Melatonin-Induced Stress Enhanced Biomass and Production of High-Value Secondary Cell Products in Submerged Adventitious Root Cultures of Stevia rebaudiana (Bert.)

Abstract

Stress is one of the important factors that directly or indirectly affects the plant architecture, biochemical pathways, and growth and development. Melatonin (MEL) is an important stress hormone; however, the exogenous addition of melatonin to culture media stimulates the defense mechanism and releases higher quantities of secondary metabolites. In this study, submerged adventitious root cultures (SARCs) of diabetically important Stevia rebaudiana were exposed to variable concentrations (0.5–5.0 mg/L) of MEL in combination with 0.5 mg/L naphthalene acetic acid (NAA) to investigate the biomass accumulation during growth kinetics with 07 days intervals for a period of 56 days. The effects of exogenous MEL on the biosynthesis of stevioside (Stev.), total phenolics content (TPC), total flavonoids content (TFC), total phenolics production (TPP), total flavonoids production (TFP), total polyphenolics content (TPPC), fresh and dry weight (FW & DW), and antioxidant potential were also studied. Most of the SARCs displayed lag, exponential, stationary, and decline phases with variable biomass accumulation. The maximum fresh (236.54 g/L) and dry biomass (28.64 g/L) was observed in SARCs exposed to 3.0 mg/L MEL and 0.5 mg/L NAA. The same combination of MEL and NAA also enhanced the accumulation of TPC (18.96 mg/g-DW), TFC (6.33 mg/g-DW), TPP (271.51 mg/L), TFP (90.64 mg/L), and TPPC (25.29 mg/g-DW). Similarly, the highest stevioside biosynthesis (91.45 mg/g-DW) and antioxidant potential (86.15%) were observed in SARCs exposed to 3.0 mg/L MEL and NAA. Moreover, a strong correlation was observed between the biomass and the contents of phenolics, flavonoids, antioxidants, and stevioside. These results suggest that MEL is one of multidimensional stress hormones that modulate the biosynthetic pathways to release higher quantities of metabolites of interest for various industrial applications.

1. Introduction

Plants are exposed to variable stresses in the environment, which cause oxidative stress and damage plant cells and tissues.¹⁻³ These stresses may be biotic or abiotic, which directly or indirectly modulate biochemical pathways, affect physiological and morphological behaviors, regulate gene expression, and ultimately affect plant growth and development.⁴⁻⁷ In response, plants release either chemicals or enzymes to prevent the cells and tissues from undergoing oxidative damage.⁸⁻¹⁰ Melatonin (N-acetyl-5-methoxytryptamine) is one of the potent stress hormones that protect plants from various stresses and also influence photosynthesis,¹¹ organogenesis,¹² and root morphogenesis.¹³ Melatonin (MEL) plays an important role in regulating several biological processes, including seed germination, plant growth, and stress responses. Exogenous melatonin positively or negatively influences adventitious root formation, which is dependent on the concentration of melatonin application.¹ MEL is the most effective scavenger of different reactive oxygen species (ROS) and reactive nitrogen species (RNS). These molecules or species include oxygen radicals, hydroxyl radicals, lipid peroxyl radicals, superoxide anions, nitric oxide, peroxynitrous acid, peroxynitrite anions, hydrogen peroxide, hypochlorous acid, and also singlet oxygen.¹⁴ All of these are hazardous reactive molecules. Different redox enzymes are regulated by melatonin in various organisms, including plants. Antioxidant enzymes such as peroxidases, superoxide dismutase, and catalases are upregulated, while pro-oxidant enzymes such as lipoxygenases and nitric oxide are downregulated.¹⁵ These enzymes act as anticancerogenic, anti-inflammatory, and also gero-protecting agents that protect against aging. The mitochondrial fluid has a high concentration of melatonin that is expelled out through the plasma in high amounts, enabling the scavenging effect of melatonin and causing the expression of antioxidant enzymes.^16,17

In plants, MEL is also responsible for the circadian rhythm.¹⁸ The circadian rhythm regulates several biological processes, including daily cycles or seasonal cycles, stability of proteins, gene expression, and metabolism.¹⁸ When the circadian rhythm is regulated, it will increase photosynthesis and the response to biotic and abiotic stresses. It will also enhance the rate of growth and seed production in crops.¹⁹ Furthermore, it is believed that the chemical structure of melatonin, which is a derivative of indole, and indole acetic acid (IAA) might be related to each other, and also, the biosynthetic pathway of melatonin is quite similar to that of IAA.²⁰ Both indole acetic acid (IAA) and melatonin are involved in several physiological functions, but the important roles of these two hormones are as growth regulators and growth promotors.²¹

MEL maintains the physiological function of plants. It also plays an important role in the regulation of the circadian rhythm in plants as well as in other animals.¹ Previously, it has been reported that in a stressful environment, MEL protects the growth and integrity of plants. MEL plays a role in the shoot and root development from any explant,²² and the involvement of melatonin in plant development was first discovered and reported in 2005. The research was conducted by Hernandez-Ruiz,²³ who observed that when the callus was incubated in a solution containing varied levels of MEL, it increased organogenesis, especially root and shoot morphogenesis. Hernandez²³ performed this experiment on grass wheat, oat, barley, and most of the older plants. According to Park,²⁴ melatonin is structurally similar to plant IAA. During germination, MEL is now known to alter the characteristics of a plant,²⁵ seedling growth, and the timing of flowering, grain yield, and senescence. Unexpectedly, MEL was found to be involved in late flowering, but it boosted seedling growth. MEL plays a major role in extending the shelf life of fruits and vegetables and also in the storage of trees, both of which may lead to lower post-loss of fresh horticultural commodities.²⁶ It is possible that MEL plays an auxin-like function in the development of roots and is sometimes involved in shoot formation because auxin is naturally synthesized in shoots. MEL binds to the auxin receptor and acts as an auxin antagonist. Hernandez-Ruiz²⁴ concluded from his experiment that MEL increases the elongation of roots in the Lupines albus plant. Another experiment conducted by Murch and Saxena²⁶ shows that MEL is involved in organogenesis and callus formation. According to Reiter et al.,²⁷ when animals consume such plants, particularly the leaves and roots containing high amounts of MEL, they show high levels of MEL in their blood. Such reports can be used for the enhancement of crop yields as the agricultural basis for the development of plants in high demand for medical, horticultural, and agronomic purposes.

