Synergistic effects of selenium nanoparticles and LED light on enhancement of secondary metabolites in sandalwood (Santalum album) plants through in-vitro callus culturing technique

Plant material collection

Fresh shoots of Sandalwood plant weighing 0.5 g were aseptically placed onto Murashige and Skoog (MS) medium in glass jars. The medium was supplemented with indole-3-butyric acid (IBA) at a concentration of 3 mg L⁻¹ and kinetin (KN) at a concentration of 1 mg L⁻¹. Additionally, the medium contained 0.8% agar-agar and 3% w/v sucrose. The pH of the medium was adjusted to 5.6 before autoclaving. The cultures were incubated under controlled conditions at a temperature of 18 ± 1 °C and a relative humidity of approximately 76%. The photoperiod was set to 16 h of daylight and 8 h of darkness. Sandalwood plants are adapted to moderate temperatures, and the chosen range of 18 ± 1 °C is close to the ideal conditions found in their native habitats. The 16-hour daylight period provides sufficient light for photosynthesis, which is crucial for growth and biomass accumulation. The 8-hour dark period allows for essential rest and recovery processes, promoting overall plant health and development. The entire procedure was carried out under aseptic conditions to prevent contamination following Sardar et al. (2023).

Elicitation experiment and treatment layout

A variety of colored 12-watt LED lamps were employed, purchased from Amazon (Seattle, WA, USA), including green, red, and blue lights (Empire brand, Tejas brand), with white fluorescent light serving as the control. Sandalwood plants were randomly assigned to each LED treatment within the plant tissue culture lab, maintained at a temperature of 15 °C ± 1 °C with a photoperiod of 16 h of light and 8 h of darkness (Gupta & Sood, 2023).

Dark orange to dark red SeNPs obtained from Sigma Aldrich (https://www.sigmaaldrich.com/specification-sheets/494/341/919519-BULK.pdf; Product No. 919519). According to suppliers, the SeNPs were characterized using inductively coupled plasma–mass spectrometry (ICP–MS) to confirm the elemental composition, verifying the presence of selenium, having size distribution of SeNPs, ranging between 70 and 90 nm. Different SeNP concentrations, including 30, 60, and 90 µg L⁻¹ was prepared for elicitation treatments, with a 0 µg L⁻¹ concentration serves as the control.

The SeNPs were dispersed in phosphate-buffered saline solution to enhance their solubility and stability. This buffer was chosen for its ability to maintain the pH and ionic strength, which are critical for stabilizing SeNPs and preventing aggregation. The stock solution of SeNPs was prepared at a concentration of 1,000 µg/L. The nanoparticles were initially dispersed in the buffer solution using ultrasonication. This process involved subjecting the suspension to ultrasonic waves for 30 min to break down any agglomerates and ensure a homogeneous suspension. For the preparation of different SeNP concentrations (30, 60, and 90 µg/L), precise volumes of the stock solution were transferred to separate containers. The following volumes of the stock solution were used:

• 30 µg/L: (3 mL of the stock solution diluted to 100 mL with the buffer)

• 60 µg/L: (6 mL of the stock solution diluted to 100 mL with the buffer)

• 90 µg/L: (9 mL of the stock solution diluted to 100 mL with the buffer)

Each diluted solution was thoroughly mixed using a vortex mixer to ensure even dispersion of the nanoparticles. This step was crucial to maintain the consistency of the SeNP concentration in each sample. The specific volumes of the prepared SeNP solutions were applied to the experimental setups as per the standardized protocol described in the methods section (Sardar et al., 2023).

