily in the morning and afternoon using a sprinkler valve system. The water valve system had three valves per bed, with each bed encompassing an area of 15 m². The experimental cultivation of plants adhered to organic practices, without the application of chemical fertilizers or pesticides. Weed control was carried out manually.

Experimental design and treatments

The experiment was conducted using a randomized complete block design (RCBD) with four replications. A total of 6 treatments (n = 25 per treatment) were applied including fresh foliar distilled water (control) an 6-BA at 5, 10, 15, 20 and 25 ppm at week 4 after sowing. 6-BA solution (adjusted to pH 7.0 with NaOH) was applied once onto the leaves of the plants at 17:00 hours using a foliar sprayer until saturation. Control plants that were sprayed with distilled water were also adjusted to pH 7.0 using NaOH. The spraying volume for each treatment was maintained at 0.5 L. After a lapse of 2 hours following the spraying, regular watering procedures were resumed.

Leaf parameters

Leaf parameters were monitored weekly after spraying.

Number of leaves: The number of leaves of marjoram was recorded and tracked weekly.

Leaf length and width (cm): The length and width of mature leaves of marjoram were measured at their largest size.

Leaf area (cm²): The leaf area was determined using a Canon Lid 210 scanner (China) and Fiji, an open-source software for image analysis (Massonnet et al., 2007).

Leaf biomass: The leaf biomass was determined by cutting 10 cm above the plant's neck and immediately weighing the fresh weight (FW). The plant was then wrapped in a clean paper bag, labelled, and oven-dried (Memmert, China) at 65°C for 48 h to determine the dry weight (DW) following the methodology established by Corell et al. (2012).

Biochemical analysis of leaf

Chlorophyll content

Approximately 0.02 g of E. ciliata leaf tissue (n = 10) was homogenized in 5 mL of ice-cold 80% acetone. The homogenate was centrifuged at 5,000 rpm for 5 min at 4°C, and the absorbance of the supernatant was subsequently recorded at 475, 645, and 663 nm using a spectrophotometer, following the method of Lichtenthaler et al. (1987). Total chlorophyll (Chl a + b) content was expressed as mg/g FW.

Soluble sugar content

To quantify soluble sugars, 0.1 g of fresh E. ciliata leaf tissue (n = 10) was transferred to a 1.5 mL centrifuge tube containing 1 mL of distilled water. The samples were homogenized and then incubated in boiling water for 10 min. After cooling, the mixtures were centrifuged at 4,000 rpm for 10 min at room temperature. Soluble sugar content was determined using a commercial kit (Nanjing Jiancheng Biological Engineering Institute, Nanjing, China), and absorbance was recorded at 620 nm. Results were expressed as mg/g FW.

Antioxidant enzyme activities

For enzyme activity assays, 0.1 g of fresh E. ciliata leaves (n = 10) was homogenized, and the activities of superoxide dismutase (SOD) and peroxidase (POD) were determined using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). SOD activity was measured at 550 nm, while POD activity was determined at 420 nm.

Essential oil isolation

To determine the essential oil content, expressed as a percentage (%), each experiment was taken from the total vegetal material of 150 g (n = 10). The subsamples were dried in a shaded area with adequate air circulation for a period of 2 days. The subsequent distillation process was carried out using a Clavenger-type distiller (Dolphin Labware, India) employing vapor entrainment. Once the essential oil was extracted, it was dried with anhydrous Na₂SO₄. It was then placed in standard airtight containers and stored at a temperature of -0.5°C prior to analysis (Zhang et al., 2015).

Gas chromatography/mass spectrometry (GC/MS) analysis and identification of components