Elicitation of culture media with different types of stressors or elicitors (biotic or abiotic) may enhance the productivity of metabolites of interest.^28,29 Elicitation strategies are gaining more industrial interest for uniform and higher productivity of commercial secondary cell products.³⁰ Elicitation strategies are commonly employed for high-value medicinal plants. MEL is considered a potent elicitor, and a combination of MEL with other auxins synergistically promotes primary and secondary metabolism, root organogenesis, and other physiological functions of cell cultures of various medicinal plants.^23,27,31

Stevia rebaudiana (often known as Stevia) is one of the active perennial plants in the Asteraceae family and contains higher levels of natural sweeteners in the leaves.^32,33 It is also known as honey plant, sweet leaf, honey leaf, or sweet herb due to its natural ability to produce sweetness.^29,34 It is spread throughout the tropical and subtropical areas.²⁹ It contains the economically significant component stevioside, which has been utilized in several nations to manage diabetes and obesity. Many nations, including Canada, China, Japan, Mexico, South America,⁸ United Kingdom, the United States, Korea, Malaysia, Brazil, and Paraguay, now regularly cultivate stevia to extract stevioside for various industrial applications.^8,35,36 The Guarani tribes of Brazil and Paraguay have been using stevia leaves for more than 1500 years to sweeten their traditional beverages and for the treatment of various common diseases.³⁶ Stevioside is 300 times sweeter than commercial sugar and contains no calories.³⁵ Stevia has a sweet flavor and is famous for its antioxidant activities.²⁹ Additionally, its bioactive chemicals include anticancer, anti-hyperglycemic, and anti-hypersensitive properties, as well as the ability to prevent dental cavities.^37,38 There are no specific receptors in the human body to absorb it, and therefore, natural stevioside cannot reach the bloodstream of the human body, which is why this plant is known to be low caloric, and the majority of the reports recommend it to obese and diabetic patients.³⁹⁻⁴² There are still no cases of a single patient having fully recovered from diabetes after using different synthetic medications. In the year 2025, 57 million individuals are predicted to be diabetic.^8,36,37 As a natural alternative to sugar and other artificial sweeteners, stevia extracts have no known diverse effects and can be used by diabetic patients to minimize their blood glucose levels.³⁹⁻⁴¹

S. rebaudiana plants are traditionally propagated via cutting, although this method does not produce a large number of plants.⁴³S. rebaudiana has tiny seeds with a very low germination rate, and the stem cuttings are unable to produce larger biomass for the current industrial demand (Ahmad et al., 2022a). In order to produce homogeneous biomass and phytochemicals, in vitro culture technologies such as cell culture and in vitro shoot, hairy root, and adventitious root cultures are utilized.^29,44 Submerged adventitious root cultures (SARCs) are powerful culture systems when compared to other cultures because they can be easily scaled up from flask cultures to bioreactors.⁴⁵ The best outcomes for the biosynthesis of bioactive substances, such as mother plants, come from SARCs. Furthermore, it has been observed that the majority of the bioactive substances are commonly obtained from the roots of different plants.⁴⁶ When compared to conventional propagation, SARCs of medicinal plants such as S. rebaudiana and others can offer uniform and homogeneous quantities of secondary metabolites and are simple to grow as compared to other cell culture technologies.^45,47 Adventitious root cultures are the best choice for the biosynthesis of biomass and secondary metabolites of interest because they provide a practical approach compared to other in vitro methods. The adventitious root culture requires a short time, develops quickly, can be scaled up, and the culture parameters, including both chemical and physical factors, are easily regulated.^8,45,48

Therefore, the overall objective of the current study was to investigate the effect of the stress hormone (MEL) on the growth kinetics, large-scale biomass, fine chemicals such as phenolics and flavonoids, antioxidant potential, and especially the stevioside content in SARCs of S. rebaudiana.

2. Materials and Methods

This research work was conducted at the Centre for Biotechnology and Microbiology, University of Swat. An independent experiment for each treatment of MEL + NAA was used in this study to develop SARCs of S. rebaudiana.

2.1. Plant Materials

The black-coated, viable, and fertile plant materials (seeds) of the medicinally important plant (Stevia) were procured from the Pakistan Council for Scientific and Industrial Research, Peshawar, and the Horticulture Department, The University of Agriculture Peshawar. The plants were authenticated by the expert taxonomist of the Medicinal Botanic Garden, MBC, PCSIR, Peshawar, Pakistan.

2.2. Surface Sterilization of Seeds

Black-coated fertile seeds were surface-sterilized according to the protocol of Ahmad et al.⁴⁹ Seeds were washed with sterile distilled water and then exposed to mercuric chloride (0.2%) for 5 min to remove surface contamination.

2.3. Melatonin and Naphthalene Acetic Acid Stock Solution Preparation

Stock solutions of MEL (Sigma; Pakistan) and naphthalene acetic acid (NAA; Sigma; Pakistan) were prepared by adding 100 mg of MEL and NAA powder separately in 100 mL of distilled water to obtain a stock solution of 1 mg/1 mL. The stock solution was placed in an aseptic jar in a refrigerator at 4 °C.

2.4. Culture Media Preparation

In this study, solid Murashige and Skoog (MS; 1962; Phyto-Tech) media was used as the primary medium for seed germination, while liquid MS media was used for the inoculum and SARC development. Here, 1000 mL of distilled water was mixed with 30 g of sucrose and 4.43 g of MS salt according to the protocol of Ahmad et al.⁸ For seed germination, MS media having no PGRs was exploited. According to the literature and our previous reports, 0.5 mg/L NAA was augmented into the culture media for inoculum root development. The media was autoclaved at 121 °C and 15 psi for 20 min, and the pH was adjusted to 5.7. Practically, 8 g of agar was used for solidification of the media, while, for SARC development, liquid media without agar was used.

2.5. Aseptic Inoculation of Seeds for Inoculum Development

The inoculation of Stevia seeds was carried out in a laminar air-flow unit (LFU) aseptically. Before inoculation, the LFU was washed with 70% ethanol (Sigma; Pakistan). Then, the media, along with all other instruments and materials, were exposed to UV light for 15–20 min according to the protocol of Ahmad et al.³⁶ After sanitization, the seeds were aseptically transferred to culture media for plantlet development.

2.6. Growth Conditions

The flasks containing multiple viable seeds were shifted to the growth chamber under luminous tube lights with a 16/8 h photoperiod with optimal light intensity. The temperature of the growth room was adjusted between 23 and 27 °C. Each treatment was prepared into three independent experiments. Each experiment follows a completely randomized design (CRD). After 1 month of seed germination, the roots were excised for stock adventitious root development.

2.7. Melatonin Concentration for the Development of SARCs

Plantlets were regenerated according to the method of Ahmad et al.,⁵⁰ and the seed-derived roots were collected after 30 days of plantlet development. At the initial stage, the roots were shifted to NAA (0.5 mg/L)-augmented MS media for stock culture development. The flasks containing roots in liquid media were placed on an orbital shaker (Thermo Scientific; Germany) in the dark for 30 days. After 30 days, a known and uniform amount of adventitious roots were exploited for each experiment and each treatment. All experiments were carried out in 250 mL Erlenmeyer flasks comprising 50 mL of MS media with different concentrations of melatonin (0.5–5.0 mg/L), 30 g/L sucrose, and a pH level of 5.6–5.8. Fresh adventitious root biomass and growth kinetics were studied at 7 day intervals for a period of 56 days. The growth curve was established for growing adventitious roots. The color and morphology of adventitious roots were studied along with the growth kinetics. The details of the experiment and their treatments are given in Table 1.