For the culture experiment, calli of sandalwood, after 30 days, were dissected into small pieces and placed on sterile filter paper. Subsequently, these pieces were cultured on MS media, and three different concentrations of SeNPs as elicitors along with LED elicitation as described above. The experiment utilized 48 autoclaved test tubes (Table 1), each containing approximately 9 mL of medium, and was sealed with a cotton plug following Sardar et al. (2023). In the first column (T1 to T4), there were 12 test tubes, with four treatments and three replicates for each treatment. The treatments in this column included no elicitation (T1) and elicitation with 30 µg/L, 60 µg/L, and 90 µg/L of SeNPs alone (designated as T2, T3, and T4, respectively). The LED elicitation conditions in this column were Control (T1) with white fluorescent light at 15 °C ± 1 °C with an intensity of 3,000 µmol m⁻² s⁻¹. Moving to the second column (T5 to T8), again consisting of 12 test tubes, there were four treatments, each with three replicates. In this column, the test tubes were exposed to 100% green LED at ∼550 nm wavelength with a bandwidth of 20 nm at 12 peak height along with one of the three concentrations of SeNPs (30 µg/L, 60 µg/L, and 90 µg/L), labelled as T5, T6, T7, and T8, respectively. In the third column (T9 to T12), another 12 test tubes were arranged with four treatments and three replicates each. These test tubes were subjected to 100% red LED at ∼660 nm wavelength with a bandwidth of 20 nm at 12 peak height along with 30 µg/L, 60 µg/L, and 90 µg/L of SeNPs, forming the treatments T9, T10, T11, and T12, respectively. Lastly, the fourth column (T13 to T16) also comprised 12 test tubes with four treatments and three replicates per treatment. These test tubes were exposed to 100% blue LED at ∼460 nm wavelength with a bandwidth of 20 nm at 12 peak height along with 30 µg/L, 60 µg/L, and 90 µg/L of SeNPs, designated as T13, T14, T15, and T16, respectively. In the LED light elicitation experiments for sandalwood callus cultures, a light intensity of 3,000 µmol m⁻² s⁻¹ was maintained, accompanied by a constant temperature of 15 °C ± 1 °C. The photoperiod was set to 16 h of continuous light followed by 8 h of darkness, and the cycling pattern employed was continuous throughout the experimental duration. Each column represented different combinations of SeNPs and LED light, with specific concentrations, creating a comprehensive experimental design with a total of 48 test tubes, each with unique elicitation conditions for further analysis and comparison. After 50 days, data on fresh weight (FW), dry weight (DW), callus moisture content, and number of shoot branches per callus were collected. To ascertain the fresh weight (FW), calli obtained from each treatment underwent cleansing with sterile distilled water. Subsequently, they were positioned on filter paper, compressed with forceps to eliminate surplus water, and weighed. Following this, each callus underwent a 24-hour drying process in an oven set at 50 °C before being re-weighed. The obtained fresh weight (FW) and dry weight (DW) data were converted to grams per litter. The percentage moisture content was determined by employing the FW and DW of the callus, utilizing the formula outlined by Rashmi & Trivedi (2014).

Percentage moisture contents = [(B-A) - (C-A)]/(B-A) X 100.

Letting A represent the weight of the empty petri dish, B denotes the weight of the petri dish with fresh callus, and C signifies the weight of the petri dish with dried callus, observations were meticulously recorded. These observations encompassed the characteristics of callus nature, response and colour. This systematic approach aimed to comprehend the effects of elicitation on the morphological health of the callus.

Extract preparation

Callus cultured in vitro (100 g) was harvested, rinsed with distilled water, freeze-dried, and finely powdered using a mortar and pestle. The resulting powder was then subjected to an 18-hour extraction in dichloromethane: methanol (1:1, v/v) at 40 °C, maintaining a ratio of 1:200 (w/v) of plant material to solvent. After extraction, solid residues were removed through filtration using Whatman No. 1 filter paper and subsequent centrifugation at 5,000 g for 10 min. The resulting supernatant was concentrated utilizing an Eyela N-N series rotary evaporator coupled with an Eyela aspirator (Model: A 3S; Rikakikai Inc., Tokyo, Japan) at 40 °C under reduced pressure. The obtained sandalwood extract was stored at −20 °C for future use. After 50 days of callus cultivation, the extract was prepared for the study of phytochemicals (Misra & Dey, 2012).

