1. INTRODUCTION

Secondary metabolites (SMs), in particular, which are remarkable sources of value-added bioactive molecules, provide the basis for the pharmacological capabilities of plants, which have been employed as medicines throughout history [1]. SMs are a huge and diverse class of compounds, some of which are probably unimportant byproducts of metabolic pathways brought on by different enzyme activity [2]. SMs are formed in response to many types of stress it faces and have a complicated chemical makeup that helps to carry out diverse physiological activities in plants [1]. About 200,000 known SMs are found in plant species and are crucial for survival, reproduction, forming symbiotic associations, climatic tolerance, and disease resistance [3,4]. Plant secondary metabolism is typically activated and reconfigured during acclimatization and adaptation to adverse environmental conditions, and stimulation of this specialized metabolism to combat these environmental stresses results in the production of various classes of plant SMs, including phenolic compounds (coumarin, lignin, furanocoumarins, flavonoids, isoflavonoids, and tannins), terpenes (monoterpenes, sesquiterpenes, diterpenes, triterpenes, and polyterpenes), nitrogen-containing SMs (alkaloids, cyanogenic glycosides, glucosinolates, and non-protein amino acids), and sulfur-containing SMs (glucosinolates, phytoalexins) [5,6]. They are having wide applications in human use and are used as pharmaceuticals, agrochemicals, flavors, fragrances, colors, biopesticides, and food additives, and have a variety of biological activities. Therefore, the majority of SMs, including terpenes, phenolics, and alkaloids, are highly sought after in the biopharmaceutical, cosmeceutical, and nutraceutical industries [7,8].

Few significant plant-based compounds with uncomplicated chemical structures can be produced using the chemosynthesis method, but many, like alkaloids, are challenging to synthesize by the chemosynthesis method, or their production costs outweigh their commercial availability [9]. From intact plants grown in nature, many of these specialized compounds can be obtained. The plant raw materials from which several of these highly valuable substances are derived are getting scarcer due to ecological, political, or geographic factors. A metabolite's production tends to be confined to a single species or genus and may only be initiated during a specific growth or developmental stage of a plant, or may be related to the season, stress, or nutrient availability [9,10]. Different types of stresses, including biotic and abiotic stresses that a plant has to encounter, make it difficult to ensure and maintain a steady supply of desirable plant SMs, and also, their subsequent processing is challenging. These drawbacks make it difficult to isolate SMs from intact plants [11]. The restricted availability of the compounds is one of the key issues with the commercial supply of molecules derived from plants. Most of the time, SMs make up less than 1% of the dry weight (DW) of plant cells, and occasionally even less, as is the case with the paclitaxel content of Taxus bark, which is only 0.01% [12].

The elevated demand in the marketplace and limited yield of pharmaceutically significant phytochemicals have sparked interest in biotechnological methods to increase the yield of SMs [13]. For SM production under controlled conditions, plant cell culture systems (cell suspension, hairy/adventitious root (AR) cultures, and callus cultures) have emerged as an appealing substitute [11]. Utilizing a plant cell culture technology may be able to lower the overexploitation of therapeutic plants that are in danger of extinction, and also those that are very rare [14]. By using plant tissue culture (PTC) techniques, cultures may be generated anywhere in the world, regardless of the conditions needed for plant growth. They are also free of pathogens and insects, preventing the need for pesticides and herbicides [9]. Recently, various techniques have been developed to increase the production of important plant SMs employing plant in vitro cultures, including cell line selection, precursor feeding, cell immobilization, biotransformation, metabolic engineering, synthetic biology, and elicitation [15].

Through elicitation employing a variety of biotic and abiotic substances, a simpler method to improve SM generation in plant cell culture systems can be achieved [16]. These elicitation strategies range from media manipulations through environmental stress and chemical elicitors, and it shows encouraging results in terms of boosting the yield of SMs in cell cultures of different medicinal plants [17]. Use of nanoparticles (NPs) as elicitors is giving assurance as more potent substitutes to other elicitors due to their customizable physicochemical properties and the physiological, molecular, biochemical, and metabolic responses they generate [18,19]. There are many previous studies reporting the use of NPs in order to positively impact the yield of useful SMs in plants. NPs consisting of metals, metal oxides, non-metallic, magnetic, polymeric, bi-metallic, carbon-based, silica, biopolymer, core-shell structures, and nanocomposites are used for the elicitation of SMs in plant cell culture systems [11]. Because of their smaller size, ease of penetration, and consequent ease in manipulating the cell's machinery to generate increased phytochemicals, nanomaterials have been used and studied widely over the past few years. PTC has utilized nanomaterials for a variety of goals, including growth, organogenesis, transformation, and the induction of SMs in many plants [13].