Table 1. Different Concentrations (0.5–5.0 mg/L) of the Stress Hormone (MEL) in Combination with Constant NAA (0.5 mg/L) and Control MS-Liquid Media Containing NAA without MEL for SARC Development.

treatments	liquid MS media	melatonin conc. (mg/L)	NAA (mg/L)
T1	full strength	MEL 0.5	0.5
T2	full strength	MEL 1.0	0.5
T3	full strength	MEL 1.5	0.5
T4	full strength	MEL 2.0	0.5
T5	full strength	MEL 2.5	0.5
T6	full strength	MEL 3.0	0.5
T7	full strength	MEL 3.5	0.5
T8	full strength	MEL 4.0	0.5
T9	full strength	MEL 4.5	0.5
T10	full strength	MEL 5.0	0.5
T11 (C)	full strength	MEL 0.5	0

2.8. Collection and Storage of SARCs of Stevia

The fresh biomass of adventitious roots was collected after full establishment. It was washed with sterile distilled water, gently pressed with filter paper, and weighed. The fresh biomass data were used for the establishment of the growth kinetics curve. The fresh biomass of roots was dried in an oven (Thermo Scientific; Germany) at 50 °C. These dried materials were powdered with the help of an electrical blender. The powdered material was used for the quantification of the stevioside content and the production of other important fine chemicals of interest.

2.9. Analytical Methods

2.9.1. Extract Preparation

The SARC from each treatment was used separately for the preparation of extracts for various activities. From each treatment, the oven-dried SA roots were subjected to electrical blending for powder formation. The dried powder of each SARC (10 mg) was added to a Falcon tube (50 mL) containing 10 mL of ethanol (HPLC grade; Merck, Pakistan). For instance, the amount of powder and ethanol was increased when needed. The mixture was incubated on an orbital shaker in the dark for 24 h. Shaking was used to release the maximum amount of intracellular metabolites into ethanol. After overnight incubation, the mixture from each treatment was subjected to centrifugation (8000 rpm, 10 min) to separate the cell debris from the solvent. The supernatant of each treated sample was stored and subsequently used to determine the phytochemical profile and antioxidant potential.

2.9.2. Estimation of the Total Phenolics Content (TPC) in SARCs

A recent protocol by Ahmad et al.⁴⁴ was used for the estimation of TPC in SARCs of S. rebaudiana exposed to varied concentrations of MEL. During the assay, solution A (2.25 mL of 2N Folin–Ciocalteu reagent) was mixed with each sample extract (0.1 mL) of SARCs with further addition of solution B (20% Na₂CO₃) and then placed in the dark to avoid oxidation and perform possible reaction. A 96-well microtiter plate reader (Thermo Scientific; Germany) was used for the determination of TPC in each treated sample of SARCs. According to the recent protocol, a wavelength of 765 nm was used to determine the optical density (absorbance) of the sample and compared with a standard such as gallic acid (GA) purchased from Sigma (Germany). For obtaining the standard curve for GA, different concentrations were applied (1.0–10 μg/mL) with a prominent R² value (0.9773). The TPC content in each treated sample of SARCs was compared with GA, and the results were calculated in mg/g-DW equivalent to GA using the basic formulas according to the protocol as follows

where “T” represents each treated sample extract of SARCs of stevia with absorbance at 765 nm using a microtiter plate reader.

The dry biomass of each treated SARC was multiplied by the TPC for the determination of TPP (mg/L), while the TPPC was obtained by adding TPC and TFC and expressed in the same unit (mg/g-DW).

2.9.3. Estimation of the Total Flavonoid Content (TFC) in SARCs

A recent protocol by Ahmad et al. (2021) was used for the estimation of TFC in SARCs of S. rebaudiana exposed to various concentrations of MEL. The aluminum chloride (5% w/v; Sigma Germany) method was used for the determination of TFC, where 0.25 mL of the ethanolic extract of each treated SARCs was added to a Falcon tube containing 0.2 mL of mercuric chloride by further addition of HPLC-grade ethanol to obtain a final volume of 25 mL. A single drop of acetic acid was mixed with the sample extract (0.25 mL) for blank preparation, and the volume was increased to 25 mL by adding ethanol. A microtiter plate reader (Thermo Scientific, Germany) was used for TFC determination at a wavelength of 415 nm. According to the recent protocol, the absorbance of the sample was compared with a standard such as rutin (RE) purchased from Sigma (Germany). The standard plot was prepared from various concentrations of RE (1.0–10 μg/mL; R² = 0.9788), and the results of the SARCs of each treatment were compared with the standard plot and expressed in mg/g-DW with the RE equivalent, and the following equation was used for the final TFC determination according to the protocol:

where “T” represents each treated sample extract of SARC of Stevia with absorbance at 415 nm using a microtiter plate reader.

The dry biomass of each treated SARC was multiplied by the TFC for the determination of TFP (mg/L), while the TPPC was obtained by adding TPC and TFC and expressed in mg/g-DW.

2.9.4. Quantification of the Steviol Glycoside (Stevioside) in SARCs of Stevia

A high-performance liquid chromatography (HPLC) system purchased from PerkinElmer was used for the quantification of the stevioside in various treated SARCs of S. rebaudiana according to the recent protocol of Ahmad et al.⁴⁵ The specifications/parameters of the HPLC system were adjusted so as to quantify the maximum amounts of stevioside in SARCs. The HPLC was attenuated with the most commonly used column (C₁₈) with a fixed size (150 × 4.6 mm) and particle diameter (5 μm), multiple wavelength detector, vacuum degasser for the solvent, and a quaternary pump with an autosampler with the maximum injection volume of 10 μL. Two different solutions (A & B) were used for the mobile phase, such as 25% pure water (A) and 75% acetonitrile (B), with a constant flow rate of 1.0 mL/min using a 10 μL injection volume of maximum stevioside quantity. A standard chromatogram was obtained by running the standard stevioside (Sigma; Germany) with similar conditions, and the chromatograms of each treated SARCs was compared with it. Stevioside quantification was carried out on the basis of the retention of the standard, and the results of the stevioside content were measured in mg/g-DW, while stevioside production was expressed in mg/L DW.

2.9.5. Antioxidant Activity in SARCs

The standard method of 2,2-diphenyl-1-picrylhydrazyl (DPPH) was used for the determination of the radical-scavenging activity in various treated samples of SARCs of S. rebaudiana according to the latest protocol of Ahmad et al.⁴⁵ The DPPH solution preparation is based on the optical density (OD), which must be >1.0 for accurate activity investigation. According to the protocol, 4 times dilution of the DPPH solution (0.05 g/40 mL ethanol) was needed to obtain the required OD at 517 nm wavelength. According to the method of Ilyas et al.,⁵⁰ a ratio of 2:1 should be used for the assay, where 200 μL of DPPH solution was mixed with 100 μL of the sample extract from each treatment and subsequently incubated in the dark for 30 min to avoid the oxidation of the DPPH solution. The OD of the mixture after incubation was tested at 517 nm wavelength using a microplate reader (Thermo Scientific; Multiskan Sky with Touch Screen, Singapore). The radical-scavenging activity in all SARCs exposed to varied MEL concentrations was determined using the standard equation as follows:

Here, “A_SARCs” represents the absorbance at 517 nm of SARCs of S. rebaudiana, while “A_DPPH” denotes the absorbance of DPPH free radicals without SARC extracts.