Determination of casein/BSA/PVPP-bound tannins

For casein/BSA/PVPP-bound tannins, 100 mg of BSA/casein or PVPP was added to 200 µl of the extract, followed by shaking for 1 h at room temperature and incubation at 4 °C for 1–2 h. The mixture was then filtered or centrifuged, and the filtrate represented unbound polyphenols. Bound tannins were calculated as the difference between total polyphenols and unbound polyphenols, and the results were expressed as milligrams of catechin per gram of the extract (Schneider, 1976)

Determination of total flavan-3-ols content

In the determination of total flavan-3-ols content, a sample solution containing 1 mg/ml of the extract in methanol was prepared. To 200 µl of this sample, 1 ml of p-dimethylamino cinnamaldehyde (DMACA) reagent (0.1% in 1 N HCl in methanol) was added. The mixture was vortexed and allowed to stand at room temperature for 10 min. During this time, a blue color developed, and the intensity was measured at 640 nm using a spectrophotometer. Catechin was used as the standard, ranging from 25 to 150 µg, and the results were expressed as milligrams of catechin per gram of the extract. This method was adapted from the reference provided (Arnous, Makris & Kefalas, 2001).

Determination of total saponin content

To determine the total saponin content, a sample solution containing 1 mg/ml of the extract in methanol was prepared. Ten microliters of this extract were mixed with 50 µl of 8% vanillin in ethanol and 500 µl of 72% H₂SO₄. The mixture was heated to 60 °C for 20 min and then cooled to 4 °C for 5 min. During this process, a yellow/green color developed, and the absorbance was measured at 544 nm using a spectrophotometer. Sapogenin was used as the standard, ranging from 10 to 100 µg, and the results were expressed as milligrams of sapogenin per gram of the extract. This method was adapted from the reference provided (Makkar, Siddhuraju & Becker, 2007).

Total terpenoid contents

In the total terpenoid content assay, 100 µg of the plant extract dissolved in methanol was used. To this, 500 µl of 2% vanillin- H₂SO₄ solution in cold methanol was added. The mixture was heated to 60 °C and kept at this temperature for 20 min, then cooled to 25 °C for 5 min. Within the next 20 min, the absorbance of the solution was measured using a spectrophotometer at a wavelength of 608 nm. The color of the solution changed to blue–green, and the absorbance at 608 nm was recorded. Linalool was used as the standard, ranging from 20 to 100 mg/l, and the results were expressed as micrograms of linalool per milligram of the extract. This method was adapted from the reference provided (Misra & Dey, 2012).

Determination of total flavonoid content and total phenolic contents

Sandalwood plant extract’s total flavonoid content (TFC) was evaluated using a modified method by Ebrahimzadeh, Pourmorad & Bekhradnia (2008). The plant extract in methanol was combined with AlCl2 and sodium acetate, and after incubation, absorbance was measured at 415 nm. Quercetin served as a standard for calibration, and the flavonoid content was expressed as milligrams of quercetin equivalent per gram of extract (mg QE g⁻¹). For total phenolic content (TPC) in sandalwood callus extracts, a modified method based on Kim, Jeong & Lee (2003) was employed. The plant extract was mixed with Folin–Ciocalteu reagent and Na₂CO₃, and after incubation, absorbance was measured at 750 nm. Gallic acid was used as a standard for calibration, and TPC was expressed as milligrams of gallic acid equivalent per gram of extract (mg GA g⁻¹).

Determination of DPPH scavenging percentage

The DPPH free radical scavenging assay was employed to assess the antioxidant activity of sandalwood culture extracts. A combination of plant extract (50 µL) and 3 mL of methanolic DPPH solution (0.004%) was incubated in darkness at room temperature for 30 min. Subsequently, the absorbance was measured at 517 nm using a UV–visible spectrophotometer. The percentage of free radical scavenging activity (%RSA) was then calculated using the following formula:

%RSA = {(Absorbance of control - Absorbance of Sample)/Absorbance of control}×100, with BHT (Sigma-Aldrich) as the standard (Sardar et al., 2023).

Determination of activities of bioactive antioxidants

To evaluate the antioxidant enzyme activity of specific callus samples, a modified version of the methods described by Khan et al. (2013), Giannopolitis & Ries (1977), Arrigoni et al. (1992), and Abeles & Biles (1991) was employed. Each sample was homogenized, and the resulting supernatants were utilized to measure the activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and phenylalanine ammonia-lyase (PAL). PAL activity was assessed through the absorbance fluctuation at 290 nm.