Gas chromatography-mass spectrometry (GC/MS) was employed to analyze the composition of essential oil components in the E. ciliata treatment that yielded the highest total essential oil content. Briefly, 1.5 mg of essential oil, previously dried with anhydrous Na₂SO₄, was dissolved in 1 mL of pure chromatographic-grade hexane. The GC/MS analysis was conducted using an Agilent Technologies HP 6890N/HP 5973 MSD coupled gas chromatography system. The GC employed a capillary column Rxi 5-MMS, with an inner diameter of 0.25 mm, a length of 30 m, and a film thickness of 0.25 µm. The detector was injected with 0.1 µL of the sample. The column furnace temperature was initially maintained at 50°C for 1 minute, after which it was gradually increased at a rate of 10°C/min to 250°C and held for 20 minutes. The temperature of the sample injection chamber was set at 250°C. Helium was used as the carrier gas, with the separation column and chromatographic conditions previously described by Liang et al. (2020). The response intensity (RI) was determined from gas chromatograms using a series of n-alkanes (C₅–C₃₆) under the same operating conditions. Based on RI, the chemical constituents were identified by comparing them with n-alkanes as a reference. The essential oil components were identified by matching their mass spectra with various computer libraries (Wiley 275 libraries, NIST 05, and RI from other literature).

Data analysis

The analysis of leaf parameters and essential oil yields was performed using SPSS 26.0 software. All data were presented as mean values accompanied by standard deviations (SD). Statistical comparisons among treatments and control groups were conducted using one-way ANOVA in OriginPro (Version 2021b, OriginLab Corporation, Northampton, MA, USA), with significance determined at a 95% confidence interval (P < 0.05). In addition, a Pearson correlation matrix was generated in OriginPro to examine the strength and direction of linear relationships between key parameters affected by 6-BA application in E. ciliata. Furthermore, principal component analysis (PCA) was performed to reduce dimensionality and to identify the major variables contributing to variation in physiological traits and essential oil yields across treatments.

RESULTS

Effect of 6-BA on leaf parameters of Elsholtzia ciliata

The experimental results demonstrated that all treatments involving BA significantly increased the number of leaves, leaf length, leaf width, and leaf area compared to the control group (Figure 1). In the treatments applied during the rapid growth stage, these increases were observed consistently across weeks, intensifying gradually from week 4 to week 8. Among the treatments, 15 ppm 6-BA resulted in the most substantial increase in leaf count. Specifically, the average number of leaves per plant rose from 31.42 to 62.54 per week, peaking at 313.08 per plant by week 8. Concurrently, at week 8, the leaf length reached 14.53 cm, the leaf width extended to 8.75 cm, and the leaf area expanded significantly to 95.66 cm². Notably, the 15 ppm 6-BA treatment showed statistically significant differences compared to the other treatments. By week 8, FW ranged from 14.50 g to 16.97 g, representing an increase of 8.76% to 22.04% compared to the control group (13.23 g). Similarly, DW ranged from 1.20 g to 1.60 g. Remarkably, the 15 ppm 6-BA treatment produced the highest biomass parameters, with these differences also being statistically significant compared to the other treatments.

Figure 1. Effects of 6-BA on leaf paramters of Elsholtzia ciliata (Thunb.) Hyland including (A) number of leaf, (B) leaf length, (C) leaf width, (D) leaf area, (E) leaf fresh weight, and (F) dry weight after 4-week srapying. Significant differences among treatments in each week are marked with letters (P < 0.05).

Effect of 6-BA on biochemical parameters of Elsholtzia ciliata leaf

Application of 6-BA significantly influenced chlorophyll content in E. ciliata leaves (Figure 2A). Chlorophyll levels increased progressively with rising concentrations up to 15 ppm, reaching a maximum of 2.54 mg/g FW, which was markedly higher than the control. Beyond this concentration, chlorophyll content declined slightly, although values at 20 ppm remained significantly higher than those in untreated plants. Similarly, soluble sugar content responded positively to 6-BA treatment (Figure 2B). The highest concentration of soluble sugars (20.46 mg/g FW) was observed at 15 ppm, which was significantly greater than in the control and comparable to levels recorded at 20 and 25 ppm. In contrast, low concentrations of 6-BA (5–10 ppm) induced moderate, non-significant increases relative to the untreated group.

Antioxidant enzyme activities were also markedly affected. POD activity increased steadily with 6-BA application, peaking at 15 ppm (555.99 U/g FW), which represented a significant enhancement compared with both control and other treatments (Figure 1C). Although activities declined slightly at higher concentrations (20–25 ppm), they remained elevated relative to the untreated plants. A similar pattern was evident for SOD activity (Figure 1D). The maximum activity (277.53 U/g FW) was detected at 15 ppm, which was significantly greater than all other treatments. At higher concentrations (20–25 ppm), SOD activity decreased but did not return to control levels.