2.10. Statistical Analysis

An independent experiment was performed for each treatment of SARC development, and to minimize errors and obtain the best results, the experiment for each SARCs was repeated twice. The mean values were calculated using one-way analysis of variance (ANOVA). Excel software was used to organize the overall data. The mean values of each treatment from independent experiments, along with the least significant differences and standard deviations, were determined by STATISTIX (v. 8.2), and the same software was used to determine the probability levels.

3. Results

3.1. Growth Kinetics of SARCs

This study was designed to investigate the pivotal role of the stress hormone MEL on the growth kinetics of SARCs of S. rebaudiana. The SARCs were investigated in the liquid MS media supplemented with different concentrations of melatonin (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 mg/L) along with 0.5 mg/L NAA. The media having NAA without melatonin was considered the control. During culture development, the growing biomass was collected at 7 day intervals for a period of 56 days. After 7 and 14 days of dark incubation, SARCs in the media containing lower MEL (0.5 mg/L) and NAA displayed 33.01 and 35.07 g/L biomass accumulation, representing the lag phase. After 21 days of culture incubation, the biomass growth was significantly enhanced until 42 days of culture, representing the log phase of growth kinetics with the maximum biomass accumulation. During this phase, the biomass gradually increases from 43.21, 59.23, and 63.45 to 68.28 g/L, respectively, as compared to the control (Figure 1). On the 49th day of culture, the fresh biomass of SARCs was 67.15 g/L in the stationary phase. On the 56th day of culture, the biomass growth was 67.89 g/L, indicating the declining phase, as compared to the control (Figure 1).

Figure 1.

Effect of various concentrations of MEL (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 mg/L) with 0.5 mg/L NAA on the growth kinetics of SARCs of S. rebaudiana. Mean values from different treatments are significant (p ≤ 0.05) using one-way ANOVA along with SE (±).

A slightly higher biomass accumulation was observed when the MEL concentration increased to 1.0 mg/L with constant NAA. In the lag phase (07–14 days), approximately 35.02–37.99 g/L fresh biomass accumulation was recorded as compared to the control. After 21 days of culture incubation, the biomass growth was significantly enhanced until 49 days in the log phase. An increase in biomass growth was observed on 28, 35, 42, and 49 days of culture, in which the biomass biosynthesis was 49.42, 59.8, 68.99, and 87.33 g/L, respectively, in comparison with the control. On day 49, the biomass gain of SARCs was 88.31 g/L, and it was in the stationary phase. On day 56 of culture, the biomass growth was 83.82 g/L in the decline phase as compared to the control (Figure 1). When the concentration of melatonin was increased from 1 to 1.5 mg/L, an incremental increase in biomass gain was recorded. In the lag phase (7 and 14 days), the higher concentration of MEL displayed 37.81–41.83 g/L biomass accumulation as compared to the control. Similarly, as the culture continued to grow, the fresh biomass accumulation was significantly enhanced until day 49 in the log phase. The biomass was increased from 51 g/L (28 days) to 67.11, 75.15, and 95.19 g/L (49 days) in comparison with the control. On day 56 of the culture, the biomass gain was 91.81 g/L in the decline phase compared to the control (Figure 1).

Increasing the concentration of MEL from 1.5 to 2.0 mg/L enhanced the growth of SARCs of S. rebaudiana. During the lag phase, from days 7 to 14, 38.6–43.52 g/L fresh biomass was biosynthesized as compared to the control. After 21 days of culture incubation, the growing cultures started abrupt accumulation of biomass until 42 days in the exponential phase, where higher quantities of biomass gain (56.58, 71.76, 87.82, and 104.82 g/L) were observed as compared to the control. On the 49th day of culture, the growing biomass of SARCs was 106.61 g/L in the stationary phase, with no further biomass gain, followed by the decline phase with a reduction in biomass accumulation from 105.61 to 101.71 g/L, as shown in Figure 1.

Furthermore, with an increase in the MEL concentration from 2.0 to 2.5 mg/L, a steady growth pattern of SARCs was still recorded in the lag phase, starting from 7 days until day 14, wherein 40.65–49.64 g/L fresh biomass was synthesized as compared to the control. After 21 days of culture incubation, the biomass was significantly enhanced until 42 days in the log phase and accumulated 61.8, 78.91, 89.99, and 109.54 g/L fresh biomass as compared to the control. The maximum biomass (111.33 g/L) was observed at the end of the log phase before the stationary phase, while the biomass decreased subsequently in the decline phase, where the biomass was 105.43 g/L as compared to the control cultures (Figure 1). The maximum biomass accumulation was observed when the full-strength MS-liquid media was augmented with 3.0 mg/L MEL along with 0.5 mg/L NAA, which further showed a strong correlation with higher productivity of fine chemicals and antioxidant potential. An overview of culture development and phytochemical profiling is given in Figure 2. In the lag phase, the MEL (3 mg/L) along with NAA showed incremental and steady growth from days 7 to 14 and displayed 43.7–53.77 g/L biomass accumulation. In this experiment, the maximum biosynthesis of fresh biomass gain in SARCs was observed in the log phase in culture media containing 3.0 mg/L MEL, which exhibited 68.02, 79.07, 91.16, and 118.27 g/L biomass accumulation; however, at the end of the log phase, the biomass reached the maximum level (120.05 g/L), and after the depletion of media particles and other components, the decline phase started, where a decrease in biomass accumulation (113.66 g/L) was observed (Figure 1).

Figure 2.

Illustration and overview of the overall experiment. Images of plant materials and in vitro cultures were obtained by Bakht Naz during the experimentation. A seed-derived plant of S. rebaudiana was used for SARC development. The culture media was supplemented with NAA [a derivative of natural auxin (IAA): an important precursor of melatonin] and melatonin, which enhanced the biomass accumulation (fresh and dry) during growth kinetics (with lag, log, stationary, and decline phases), phytochemicals [total phenolics content (TPC), total flavonoids content (TFC), total phenolics production (TPP), total flavonoids production (TFP), and total polyphenolics content (TPPC)], antioxidant activity, and steviol glycosides (stevioside) in SARCs of S. rebaudiana.

Concentrations of the stress hormone (MEL) lower or higher than 3.0 mg/L decreased the biomass of SARCs during growth kinetics analysis. During the lag phase (7 and 14 days), the SARCs exposed to a combination MEL (3.5 mg/L) and NAA (0.5 mg/L) exhibited 32.93 and 37.4 g/L fresh biomass gain, respectively, as compared to the control. After a 21 day culture period, the growing biomass abruptly increased in the log phase, starting for days 21 to 42, but actually ended on day 49, which significantly enhanced the biomass of SARCs from 43.19 to 57.57, 69.63, and 97.22 g/L, respectively, as compared to the control, with minor variations. On day 49 of culture, the biomass was observed to be 99.67 g/L in the stationary phase of growth kinetics. Moreover, the growing biomass decreased (90.44 g/L) due to the depletion of media particles/cell death and exhibited the decline phase (Figure 1).