Determination of anthocyanin and tocopherol contents

The callus was dried and pigments were extracted. Chromatography was performed using butanol-acetic acid-water (BAW) in a 4:1:5 ratios, butanol-hydrochloric acid (Bu-HCl) in a 1:1 ratio, and 1% hydrochloric acid (HCl) as described by Yashin & Yashin (2006). Anthocyanin content was measured at a wavelength of 525 nm.

The alpha-tocopherol content was determined by first extracting the compound from the sample using hexane as the solvent. The sample was weighed and homogenized with hexane, and the resulting mixture was filtered to separate the liquid phase containing alpha-tocopherol. The hexane was then evaporated using a rotary evaporator to concentrate the extract. Chromatographic separation was performed with a C18 reversed-phase column, employing a mobile phase consisting of hexane and isopropanol as specified by Baker Jr (1981). Alpha-tocopherol was detected using a UV-Vis spectrophotometer set to 295 nm. A calibration curve was prepared with standards of known alpha-tocopherol concentration, and this curve was used to quantify the alpha-tocopherol content in the sample.

Experimental design and statistical analysis

A randomized experimental design was adopted for all investigations, ensuring a comprehensive and unbiased approach. Each treatment underwent replication three times to enhance the reliability of the results. The collected data were subjected to rigorous analysis through a two-way analysis of variance (ANOVA), utilizing Costat software version 6.3. To determine mean values and assess statistically significant differences (p < 0.05), the Turkey Honestly Significant Difference (HSD) test was applied (Mazhar, Akram & Shahid, 2022; Mazhar et al., 2023). The Turkey HSD test is widely used for post hoc analysis in situations where multiple pairwise comparisons are needed, providing a robust method to identify differences between treatment means while controlling the experiment-wise error rate.

Influence on callus formation and appearance

Under white fluorescent light (WFL) exposure as a control treatment, the callus exhibited a vulnerable nature, with a somewhat lackluster response and a dull yellow coloration. Conversely, the green LED Light (G) treatments showcased a range of callus natures, transitioning from fragile to partially firm and moderately dense. The callus response improved significantly, spanning from poor to very good, and the colors evolved from a golden hue with a touch of brown to yellow-greenish and moderately greenish shades. In the context of red LED Light (R) exposure, the callus displayed a tender nature in lower concentrations of SeNPs (30 µg/L) and a resilient nature in higher concentrations of SeNPs. The callus response exhibited a notable enhancement, progressing from poor to very good, with color variations from yellow-brown to yellow-greenish and finally to bright greenish hues. The callus response, initially poor, showed remarkable improvement, reaching an excellent rating. The color spectrum also shifted from yellow-brown to bright greenish and brilliantly greenish shades upon elicitation with SeNPs and blue light. Specifically, the higher doses of SeNPs (60 and 90 µg/L) were better in callus formation. Similarly, red and blue LED lights were more beneficial in improving callus appearance and formation (Table 2).

The presence and concentration of SeNPs in the experimental treatments played a significant role in shaping the callus formation and appearance in sandalwood. As observed in the results, treatments with higher concentrations of SeNPs generally led to more favorable callus responses. For instance, under green LED light (G) exposure, callus treated with 90 µg/L of SeNPs exhibited a moderately dense nature and an excellent callus response, indicating that the presence of SeNPs promoted the formation of healthier and more robust callus tissue. Similar trends were observed in red LED light (R) and blue LED light (B) treatments, where higher SeNP concentrations resulted in resilient callus with significantly improved responses, ranging from very good to excellent (Table 2). A pictorial description of the callus has been presented in Fig. 1.