Figure 2. Effect of different concentrations of 6-BA on biochemical parameters of Elsholtzia ciliata leaves. (A) Chlorophyll content, (B) Soluble sugar content, (C) POD activity, and (D) SOD activity. Different letters above bars indicate significant differences among treatments according to one-way ANOVA (P < 0.05).

Effect of 6-BA on essential oil accumulation and chemical components of Elsholtzia ciliata leaves

The experimental results indicate a consistent upward trend in essential oil content over time, particularly in the treatments involving varying concentrations of 6-BA (Figure 3). The essential oil content in the control group reached 0.71% by 4-wps. However, when 6-BA was applied at concentrations ranging from 5 to 25 ppm, the essential oil content increased significantly compared to the control treatment, ranging from 0.91% to 3.27%. These differences were statistically significant. Notably, the highest essential oil concentration was observed at a 6-BA concentration of 15 ppm, reaching an impressive 3.27%, which is 4.61 times higher than the content in the control group (0.71%).

Figure 3. Essential oil accumulation in Vietnamese balm after 4 week spraying with 6-BA at different concentrations. Different letters indicate significant differences (P < 0.05).

A comparison of the total essential oil content obtained through 6-BA treatments revealed that E. ciliata treated with 15 ppm 6-BA yielded the most promising results. Consequently, the study focused on analyzing the essential oil composition of this treatment in comparison to the control group. In the 15 ppm 6-BA treatment, 26 components were identified, collectively accounting for 95.88% of the E. ciliata essential oil (Figure 4A). The principal constituents of essential oils were (Z)-β-farnesene (21.43%), neral (16.22%), geranial (16.22%), β-ocimene (12.99%), and caryophyllene (9.55%). Apart from these primary components, smaller constituents were also detected in the sample, including compounds like 1-octen-3-ol (4.03%), D-limonene (2.14%), acetophenone (1.64%), germacrene D (1.79%), nerol (1.62%), and humulene (1.16%). Additionally, the presence of rosefuran (0.28%) and (Z)-geranylacetate (1.33%) was observed in Vietnamese balm essential oil when treated with BA at a concentration of 15 ppm, as detected during the analysis of essential oil composition. This composition exhibited variations when compared to the essential oil samples from the control treatment (Figure 4C). The primary components belonged to two categories, including oxygenated monoterpenes and sesquiterpene hydrocarbons. Notably, this treatment's content of oxygenated monoterpenes was significantly higher than in the control group, with statistical significance observed (P < 0.05) (Figure 4D).

Figure 4. GC/MS chromatogram and chemical composition of the essential oil of Elsholtzia ciliata under 6-BA treatments (0 ppm (A) and 15 ppm (B)). Peak area of essential oil components (C), Monoterpene hydrocarbons (D), Oxygenated monoterpenes (E), Sesquiterpene hydrocarbons (F), and Oxygenated sesquiterpenes (G). Significant differences are marked with asterisks (P < 0.05).

Pearson correlation and principle component analysis

Pearson’s correlations revealed a coherent response of morphological and biochemical traits to 6-BA (Figure 5A). The 6-BA dose was positively associated with the number of leaves (r = 0.73, P ≤ 0.05) and with antioxidant capacity, as indicated by POD (r = 0.71, P ≤ 0.05) and SOD activities (r = 0.58, P ≤ 0.05). Likewise, chlorophyll content correlated positively with 6-BA (r = 0.63, P ≤ 0.05). By contrast, its association with essential-oil yield was weak (r = 0.16). Leaf size traits were tightly intercorrelated (leaf length–width–area; r typically ≥ 0.95) and were strongly linked to fresh weight. Antioxidant enzyme activities showed moderate positive associations with chlorophyll and biomass variables.

The PCA provided an integrated view of these relationships (Figure 5B). PC1 explained 74.0% of the variance and separated the treatments along the 6-BA gradient, with untreated and low-dose plants (0–10 ppm) scoring negatively, whereas higher doses, especially 15 ppm, clustered on the positive side. Traits loading positively on PC1 included SOD and POD activities, chlorophyll content, soluble sugars, number of leaves, and essential-oil yield. PC2 (8.7%) accounted for secondary contrasts, with biochemical variables tending toward the positive quadrant and leaf size/biomass traits projecting more toward the negative PC2 direction. Consistently, the 15-ppm group aligned most strongly with vectors for chlorophyll, antioxidant enzymes, and soluble sugars.