2. SECONDARY METABOLITE PRODUCTION BY PLANT TISSUE CULTURE TECHNIQUES

The demand for natural and organic products has grown as consumers become more aware of lifestyle choices. Due to its health advantages over synthetic medications, as well as in terms of cost and safety, fascination with medicinal plants has grown over the past two decades [1]. Due to limitations in topography and environment, isolation of plant SMs on a large scale from naturally existing intact plants is restricted [15]. Traditional SM isolation techniques take a long time since it takes plants years to develop to a stage where they can produce the desired metabolites [20]. It also has a number of drawbacks, including low yields and concentration changes brought on by regional, seasonal, and environmental variations [7].

Utilizing PTC procedures for the quick and effective production of SMs for commercial use is an alternative strategy to overcome such a dilemma. A need to concentrate research on raising output by applying PTC approaches and improving their large-scale production using bioreactors has resulted from the extensive usage of SMs in numerous industrial sectors [1]. In vitro propagation of plants or the in vitro culture of plant organs (usually roots) or callus are reported to be highly capable of providing plant materials that produce SMs [21]. It facilitates the bulk propagation of plants in controlled environmental conditions without regard to the seasons [22]. In addition, it facilitates the year-round production of huge numbers of homogeneous plants, disease-free propagules, and a significant increase in multiplication rates over traditional horticultural propagation approaches [21].

The process for producing in vitro SMs involves two steps: aggregation of biomass and the synthesis of SMs [1]. The following are procedures for producing bioactive SMs using plant in vitro culture: A) surface sterilization of the plant material, followed by establishing in vitro cultures. Organ culture material or callus can also be used. Agrobacterium rhizogenes is used to produce hairy roots by infecting sterilized donor plants or in vitro cultured materials. B) Multiplication of obtained primary callus/organs/roots, their initial selection, followed by establishing liquid cultures. C) High-yielding lines are chosen, and culture conditions are optimized (nutrient medium constituents, inoculum concentration, temperature, light, agitation, and aeration). To increase productivity even further, techniques like elicitation, precursor feeding, or immobilization are used. D) Bioreactor types depending on the culture: stirred tanks, airlift, and bubble column reactors for cell suspensions; mist or spray reactors and temporary immersion systems for organ cultures, including hairy roots, are used for further scale-up of SM production [23].

The entire procedure must be carried out in an aseptic environment utilizing a laminar flow cabinet equipped with a high-efficiency particulate air (HEPA) filter filter in order to accomplish a successful culture. The environment has extensively contaminated the source of the explants; thus, the portion that is to be removed for use is disinfected in a disinfectant, often alcohol, sodium, or calcium hypochlorite or mercuric chloride [24]. The cultures can be initiated from either seeds that have been germinated aseptically or from pieces of the entire plant. The explants are then inoculated on a nutrient-rich semi-solid medium that contains plant growth regulators and are grown in a controlled environment [23]. Two key characteristics of plant cells—cell plasticity and totipotency—support the growth of explants. These two characteristics of plant cells probe the ability of living cells to differentiate into new, genetically similar cells that can then become tissues, organs, and whole individuals [25].