Influence on callus growth and moisture contents

Table 3 provides detailed information on callus growth and moisture content in sandalwood as influenced by different concentrations of SeNPs and LED light exposure. The data is presented in terms of callus fresh weight (in grams), callus dry weight (in grams), callus moisture content (in percentage), and the number of shoots per callus. The results show that as the concentration of SeNPs increases, there is a general trend of increased callus fresh weight across all types of LED light exposure. For example, under blue LED light (B) exposure, the callus fresh weight ranges from 110.77 g (for 0 µg/L + B) to 149.33 g (for 90 µg/L + B), demonstrating a significant increase with higher SeNPs concentration. Similar to callus fresh weight, the data indicates an increase in callus dry weight with higher concentrations of SeNPs. For instance, under red LED light (R) exposure, the callus dry weight varies from 23.87 g (for 0 µg/L + R) to 35.50 g (for 90 µg/L + R), showing a substantial rise with increasing SeNPs concentration. All the treatments improved callus growth and moisture content (Table 3). However, 90 µg/L + B was the most effective. The number of shoots per callus indicates the number of shoots that have developed from each callus tissue. Higher SeNP concentrations, especially under red LED light (R) and blue LED light (B) exposure, result in an increased number of shoots per callus. For example, under red LED light, the number of shoots per callus ranges from 18.00 (for 0 µg/L + R) to 21.46 (for 90 µg/L + R), demonstrating a positive effect of SeNPs on shoot development.

Influence of LED and SeNPs elicitation on bioactive antioxidants

Table 4 presents data on the influence of LED elicitation and SeNP treatments on the activities of bioactive antioxidants in the callus extracts of sandalwood. The measured parameters include PAL (phenylalanine ammonia-lyase) activity, superoxide dismutase activity, peroxidase activity, and catalase activity, all expressed in units per gram fresh weight (U/g FW). The data shows that PAL activity increased with higher concentrations of SeNPs and varied with different LED light exposures. For instance, under blue LED light (B) exposure, PAL activity increased from 2.99 U/g FW (for 0 µg/L + B) to 6.46 U/g FW (for 90 µg/L + B), indicating a significant enhancement in the phenylpropanoid pathway due to SeNPs treatment. The results demonstrate an increase in SOD activity with higher SeNP concentrations, especially under green LED light (G) and blue LED light (B) exposures. This suggests that SeNP treatment enhances the plant’s ability to counteract oxidative stress by increasing SOD activity. The data shows that peroxidase activity increased significantly with SeNP treatment, particularly under red LED light (R) and blue LED light (B) exposures. Higher SeNP concentrations led to elevated peroxidase activity, indicating an enhanced capacity for oxidative stress management in the treated callus tissues. The results reveal an increase in catalase activity with higher SeNP concentrations, especially under green LED light (G) and blue LED light (B) exposures. This implies that SeNP treatment enhances the plant’s ability to mitigate oxidative damage by increasing catalase activity.

Impact of elicitation treatments on total phenolics, flavonoids, saponins, and anthocyanin accumulation

Table 5 presents the influence of LED elicitation and SeNP treatments on the accumulation of total phenolic, flavonoids, saponins, and anthocyanin in the callus extracts of sandalwood. The data is expressed in milligrams of quercetin equivalents per gram (mg QE/g) for flavonoids, milligrams of gallic acid equivalents per gram (mg GA/g) for phenolics, micrograms of saponin equivalents per milligram (µg saponin E./ mg) for saponins, and milligrams per 100 grams (mg/100 g FW) for anthocyanins. All the elicitation treatments significantly enhanced the mentioned variables. The individual effect of LED and SeNPs was more pronounced when used in synergism. The treatments with the highest flavonoid content include 90 µg/L + B (blue LED light, 90 µg/L SeNPs) with 0.10 mg QE/g, indicating a significant increase in flavonoid accumulation. Phenolics are diverse compounds with various biological activities. The treatments with the highest phenolic content include 90 µg/L + B (blue LED light, 90 µg/L SeNPs) with 19.11 mg GA/g, suggesting a substantial enhancement in phenolic compound accumulation. Similarly, the treatment with the highest saponin content includes 90 µg/L + G (green LED light, 90 µg/L SeNPs) with 6.79 µg saponin E./ mg, indicating a notable increase in saponin accumulation. The treatments with the highest anthocyanin content include 90 µg/L + B (blue LED light, 90 µg/L SeNPs) with 5.96 mg/100 g FW, indicating a significant increase in anthocyanin accumulation. The table shows that the red and blue lights are better at increasing phytochemicals.