Figure 5. Pearson correlation matrix and principal component analysis of morphological and biochemical traits in Elsholtzia ciliata leaves under 6-BA treatments. (A) Correlation coefficients (circle size and color indicate magnitude and sign); asterisks denote significant correlations (P ≤ 0.05). (B) PCA biplot showing sample scores by 6-BA concentration (95% confidence ellipses) and variable loadings; PC1 and PC2 explain 74.0% and 8.7% of total variance, respectively.

DISCUSSION

Plant growth regulator as cytokinins influence various metabolic pathways, morphological attributes, growth, and plant development (Taiz and Zeiger, 2002). This influence is due to enhanced processes such as protein synthesis, nitrate reductase activity, enzyme function, water uptake, mineral nutrition, photosynthesis, carbohydrate transportation, and pigment biosynthesis (Jan et al., 2023). The primary role of 6-BA is to enhance plant growth parameters. This is achieved through the augmentation of nutrient assimilation, stimulation of cell division, facilitation of shoot formation and elongation, and the delay of senescence (Mangena, 2022). In this study, when applying varying concentrations of 6-BA, enhanced leaf parameters such as number of leaves, length, width, leaf area, and leaf biomass outperformed when compared to the control. These findings are consistent with previous studies that found foliar BA increased plant height, branches, and overall plant dry weight (Abdel-Rahman and Abdel-Kader, 2020; Mangena, 2022; Jan et al., 2023). Research on Melissa officinalis revealed a similar trend, with BA treatment leading to increased side branches, chlorophyll content, number of leaves, plant height, stem diameter, fresh biomass, and various other parameters (Valiyari and Nourafcan, 2018). Plants exhibited accelerated growth in 6-BA treatments at 10 and 15 ppm, achieving higher indices than treatments supplemented with 6-BA at 5, 20, and 25 ppm concentrations. This highlights that an appropriate 6-BA concentration contributes to optimal plant growth. Notably, an excessive concentration of 6-BA can lead to growth retardation (Toscano et al., 2019). According to the plant development cycle, from the time a seed germinates into a seedling, the plant progresses through several stages: growth, flowering transition, flower formation, and blooming (Tho et al., 2023; Tho et al., 2024). In week 4, the plant enters a rapid growth stage. Therefore, 6-BA treatment aids the plant in increasing cell size, synthesizing protein, stimulating the growth of leaf cells, and mobilizing nutrients (Ahmadirad et al., 2024), promoting better growth and prolonging the growth phase.

6-BA markedly increased chlorophyll content in E. ciliata leaves, peaking at 15 ppm before a slight decline at higher doses. Mechanistically, cytokinins like 6-BA delay leaf senescence, thereby preserving chlorophyll and the photosynthetic apparatus (Wang et al., 2022). 6-BA inhibits chlorophyll degradation by downregulating senescence-associated genes and chlorophyll-catabolic enzymes (Zhang et al., 2023). For example, postharvest 6-BA treatment in Brassica leaves suppressed the expression of chlorophyll breakdown genes (such as pheophorbide a oxygenase, pheophytinase, Stay-Green protein) and of a senescence marker (SAG12), resulting in reduced leaf yellowing and prolonged greenness (Zhang et al., 2023). Concurrently, cytokinins can promote chlorophyll biosynthesis and chloroplast development. Cytokinin signaling has been shown to activate genes encoding photosynthetic components (Hudeček et al., 2023). Through these molecular actions, optimal 6-BA treatment maintains higher chlorophyll content in E. ciliata, thereby enhancing light capture and photosynthetic output relative to untreated plants. Soluble sugar levels in E. ciliata leaves also rose substantially under 6-BA, with a maximum (20.46 mg/g FW) at 15 ppm. This positive response is linked to cytokinin’s ability to boost photosynthesis and alter carbohydrate metabolism (Gujjar et al., 2021). By preserving chlorophyll and leaf function (as described above), 6-BA likely increases carbon fixation and thus sugar production. There is evidence that exogenous 6-BA enhances photosynthetic activity, resulting in greater assimilate accumulation. Beyond simply making more sugar, cytokinins also affect sugar distribution and retention. 6-BA is known to modify source–sink relationships by inducing apoplastic invertase and sugar transporters in leaves (McIntyre et al., 2021; Song et al., 2025). In senescing tissues, endogenous cytokinin normally declines, permitting nutrient export; but adding cytokinin reverses this, causing leaves to retain and import sugars (functioning more like sink tissues) (Letham, 2019). Indeed, extracellular invertase is required for cytokinin-mediated delay of senescence, highlighting that source–sink modulation is a key mechanism (Balibrea Lara et al., 2004). By upregulating cell wall invertase, 6-BA promotes sucrose hydrolysis and hexose uptake in leaves, thereby accumulating sugars in the treated foliage (Gulzar et al., 2024; Wei et al., 2024). This results in higher soluble sugar content, as observed in E. ciliata.