Cell cultures (cell suspensions, protoplasts, or gametic cells), tissue cultures (callus or differentiated tissues), or organ cultures (shoots, roots, or zygotic embryos) can be created based on the starting material and the nutrient medium [26]. For the development of SMs for commercial use, callus, cell suspension, shoot, and root cultures have been used most frequently [1]. The sequential steps involved in SM production by PTC techniques are schematically represented in Figure 1. Several research reports have showcased successful outcomes regarding the production of important SMs using PTC techniques, as listed in Table 1. A comparative analysis for the enhancement of bioactive compounds in Ocimum basilicum, Ocimum sanctum, and Ocimum gratissimum by cell culture was carried out, and found that O. basilicum produced 33 mg GAE g⁻¹ DW, 220 mg g⁻¹ DW, and 450 mg g⁻¹ DW of total phenolic, alkaloid, and terpenoid content, respectively. Likewise, O. sanctum synthesized 33.3 mg GAE g⁻¹ DW, 330 mg g⁻¹ DW, 430 mg g⁻¹ DW, and O. gratissimum synthesized 44.0 mg GAE g⁻¹ DW, 450 mg g⁻¹ DW, 420 mg g⁻¹ DW of total phenolic, alkaloid, and terpenoid content, respectively [27]. Cell suspension culture of Tinospora cordifolia showed a 5.57-fold (3.077 mg g⁻¹ DW) increase in berberine production, while cell suspension culture of Vernonia anthelmintica showed a 9.4-fold (1.104 mg g⁻¹ DW) increase in rhamnetin content [28,29]. Kuo et al [30] studied the effect of in vitro cultured plants on cucurbitacin I and cucurbitacin E production in Aquilaria agallocha and found that it enhanced production and observed 0.356 and 0.972 mg g⁻¹ yield, respectively. Otari et al. [34] studied in vitro plantlets of Bacopa floribunda and reported an increase of Bacoside A3 (1.01 mg g⁻¹ DW), Bacopaside X (1.23 mg g⁻¹ DW), Bacopaside II (43.62 mg g⁻¹ DW), Bacosaponin C (0.19 mg g⁻¹ DW), and Stigmasterol (7.69 mg g⁻¹ DW). Enhancement of total phenolic content (TPC) and total flavonoid content (TFC) was studied by various scientists and reported 4.5-mg GAE g⁻¹ DW and 3.5-mg QE g⁻¹ DW of TPC and TFC, respectively, in shoot culture of Ajuga bracteosa; 55.954-mg GAE g⁻¹ DW, 3.9355-mg QE g⁻¹ DW, respectively, in hairy root culture of Ficus carica; 5.43-mg g⁻¹ DW, 3.27-mg g⁻¹ DW, respectively, in callus culture of Digitalis purpurea; 3.0-mg g⁻¹ DW, 1.8-mg g⁻¹ DW, respectively, in callus culture of Caralluma tuberculata and 57.8-mg g⁻¹ DW, 11.1-mg g⁻¹ DW, respectively, in callus culture of Eclipta alba [31,44,42,39,43]. Berberine (5.05 mg g⁻¹) and palmatine (3.00 mg g⁻¹) were obtained from in vitro T. cordifolia plantlets [54]. AR culture of Hyoscyamus niger exhibited increased production of Scopolamine (0.64 mg g⁻¹), Hyoscyamine (0.46 mg g⁻¹), and TPC (19.33 mg g⁻¹) [46]. Escrich et al[53]. observed a 15-fold increase in taxane production by cell culture of Taxus media. Improved production of centellosides by cell culture of Centella asiatica, berberine by callus culture of Argemone mexicana, and t-resveratrol by cell suspension culture of Silybum marianum were reported [41,52,32]. Increased total phenolic content (147.98, 1.02 mg GAE g⁻¹ DW) was obtained by callus culture of Artemisia annua and Urginea maritima, respectively [33,55]. 2.44, 1.81, and 1.25 times greater amounts of pinostrobin, total phenolics, and flavonoids were reported by Jirakiattikul et al. [37] from shoot culture of Boesenbergia rotunda. Alkaloid content (5.67 mg g⁻¹ DW) was found to be enhanced by cell suspension culture of Catharanthus roseus [40]. 1.05 mg g⁻¹ DW of p-hydroxybenzyl alcohol, 19.0 mg g⁻¹ DW of Dactylorhin A, 6.25 mg g⁻¹ DW of Militarine, and 0.3323 mg g⁻¹ DW of Coelonin were obtained by Bletilla striata callus suspension culture [35]. Park et al. [47] synthesized 0.158 mg g⁻¹ DW of galantamine and 1.070 mg g-¹ DW of TPC by shoot culture of Narcissus tazetta. Rawat et al. [50] reported increased production of TPC (49.66 GAE g⁻¹ DW), TFC (34.67 QE g⁻¹ DW), and total tannin content (TTC) (57.66 TAE g⁻¹ DW) in callus suspension of Paris polyphylla. Total phenolics and rosmarinic acid contents were enhanced by hairy root culture of O. basilicum [48]. Production of boeravinone-B, total phenolics, and flavonoids was reported to be enhanced by callus culture of Boerhavia diffusa [36]. Resveratrol and ?-viniferin yield were increased by 26 and 620-fold in cell suspension culture of Vitis vinifera by Wang et al. [56]. Capsicum chinense cell suspension culture improved the quantity of capsaicin (2.87 mg g⁻¹ FW) [38] and dihydrocapsaicin (1.03 mg g⁻¹ FW) . A 10-fold increase in isoflavones was reported in studies of Jeong et al. [45] by cell suspension culture of Glycine max. They also reported an enhanced production of total phenolics (0.1108 mg g⁻¹ FW). Fazal et al. [51] reported that cell suspension of Prunella vulgaris produced 7.62 mg g⁻¹ DW of polyphenolics . 1.66 times higher flavonoid content and 1.49 times higher phenolics were obtained by AR culture of Oplopanax elatus [49].