As the concentration of MEL was increased in the culture media, the biomass of SARCs decreased. This means that the higher MEL level negatively influenced the biomass accumulation in the lag, log, stationary, and decline phases. The addition of 4.0 mg/L MEL to the culture media along with NAA in the lag phase (7 and 14 days) decreased the biomass gain of SARCs and displayed 31.17 and 35.03 g/L biomass, respectively, as compared to the control. From days 21 to 42 in the log phase, somehow, an exponential increase in biomass accumulation from 42.37 to 52.08 g/L, followed by 66.1 and 89.18 g/L, respectively, were recorded, as compared to the control. At the end of the exponential phase and initiation of the stationary phase, the biomass gain was recorded as 91.29 g/L in SARCs of S. rebaudiana. It is noted that after 56 days of culture, the biomass gain of SARCs was 85.23 g/L in the decline phase as compared to the control (Figure 1). Similarly, as the concentration of MEL was further increased to 4.5 mg/L in the lag phase of growth kinetics, starting from day 1 and to day 14, these phases displayed the minimum gain in biomass (32.6 and 36.05 g/L) of SARCs of the medicinally important S. rebaudiana. The exponential or log phases initiated from 21 days and reached 42 days and then finally 49 days, in which the maximum biomass accumulations with 07 day intervals were 45.93, 59.30, 70.32, and 87.4 g/L, respectively, as compared to control cultures, and finally reached 89.47 g/L at the end of the log phase.

The biomass gain (83.93 g/L) was comparatively lower in various phases of the growth kinetics upon exposure to 4.5 mg/L MEL in the culture media (Figure 1). In this study, the highest concentration of MEL (5.0 mg/L), along with constant NAA in full-strength MS-liquid media, further reduced the accumulation of fresh biomass of SARCs of stevia. During the lag phase (7 and 14 days), the exposure of SARCs to 0.5 mg/L NAA and 5 mg/L MEL induced 31.03 and 34.08 g/L biomass accumulation, respectively. However, after 21 days of culture incubation in the dark, the biomass of SARCs significantly increased until 42 days of culture and displayed incremental increase in biomass accumulation of 41.49, 61.53, 68.54, and 93.62 g/L in the exponential phase and reached 95.65 g/L in the log vs stationary phases, but the biomass of SARCs suddenly decreased to 86.13 g/L in the decline phase as compared to the control (Figure 1).

In this experiment, as the concentration of melatonin was increased from 0.5 to 3 mg/L with a constant concentration of NAA (0.5 mg/L), the combination significantly enhanced the growth kinetics of SARCs of S. rebaudiana and displayed an incremental increase in biomass gain. Further increase in the concentration of MEL from 3.5 to 5 mg/L had no significant effect on the biomass gain during the lag, log, stationary, and decline phases of growth kinetics. Furthermore, the exponential phase, which started from 42 days up to 49 days, was the most suitable phase for biomass gain of SARCs of S. rebaudiana.

3.2. Fresh and Dry Biomass of SARCs in Response to Varied Concentrations of Melatonin

In this experiment, biomass accumulation, both fresh weight (FW) and dry weight (DW), was studied using different concentrations of MEL (0.5–5.0 mg/L) along with NAA (0.5 mg/L) in submerged adventitious root cultures (SARCs) of S. rebaudiana. The results showed a significant trend in the influence of MEL on the FW and DW of cultures (Figure 3). Melatonin, when supplemented with NAA, increases the biomass production, but it was clearly observed that the addition of certain concentrations (0.5, 1.0, 1.5, 2.5, and 3 mg/L) to culture media influenced and increased the fresh and dry biomass accumulation, but further increase in the MEL concentration (3.5, 4.0, 4.5, and 5.0 mg/L) resulted in reduced biomass accumulation (either FW or DW), as shown in Figure 3. The maximum fresh (236.54 g/L) and dry (28.64 g/L) biomass were noted in SARCs having the optimal concentration of MEL (3.0 mg/L); however, the minimum fresh (136.56 g/L) and dry (16.54 g/L) biomass were observed in culture media having 0.5 mg/L MEL along with a constant NAA concentration. It was also noted that MEL at lower concentrations (0.5 and 1.0 mg/L) yielded inferior results in biomass accumulation as compared to the control (only MS media with NAA). The recorded biomass production of SARCs supplemented with 0.5 mg/L MEL was 136.56 g/L, while the dry biomass was 16.54 g/L, followed by 1.0 mg/L MEL (FW 174.66 and DW 21.14 g/L), 1.5 mg/L MEL (FW 190.38 and DW 23.06 g/L), 3.5 mg/L MEL (FW 194.45 and DW 23.54 g/L), 4.0 mg/L MEL (FW 178.36 and DW 21.62 g/L), 4.5 mg/L MEL (FW 174.8 and DW 21.18 g/L), and 5.0 mg/L MEL (FW 187.24 and DW 20.24 g/L) as compared to the control without MEL (FW 177.08 and DW 21.44 g/L), as shown in Figure 3. This pattern of response indicates that the effect of MEL on biomass accumulation in S. rebaudiana SARCs is concentration-dependent (Figure 3). These findings suggest that excessive MEL augmentation beyond a certain limit to SARCs of S. rebaudiana can lead to reduced biomass yield, and therefore, optimizing the MEL concentration is of key importance for higher biomass and maximum productivity of fine chemicals. The results suggest that the concentration of MEL must not exceed the threshold of 3.0 mg/L to avoid inhibitory effects on biomass accumulation.

Figure 3.

Effect of multiple concentrations of MEL (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 mg/L) with 0.5 mg/L NAA on fresh and dry biomass biosynthesis in SARCs of S. rebaudiana. Mean values from different treatments are significant (p ≤ 0.05) using one-way ANOVA along with SE (±).

3.3. Melatonin-Concentration-Dependent Biosynthesis of Phenolics and Flavonoids in SARCs

In this experiment, various MEL concentrations in the culture media were correlated with TPC, TPP, TFC, TFP, and TPPC, and linear and positive correlations were observed in SARCs of S. rebaudiana (Figure 4a–c). As the concentrations of MEL (0.5–3.0 mg/L) were increased to a specific level, the biosynthesis of TPC, TFC, TPP, TFP, and TPPC also increased, but as the concentration of MEL was increased from 3.5 to 5.0 mg/L, the biosynthesis of phenolics and flavonoids also decreased (Figure 4a–c). The Pearson correlation coefficient (r) of MEL concentration with phenolics and flavonoids was observed to be TPC (r = 0.833), TPP (r = 0.783), TFC (r = 0.677), TFP (r = 0.599), and TPPC (r = 0.81). The full strength-liquid MS media supplemented with 0.5 mg/L MEL with constant NAA accumulates 11.02 mg/g-DW of TPC and 4.08 mg/g-DW of TFC as compared to the control. When the concentration of MEL was increased to 1.0 mg/L, the accumulation of phytochemicals also increased (Figure 4a). The biosynthesis of TPC and TFC also increased to 12.34 and 4.13 mg/g-DW as compared to the control. Further, when the concentration of melatonin was increased to 1.5 mg/L, the biosynthesis of phytochemicals linearly increased, and TPC and TFC reached 13.88 and 4.65 mg/g-DW, respectively. The same concentration-dependent enhancement in the biosynthesis of fine chemicals was observed with 2.0 mg/L MEL, which displayed 15.34 and 5.2 mg/g-DW of TPC and TFC production (Figure 4a). Furthermore, the higher concentration of MEL showed a positive effect on the accumulation of phytochemicals. As the concentration of MEL was increased to 2.5 mg/L, it promoted the TPC and TFC (16.22 and 5.43 mg/g-DW, respectively) as compared to the control. Further, as the concentration of melatonin was increased to 3.0 mg/L, it showed a positive effect on the accumulation of phytochemicals. In this study, 3.0 mg/L MEL displayed the highest biosynthesis of TPC and TFC (18.96 and 6.33 mg/g-DW, respectively). Moreover, as the concentration of melatonin was increased to 3.5 mg/L, it had a negative effect on the biosynthesis of phytochemicals and displayed 17.56 and 5.87 mg/g-DW of TPC and TFC, respectively, in SARCs of S. rebaudiana as compared to the control (Figure 4a). Furthermore, when the concentration of melatonin was increased to 4.0 mg/L, 14.27 and 4.77 mg/g-DW of TPC and TFC were synthesized, respectively, while further increase in the MEL concentration (4.5 mg/L) decreased the phenolic and flavonoid content (11.06 and 3.71 mg/g-DW, respectively). Finally, when the concentration of melatonin was increased to 5.0 mg/L, it showed a negative effect on the accumulation of phytochemicals. The accumulations of TPC and TFC were 8.09 and 2.7 mg/g-DW, respectively, as compared to the control. These results show that an increase in the concentration of melatonin from 0.5 to 3.0 mg/L increases the accumulation of phytochemicals, and the biosynthesis is MEL-concentration-dependent.