Cytokinins also fortify the antioxidant defense system of plants. In E. ciliata, POD and SOD activities increased steadily with 6-BA concentration, peaking at 15 ppm (with significant gains over the control) before a slight reduction at 20–25 ppm. This pattern indicates that moderate 6-BA doses optimally activate antioxidative enzymes. The enhancement of SOD/POD by 6-BA is a well-documented phenomenon associated with its anti-aging and stress-protective roles (Hönig et al., 2018). Molecularly, 6-BA upregulates antioxidant enzyme genes, resulting in higher enzyme protein levels and activities (Gulzar et al., 2024). By boosting SOD and POD, 6-BA helps scavenge reactive oxygen species (ROS) that accumulate during stress or senescence. SOD catalyzes the dismutation of superoxide radicals to H₂O₂, and POD (as well as CAT) then detoxify H₂O₂; together these enzymes mitigate oxidative damage (Hönig et al., 2018; Yang et al., 2018; Gulzar et al., 2024). This antioxidant boost is closely tied to chlorophyll preservation. ROS normally accelerate chlorophyll breakdown and senescence (Hönig et al., 2018; Yang et al., 2018; Gulzar et al., 2024), but 6-BA’s suppression of ROS through SOD/POD activity helps protect chloroplasts and maintain pigment integrity. Empirical studies across species reinforce this link. In maize and wheat, foliar 6-BA applications elevated antioxidant enzymes and improved photosystem II stability under stress (Gulzar et al., 2024). Likewise, in Lamiaceae herbs, maintaining strong antioxidant defenses via cytokinins likely contributes to their stress resilience and extended leaf lifespan (Avasiloaiei et al., 2023; Ismail et al., 2024).

Numerous studies have explored the effects of plant growth regulators on essential oil accumulation using methods such as foliar spraying or in vitro culture (Prins et al., 2010). The underlying mechanisms of these effects on plant growth, such as foliar induction, flower production, or an overall increase in biomass, often result in higher essential oil yields when compared to treatments without plant growth regulators (Prins et al., 2010). In this study, essential oil yields of 6-BA treatment were higher than those of the control. This is substantiated by the work of Povh and Ono (2006), who noted a higher essential oil content in Salvia officinalis following growth regulator treatment, mainly due to an increase in leaf count, which is essential for essential oil synthesis. Furthermore, specialized structures synthesize and store essential oils (Sundar and Parikh, 2023). Cytokinin treatments often boost vegetative growth (more leaves and branches) and stimulate the development of glandular trichomes, thereby providing more sites for oil biosynthesis and storage (Khetsha et al., 2021; Sharma et al., 2022; Dong et al., 2023). Such morphological and physiological changes enhance the plant’s capacity to synthesize and accumulate volatiles. Cytokinins also delay leaf senescence and maintain chlorophyll content, prolonging photosynthetic activity. This improved source-sink status ensures a greater supply of assimilates (sugars and precursor molecules) available for terpene biosynthesis (McIntyre et al., 2021; Dani et al., 2022). The study on the influence of cytokinins on Thymus mastichina by Fraternale et al. (2003) observed a similar effect on secretory structure formation, where an addition of 6-BA at a concentration of 0.1 mg/L in in vitro culture conditions led to increased essential oil yields. In addition to influencing the number of leaves and secretory cell density, BA can affect the essential oil biosynthesis pathway by influencing various steps within the metabolism (Prins et al., 2010). Decendit et al. (1993) and Papon et al. (2005) suggested that BA enhances the activity of the geranyl 10-hydroxylase. Previous studies have shown that adding cytokinins through leaves stimulates essential oil accumulation (El-keltawi and Croteau, 1987), which is consistent with the results of foliar spray methods. This study mainly investigated the effect of BA on the accumulation of essential oil in Vietnamese balm leaves because this is the main part that accumulates essential oil yields of this plant species (Pudziuvelyte, Liaudanskas, et al., 2020; Pudziuvelyte, Marksa, et al., 2020; Luu et al., 2023). The experimental results show that the BA-treated samples yielded higher essential oil content under the same initial extraction as the control. This suggests that BA could influence the density of secretory cells, thereby enhancing essential oil production.