Influence of elicitation on casein/BSA/PVPP-bound tannins (TCTC), flavan-3-oils, terpenoids, and tocopherol contents

Table 6 presents the influence of LED elicitation and selenium nanoparticles (SeNPs) treatments on the content of casein/BSA/PVPP-bound tannins (TCTC), flavan-3-ols, terpenoids, and tocopherols in the callus extracts of sandalwood. All the treatments significantly enhanced the contents of TCTC, flavan-3-ols, terpenoids, and tocopherols in the callus extracts of sandalwood. However, higher doses of SeNPs and blue and red LED light elicitations were more effective, specifically when they were employed in synergism. Treatments with the highest terpenoid content include 90 µg/L + B (blue LED light, 90 µg/L SeNPs) with 14.88 µg linalool E./mg, indicating a significant increase in terpenoid accumulation. The treatments with the highest flavan-3-ol content include 90 µg/L + B (blue LED light, 90 µg/L SeNPs) with 7.54 µg catechin E./mg, suggesting a substantial enhancement in flavan-3-ol accumulation. The treatments with the highest tannin content include 90 µg/L + B (blue LED light, 90 µg/L SeNPs) with 3.45 µg catechin E./mg, indicating a notable increase in tannin accumulation. Treatments with the highest α-tocopherol content include 90 µg/L + B (blue LED light, 90 µg/L SeNPs) with 4.90 µg/g FW, demonstrating a significant enhancement in tocopherol accumulation. The treatments with 90 µg/L of SeNPs combined with blue LED light (90 µg/L + B) appear to be the most effective in promoting the accumulation of terpenoids, flavan-3-ols, tannins, and tocopherols in sandalwood callus extracts. These treatments demonstrate significant enhancements in the levels of these bioactive compounds, indicating their potential applications in the pharmaceutical, cosmetic, and food industries due to their medicinal and antioxidant properties.

Figure 2 shows the DPPH scavenging percentage of the callus extract of sandalwood elicited with LED light and various elicitation doses of SeNPs. The DPPH scavenging percentage is a measure of the ability of the extract to scavenge DPPH, a free radical. A higher DPPH scavenging percentage indicates that the extract is more effective at scavenging free radicals. The DPPH scavenging percentage of the extract increased with increasing elicitation doses of SeNPs. This suggests that SeNPs can enhance the antioxidant activity of sandalwood extracts. The figure also shows that the DPPH scavenging percentage of the extract was higher when the callus cultures were elicited with LED light than when they were not elicited with LED light. This suggests that LED light can also enhance the antioxidant activity of sandalwood extracts. Treatment 16 involving the combined use of 90 µg/L SeNPs and blue light was most significant in increasing the DPPH scavenging capacity of the callus extract.

Statistical explanation of correlation matrix, ANOVA, and principal component analysis

The results from the two-way ANOVA (Table 7) indicate that both SeNP concentrations and the type of LED light exposure have significant effects on various biochemical and physiological parameters in sandalwood callus cultures. The interaction between SeNPs and LED light further contributes to the observed variations, highlighting the complex interplay between these factors in shaping the biochemical composition and antioxidant potential of sandalwood callus extracts. Kendell’s correlation matrix is shown in Table 8. There are strong positive correlations (ranging from 0.90 to 1) between various biochemical parameters indicating that these variables tend to increase or decrease together in response to the treatments. A strong positive correlation (0.94 to 0.99) was observed for growth variables, suggesting that these growth-related parameters are closely related and respond similarly to the experimental conditions. There are no significant negative correlations among the studied variables, indicating that none of the variables move in the opposite direction under the experimental conditions.

Figure 3 is a principal component analysis (PCA) biplot, designed to visualize the relationships between variables in a dataset. The biplot displays two principal components (PCs), which are represented by the axes of the biplot, and the variables are represented by vectors. The length of a vector indicates the variable’s contribution to the corresponding PC, and the angle between two vectors indicates their correlation. In this particular biplot, most of the variables are located in the positive quarter of the plot. This indicates that they tend to increase or decrease together. For example, CAT, SOD, and POD all tend to increase together. The two PCs that are displayed in the biplot account for 98.32% of the variance in the data. This means that they can capture most of the important information in the dataset.