Figure 4.

Effect of multiple concentrations of MEL (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 mg/L) with 0.5 mg/L NAA on polyphenolics biosynthesis: (a) TPC and TFC, (b) TPP and TFP, and (c) TPPC in SARCs of S. rebaudiana. Mean values from different treatments with LSDs are significant (p ≤ 0.05) using one-way ANOVA along with SE (±).

The induction of 0.5 mg/L melatonin into liquid media helps in the biosynthesis of TPPC, TPP, and TFP (15.1, 91.13, and 33.74 mg/g-DW, respectively) as compared to the control. The Pearson correlation coefficient (r) also exhibits a positive correlation with TPPC, TPP, and TFP, which shows that the biosynthesis of phytochemicals is dependent on the MEL concentrations. The addition of 1.0 mg/L MEL to the culture media increased the synthesis of TPPC, TPP, and TFP to 16.47, 130.43, and 43.65 mg/g-DW, respectively, as compared to the control (Figure 4b,c). The MEL concentrations (1.5, 2.0, and 2.5) also enhanced the accumulation of TPPC, TPP, and TFP (18.53, 160.03, and 53.61 mg/g-DW; 20.54, 195.73, and 66.35 mg/g-DW; and 21.65, 215.23, and 72.05 mg/g-DW, respectively) in SARCs of stevia. Further, when the concentration of melatonin was increased to 3.0 mg/L, it showed a positive effect on the accumulation of phytochemicals. The highest TPPC, TPP, and TFP contents were observed in SARCs after the addition of 3.0 mg/L MEL to the culture media, which displayed 25.29, 271.50, and 90.64 mg/g-DW as compared to the control (Figure 4b,c). Furthermore, as the concentration of MEL was increased to 3.5 and 4.0 mg/L, it showed a negative effect on the accumulation of phytochemicals and displayed biosynthesis of TPPC, TPP, and TFP of 23.43, 206.68, and 69.08 mg/g-DW and 19.04, 154.25, and 51.56 mg/g-DW, respectively, as compared to the control. This means that the biosynthesis of phytochemicals was found to be MEL-concentration-dependent, and a further increase in MEL drastically decreased the synthesis of TPPC, TPP, and TFP, respectively. The supplementation of 4.5 and 5.0 mg/L to the culture media negatively affected the biosynthesis of TPPC, TPP, and TFC to 14.77, 117.12, and 39.28 mg/g-DW and 10.45, 81.87 and 27.32 mg/g-DW, respectively, as compared to the control (Figure 4b,c).

3.4. Phenolics and Flavonoids and their Correlation with the Biomass of Growing SARCs

In this study, the biomass-dependent biosynthesis of phenolic and flavonoids was investigated. Phenolics and flavonoid biosyntheses have shown a strong correlation with the fresh and dry biomass of SARCs of S. rebaudiana. As the biomass growth of SARCs increased, the biosynthesis of phenolics and flavonoids (TPC, TFC, TPPC, TPP, and TFP) also increased (Figure 5a–c). A strong linear correlation was observed according to the Pearson correlation coefficient (r) between phytochemicals and the growing biomass of SARCs. The growing biomass increased along with the MEL concentration (0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg/L) with increased levels of phenolics and flavonoids up to some extent, but a gradual biomass-dependent decline was observed in the synthesis of phenolics and flavonoids. The biomass growth of SARCs increased step by step along with MEL (0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg/L) and displayed fresh and dry biomass in an exponential pattern, i.e., fresh biomass 136.56, 174.66, 190.38, 209.64, 219.09, and 236.54 g/L along with a dry biomass of 16.54, 21.14, 23. 06, 25.52. 26.54. and 28.64 g/L, respectively, as shown in Figure 5a–c. Similarly, as the biomass increased, the TPC (11.02, 12.34, 13.88, 15.34, 16.22, and 18.96 mg/g-DW) and TFC (4.08, 4.13, 4.65, 5.2, 5.43, and 6.33 mg/g-DW) also increased and displayed a strong correlation with the fresh and dry biomass. Here, the highest fresh and dry biomass accumulation was observed (236.54 and 28.64 g/L) when the culture media was augmented with 3.0 mg/L melatonin in combination with NAA and also displayed the maximum biosynthesis of TPC and TFC in mg/g-DW. However, further incremental increase in the MEL concentration (3.5–5.0 mg/L) negatively influenced the correlation between the fresh and dry biomass and the TPC and TFC (Figure 5a). Here, a decreasing trend in phenolics (TPC: 17.56, 14.27, 11.06, and 8.09 mg/g-DW) and flavonoids (TFC: 5.87, 4.77, 3.71, and 2.7 mg/g-DW) was observed (fresh biomass decrease: 194.45, 178.36, 174.8, and 187.24 g/L; C: 177.08 g/L; and dry biomass decrease: 23.54, 21.62, 21.18, and 20.24 g/L; C: 21.44 g/L, respectively) (Figure 5). The Pearson correlation coefficient (r) also showed a positive correlation of TPPC, TPP, and TFP, wherein the biosynthesis of phytochemicals is dependent on the fresh and dry biomass of SARCs. As the fresh biomass of SARCs increased from 136.56, 174.66, 190.38, 209.64, 219.09, to 236.54 g/L, the dried biomass also increased (16.54, 21.14, 23.06, 25.52, 26.54, and 28.64 g/L) in culture media containing varied MEL concentrations (0.5–3.0 mg/L), which displayed a strong correlation with TPPC (15, 16.47, 18.53, 20.54, 21.65, and 25.29 mg/g-DW), TPP (91.13, 130.43, 160.03, 195.73, and 215.23 mg/L), and TFP (33.74, 43.65, 53.61, 66.35, and 72.05 mg/L) biosynthesis, respectively (Figure 5b,c).