Beyond simply increasing yield, 6‑BA profoundly influenced the composition of the essential oil, skewing it towards certain high-value monoterpenoids. GC–MS analysis revealed that BA-treated E. ciliata (15 ppm) oil was dominated by oxygenated monoterpenes, notably neral and geranial (each 16.22% of total oil), alongside substantial amounts of (Z)-β-farnesene, β-ocimene, and β-caryophyllene. In fact, the total fraction of oxygenated monoterpenes in the 15 ppm BA oil was significantly higher than in control plants (P < 0.05). Cytokinin can modify the metabolic flux of terpene pathways, favoring the production of certain monoterpene derivatives (Danova et al., 2018). Monoterpenes (C₁₀ isoprenoids) are synthesized via the MEP (methylerythritol phosphate) pathway in plastids, whereas sesquiterpenes (C₁₅) derive from the mevalonate pathway (Semmar, 2024; Cheng et al., 2025). Hormonal treatments like cytokinins likely influence these pathways’ enzyme activities, leading to compositional shifts. Indeed, studies have demonstrated that cytokinin application directly enhances the activity of monoterpene-synthesizing enzymes. In Salvia officinalis and Mentha piperita, a cytokinin treatment (diphenylurea, a urea-type CK) significantly upregulated bornyl diphosphate cyclase and limonene cyclase enzymes, respectively, resulting in higher monoterpene accumulation (Radwan et al., 2017; Fiasal and Al-Rekaby, 2023).

CONCLUSION

Foliar application of 6-BA elicited coordinated, dose-dependent enhancements in the growth and physiology of E. ciliata, with 15 ppm emerging as the optimal concentration under the present conditions. Relative to the control, plants receiving 6-BA displayed sustained increases in leaf number, lamina dimensions, and foliar area that translated into significant gains in fresh and dry biomass by week 8. Concomitantly, biochemical performance improved: total chlorophyll and soluble sugars reached their maxima at 15 ppm, while antioxidant capacity, indexed by POD and SOD activities, was likewise strengthened. These physiological improvements were mirrored at the metabolite level, as essential-oil yield rose sharply with treatment and the 15 ppm dose produced both the highest total oil content and a shift toward oxygenated monoterpenes (notably neral and geranial). Correlation analysis and PCA jointly reinforced this pattern, with the 15 ppm cohort aligning with the principal axis of variation that captured elevated pigment status, carbohydrate accumulation, antioxidant activity, and leaf production.

ACKNOWLEDGEMENTS

The authors would like to express their sincere gratitude to the Faculty of Biology, Ho Chi Minh City University, for their invaluable support and assistance throughout this study.

AUTHOR CONTRIBUTIONS

Luong Thi Le Tho: Conceptualization (Lead), Data Curation (Equal), and Project Administration (Lead); Tran Thi Phuong Dung: Formal Analysis (Equal), Writing-Original Draft (Equal); Luu Tang Phuc Khang: Investigation (Lead), Formal Analysis (Equal), Writing-Original Draft (Equal), Software (Lead), Writing-Review & Editing (Lead).

CONFLICT OF INTEREST

The authors declare that they hold no competing interests.

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