Figure 1. Secondary metabolite production by plant tissue culture techniques. [Click here to view]

Table 1. Some bioactive SMs obtained via PTC systems. [Click here to view]

3. STRATEGIES TO INCREASE SECONDARY METABOLITES YIELD

Despite being widely established, PTC approaches for the generation of SMs are still only applied to a few processes in large-scale manufacturing. Key strategies (Fig. 2) used to boost production of SM in plant cell cultures are: selection of high-yielding cell lines through screening and optimization to identify the most productive strains; optimization of culture conditions, including medium composition, pH, temperature, light, and oxygen levels to create an ideal environment for metabolite production; addition of elicitors, such as fungal extracts, signaling molecules, and abiotic stressors, to trigger defense responses and metabolite production; feeding of precursors and intermediates to provide building blocks and boost biosynthetic pathways; employing permeabilization techniques, such as ultrasonication, to increase cell permeability and metabolite release; use of two-phase culture systems to continuously remove and sequester products avoiding feedback inhibition; addition of adsorbents like activated charcoal or resins to capture and remove metabolites from the medium; application of metabolic engineering to enhance biosynthetic pathways or block competing pathways; in situ removal of the products from culture to increase the accumulation of SMs which may be limited by feedback inhibition; immobilization technique by using a simple gel matrix which have reported to increase metabolite production, as well as ease the process of extraction and purification of the resulted products; and scale-up using bioreactors with optimized designs for plant cell culture at a larger scale. Another method that can be used for enhancing SM production is genome editing using CRISPR/Cas9 technology. These strategies aim to overcome the challenges of low productivity and enable commercial-scale production of valuable plant-derived pharmaceuticals using cell suspension cultures [15,20,57,58].

Figure 2. Strategies to increase SM yields. [Click here to view]

4. ELICITATION

Plant cells generate SMs as a defense mechanism against infectious pathogenic elicitors, as well as in response to environmental stresses during in vivo growth. As a result, there is growing interest in using substances to help develop plant cells, tissues, or organs to mount a defense response in order to increase the production of bioactive chemicals under in vitro cultures. This process is referred to as elicitation [57]. The technique of SM elicitation involves the exogenous administration of elicitors in cell and tissue cultures. Changes to the culture media, such as the exogenous administration of elicitors or precursors or modifying external factors, can result in the induction of the stress response and an increase in the synthesis of essential metabolites [18].

Chemical, physical, or biological factors that can cause physiological as well as biochemical alterations by inducing stress in the target living things are known as elicitors [59]. On application of such elicitors into the target system, it triggers and enhances the buildup of the specified metabolites even when it is applied in a very small amount [60]. They have been used exogenously and have been demonstrated to stimulate secondary metabolism and encourage the accumulation of desirable metabolites in a variety of plant species. The biosynthetic pathways can be affected by a variety of elicitors in different ways [20].