Figure 5.

Effect of multiple concentrations of MEL (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 mg/L) with 0.5 mg/L NAA on the correlation of biomass with polyphenolics biosynthesis: (a) TPC and TFC, (b) TPP and TFP, and (c) TPPC in SARCs of S. rebaudiana. Mean values from different treatments with LSD are significant (p ≤ 0.05) using one-way ANOVA along with SE (±).

As the MEL concentration was decreased (from 3.0 to 5.0 mg/L), a correlated reduction in biomass and TPPC, TPP, and TFP was observed. Here, a gradual decrease in the biosynthesis of TPPC (23.43, 19.04, and 14.77 mg/L), TPP (206.68, 154.25, and 117.12 mg/L), and TFP (69.08, 51.56, and 39.28 mg/g-DW) was observed as the fresh (194.45, 178.36, 174.8, and 187.24; C: 177.08 g/L) and dry (23.54, 21.62, 21.18, and 20.24; C: 21.44 g/L) biomass decreased, respectively (Figure 5a–c).

3.5. Stevioside Content and Production, Antioxidant Potential, and Their Correlation with the Biomass

The DPPH-based radical-scavenging activity (RSA) or antioxidant activity (AA) was investigated in SARCs of S. rebaudiana exposed to varied concentrations of MEL (0.5–5.0 mg/L), while the media without melatonin was considered as the control. A MEL-concentration- and biomass-dependent antioxidant potential was noticed in SARCs of stevia (Figure 6a). As the MEL concentration increased from 0.5 to 3.0 mg/L, a positive correlation was observed between biomass and RSA (antioxidant activity vs biomass; r = 0.779). As the fresh biomass (136.56, 174.66, 190.38, 209.64, 219.09, to 236.54 g/L) and dry biomass (16.54, 21.14, 23.06, 25.52, 26.54, and 28.64 g/L) increased, a strong correlation was observed, with an exponential increase in the antioxidant potential (47.56, 57.27, 66.97, 76.87, 81.77, and 86.15%); however, as the MEL concentration was increased (3.5–5.0 mg/L), a declining pattern of fresh and dry biomass and antioxidant potential was observed. A declining correlation between the fresh biomass (194.45, 178.36, 174.8, and 187.24 g/L) and dry biomass (23.54, 21.62, 21.18, and 20.24, g/L) with the antioxidant potential (79.53, 65.66, 65.78, and 59.22%) was observed in SARCs of S. rebaudiana, as shown in Figure 6a.

Figure 6.

Effect of multiple concentrations of MEL (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 mg/L) with 0.5 mg/L NAA on the correlation of biomass with antioxidant activity (a) and the correlation of biomass with the stevioside content and production (b) in SARCs of S. rebaudiana. Mean values from different treatments with LSD are significant (p ≤ 0.05) using one-way ANOVA along with SE (±).

In the current experiment, 0.5 mg/L NAA, along with different concentrations of MEL, was used to explore its impact on the stevioside content and production during its biosynthesis. The stevioside content and production in the control (without MEL) were 65.44 mg/g-DW and 1403.0336 mg/L, respectively. The results revealed that higher stevioside contents and production (91.45 and 2619.128 mg/L DW, respectively) were found with 3.0 mg/L MEL along with NAA, while the lowest stevioside contents and production (45.77 and 757.0358 mg/L DW, respectively) were observed by the addition of 0.5 mg/L MEL to culture media (Figure 6b). The stevioside content and production during biosynthesis increased with an increasing concentration of melatonin up to 3.0 mg/L, but after that, it began to decline. Exposure of SARCs of stevia to increasing concentrations of MEL (1.0, 1.5, 2.0, and 2.5 mg/L) influenced the stevioside content and production during biosynthesis as follows: 52.66 mg/g-DW and 1113.2324 mg/L; 61.78 mg/g-DW and 1424.6468 mg/L; 74.29 mg/g-DW and 1895.8808 mg/L; and 85.23 mg/g-DW and 2262.0042 mg/L, respectively.

Furthermore, a strong correlation of the stevioside content and production with the fresh and dry biomass was observed (stevioside content vs biomass: r = 0.877; and stevioside production vs biomass: r = 0.912). It is clearly observed that as the fresh biomass (136.56, 174.66, 190.38, 209.64, 219.09 to 236.54 g/L) and dry biomass (16.54, 21.14, 23.06, 25.52, 26.54, and 28.64 g/L) increases, the stevioside content (52.66, 61.78, 74.29, 85.23, and 91.45 mg/g-DW) and production (1113.23, 1424.64, 1895.88, 2262.42, and 2619.128 mg/L) also increase and display a strong correlation with each other (Figure 6b).

As the fresh biomass (194.45, 178.36, 174.8, and 187.24 g/L) and dry biomass (23.54, 21.62, 21.18, and 20.24 g/L) decreased with MEL (3.5, 4.0, 4.5, and 5 mg/L), a linear decrease in the stevioside content and production during biosynthesis was noticed as follows: 82.33 mg/g-DW and 1938.0482 mg/L; 77.74 mg/g-DW and 1680.7388 mg/L; 71.92 mg/g-DW and 1523.2656 mg/L; and 63.33 mg/g-DW and 1281.7992 mg/L, respectively (Figure 6b). A positive correlation exists between the biomass and biosynthesis of stevioside in SARCs of stevia.

4. Discussion

Various environmental factors (biotic and abiotic) compromise the biosynthesis of pharmaceutically important secondary cell products in high-value medicinal plants in the wild habitat and cannot overcome market demand.⁵¹ To avoid such biotic and abiotic stresses and to produce optimum and uniform quantities of metabolites of interest, advanced culture systems such as biotechnological cell and root culture technologies are needed.⁵² Traditional approaches for the cultivation of Stevia (seeds and stem cuttings) and the isolation of uniform secondary cell products are correlated with variations in environmental factors and some pathogenic attacks that reduce productivity and plant growth and development.^33,50,53,54 Nevertheless, there is industrial demand for the use of stress-mediated alterations of biotic and abiotic elicitors to achieve a feasible strategy (optimization) for cell and root culture technology to enhance the production of phytochemicals.⁵⁵ It has been reported that cell and root cultures technologies surpass wild-grown Stevia in the biosynthesis of high levels of stevioside and can be upgraded to bioreactor technology.⁵⁶ Therefore, SARCs of stevia have been considered favorable culture systems for the accumulation of intracellular metabolites (stevioside), and their optimization can overcome the market demand after scaling up to a bioreactor.^55,57

In this study, MEL was used as a stress inducer during growth kinetics to investigate its effect on the growing biomass in the lag, exponential, stationary, and decline phases in SARCs of stevia. 3.0 mg/L MEL with NAA (0.5 mg/L) was the most suitable candidate for the highest biomass accumulation in the lag (43.7–53.77 g/L), exponential (68.02, 79.07, 91.16, and 120.05 g/L), stationary (118.27 g/L), and decline (113.66 g/L) phases compared to other treatments and the control. At the end of the experiment, the fresh and dry biomass were collected, and the same combination displayed the fresh (236.54 g/L) and dry biomass (28.64 g/L) biosynthesis in SARCs of stevia. In the literature cited, limited reports are available on the application of MEL on SARCs of stevia; however, the effect of other stress inducers are widely reported by Kazmi et al.,⁵⁸ who observed that a combination of elicitors such as M-jasmonate, PAA, BA, and MEL promoted the development of adventitious roots in Stevia from leaf explants. The exposure of calli cultures of Fagonia indica to white LEDs and MEL enhanced the fresh and dry biomass⁵⁹ (320 and 20 g/L). In another study, Mao et al.⁶⁰ observed that MEL, along with IAA, enhanced adventitious root formation in transgenic apple lines (MdWOX11). The current results are in agreement with the report of Sarropoulou et al.,⁶¹ who observed that the addition of MEL up to a specific level promoted the growth of adventitious roots in sweet cherry, but a decline was noticed with a further increase in the MEL concentration. The report of Adil et al.⁶² also suggested that, in the dark, MEL enhanced the development of adventitious roots in Withania somnifera. MEL is one of the best stress inducers for adventitious root formation in Solanum lycopersicum.⁶³ Furthermore, high sucrose feeding to culture media boosted the fresh and dry biomass (175 and 11 g/L) of SARCs of stevia.⁴⁵ In another study, Ahmad et al.⁸ noticed that a higher fresh and dry biomass (112.86 and 8.3 g/L) of SARCs of stevia was achieved by optimizing the pH level (pH 6) of the culture media along with NAA. The addition of metal nanoparticles to culture media enhanced the biomass biosynthesis (1.5 g/flask) in SARCs of stevia.⁴⁹ Similarly, Idrees et al.³⁶ incubated a root inoculum under violet light for a specific period and observed that color-light incubation enhanced the biomass accumulation (80 g/L) in SARCs of stevia. Recently, Ahmad et al.²⁹ found in their experiment that not only do abiotic stress inducers enhance phytochemicals, but the addition of biotic elicitors (Cuscuta reflexa extracts) also improved the biomass accumulation (86.2 g/L) in SARCs of S. rebaudiana.

Furthermore, in this study, a concentration (MEL) and biomass-dependent biosynthesis of TPC (18.96 mg/g-DW), TFC (6.33 mg/g-DW), TPP (271.50 mg/g-DW), TFP (90.64 mg/g-DW), TPPC (25.29 mg/g-DW), and DPPH-RSA (86.15%) was noticed in SARCs of stevia exposed to 3.0 mg/L MEL with NAA (0.5 mg/L). All of the phytochemicals and antioxidant activity displayed a strong and linear correlation with each other. Wild plants or in vitro cultures of medicinal plants are one of the rich sources of natural antioxidants that prevent cells and intact plants from oxidative damage caused by reactive oxygen species, and therefore, natural antioxidants are considered potent scavengers.⁶⁴ Various medicinal plants such as stevia or their in vitro cultures have proven the scavenging potential of toxic radicals due to the presence of special compounds in plants such as phenols and flavonoids.⁶⁵ A linear correlation has been reported between phytochemicals (phenolics and flavonoids) and the antioxidant potential in the medicinally important S. rebaudiana.^38,66,67 Moreover, previous reports highlighted that MEL is one of the potent scavengers of toxic chemicals, and it protects plants from various biotic and abiotic stresses and acts as a natural antioxidant.^68,69 Intact plants of lupin, barley, red cabbage, pea plants, and suspended cells of Daucus carota have widely been protected from various stresses by producing polyphenolics by the application of MEL as a natural antioxidant.⁷⁰⁻⁷² It is believed that in stress conditions, MEL stimulates the enzymatic (various tress enzymes) and nonenzymatic (polyphenols) plea system in plants, protects them from oxidative damage caused by oxygen, nitrogen, and other toxic free radicals, and also helps in the cryopreservation of cells and tissues.⁷³⁻⁷⁶

In this study, the highest stevioside content (91.45 mg/g-DW) and production (2619.128 mg/L) were observed in culture media augmented with 3.0 mg/L MEL along with 0.5 mg/L NAA, and they exhibited a strong correlation with the fresh and dry biomass and also displayed a concentration-dependent behavior in SARCs of stevia. Different elicitors are widely exploited to increase the production of Steviol glycosides (stevioside) using various in vitro cell culture technologies. Ahmad et al.⁴⁵ observed that the elicitation of culture media with 10 g/L sucrose induced the highest biosynthesis of stevioside (74 mg/g-DW) in SARCs of S. rebaudiana. The current biosynthesis of stevioside is in agreement with previous reports of Ahmad et al.⁸ and Ahmad et al.⁷⁷ The supplementation of spermidine along with various cytokinins enhanced stevioside production in shoot cultures of Stevia.³⁰ In another study, Aman et al. (2013) observed the highest quantities of stevioside (83 mg/g-DW) in culture media augmented with 7.0 g/L agar as compared to culture media containing 3.5 g/L agar. In contrast, Khalil et al.³³ observed that γ irradiation negatively influenced the biosynthesis of stevioside in Stevia. The SARC system was the best culture system for the production of stevioside, and it was superior to other cell culture technologies. The stevioside content (6.38% and 1.36–5.14%) was reduced in suspended cells upon exposure to PEG and NaCl,⁵⁷ and the difference in data is due to the type of culture and elicitors used for culture development. Reduced levels of stevioside (0.06 and 0.28 mg/g-DW) were observed in calli/shoot cultures of Stevia.⁷⁸ The mineral salts also reduced the stevioside content (6.23 mg/g-DW) in shoot cultures of Stevia.⁷⁹

5. Conclusions

Submerged adventitious root cultures (SARCs) are one of the best and feasible systems for the industrial production of high-value secondary cell products as compared to other in vitro cell culture technologies. Here, among various concentrations of MEL, the addition of 3.0 mg/L MEL along with NAA was found to be the most effective for biomass accumulation in the lag, exponential, stationary, and decline phases of growth kinetics, promoted fresh and dry biomass gain, enhanced the phytochemical profile (TPC, TFC, TPP, TFP, and TPP), improved the production of SGs (stevioside) compared to previous reports, and displayed a higher antioxidant potential. These results suggest that SARCs are the best candidates for health-promoting phytochemicals, especially for obese and diabetic patients. Furthermore, SARCs are dominant over other culture systems due to their rapid, uniform, and homogeneous intracellular phytochemical biosynthesis in liquid media in a very short duration using batch or continuous cultures and may possibly be upgraded to higher-level bioreactors to cover the escalating demand of the metabolites of interest.

Author Contributions

N.A. and H.F.: designed the experiment and supervised the work; B.N., Aftab Afzal, and H.F.: performed the experiments; B.N.: obtained images of all cultures during experimentation; Z.U., Ahmad Ali, and S.D.: analyzed the data and helped review the manuscript; H.F., N.A., Aftab Afzal, and M.A.: conceptualized the study, edited the manuscript, and performed the experiments; H.A., R.U., and E.A.A.: performed formal analysis and wrote the original draft of paper. All authors approved the final version.

This research work was supported by the Researchers Supporting Project Number (RSP2024R110), King Saud University, Riyadh, Saudi Arabia.

The authors declare no competing financial interest.