Elicitors can be classified as biotic or abiotic, as well as general and race-specific elicitors [10]. Abiotic elicitors are components of non-biological sources, including the majority of inorganic chemicals, such as salts of heavy metals, metal ions, and metal oxides, as well as physical stresses like cold shock, UV, osmotic, water stress, light, temperature shift, and others [61,62]. Biotic elicitors are derived from biological sources, such as living bacteria, yeasts, fungal polysaccharides, lipids, and glycoproteins, as well as intracellular molecules, proteins, salicylic acid (SA), jasmonic acid (JA), methyl jasmonate (MeJA), and components of microbial or plant cell walls, such as chitosan (CS) and chitin [1,63].

Studies on the signaling, gene expression, and enzyme activity involved in the manufacture of significant medicinal molecules like SA, paclitaxel, resveratrol, etc. have become more and more prevalent. These are the stages of the focused metabolic engineering process that increase the accumulation of therapeutic SMs [64–65]. Crucial variables affecting the successful generation of biomass and the accumulation of SMs include elicitor concentrations, exposure time, and the age or phase of the culture at the point of elicitor administration [67].

The outcomes of α-NAA treatment along with different spectral lights were conducted by Ali et al. [68] to estimate the growth, secondary metabolic activity, and phenolic acid biosynthesis in A. bracteosa AR cultures and found out highest AR induction frequency which was 88% and biomass formation (72-g/l FW and 22-g/l DW) were seen in explants incubated with 1.5-mg/l NAA under yellow light, maximum production of polyphenols (TPC;44.2 mg) and flavonoids (TFC;2.51 mg) were found in the AR cultures grown in response to blue light. The highest total protein content (401.6 μg) was reported in the AR grown in the presence of normal white light, and blue spectral light brought about maximum superoxide dismutase (SOD; 2.5 nM) and peroxidase activity (POD;0.85 nM), respectively [68]. Jeet et al. [69] studied the effects of CS, MeJA, SA, KCl, NaCl, and polyethylene glycol on indole alkaloids accumulation in callus culture of Alstonia scholaris [69]. Addition of yeast extract and CS in liquid hairy root culture of Astragalus membranaceus resulted in the elicitation of astragaloside and isoflavones accumulation, respectively [70]. Corylus avellana cell suspension culture was exposed to cell extract and culture filtrate derived from Camarosporomyces flavigenus fungus for paclitaxel accumulation [71].

NPs have the potential to take the place of biotic elicitors in the elicitation of secondary metabolism. Designing a particular form of monodispersed and stimulus-responsive NPs based on the target metabolite/type of plant cell growth is possible owing to their adaptable physicochemical properties [11]. Also, NPs are frequently utilized as nanocarriers of different biotic and abiotic elicitors to promote the synthesis of specific metabolites in vitro, in addition to being used as nano-elicitors [19].

Distinct features of NPs, like their chemical reactivity, binding properties, optical activity, and morphology, make them vary from their corresponding bulk materials. NPs will also have more enhanced properties owing to their size, shape, and structures [72]. Due to their special qualities—like their small size, high surface-to-volume ratio, capacity to engineer electron exchange, and high surface reactive capabilities—NPs readily penetrate and interact with plant cells and tissues' constituent components [5]. Basic methods for intact plants to uptake NPs are foliar absorption either through the stomata or stratum corneum pores of the leaves and root uptake through primary roots, root cell wall pores, or damaged areas from soil [73]. It can also enter through other aerial parts like hydathodes, cuticles, trichomes, or damaged tissues. In vitro cultured cells, organs, or plantlets, uptake NPs through cell pores from the NPs-treated medium [74]. The length and width of the stomata and stomatal pores are usually in micrometer scale. Therefore, entry of NPs through stomatal pores is relatively easy and, therefore, is the primary way of foliar uptake. But due to the smaller radii of cuticular pores with approximately 2.4 nm, only smaller NPs pass through cuticles. But NPs of about 50 nm have also been found to enter through cuticles [75]. In case of root uptake, various studies proved that in different plant species, the size exclusion limit varies and, therefore, NPs varying from 3.5 to 140 nm or above can be entered through this pathway in different species [76]. It has been found that in certain instances, NPs larger than the plant cell wall pores were also observed to enter the cells, either by enlarging pre-existing plant cell wall pores or by stimulating the formation of new, larger plant cell wall pores [5,77]. There are many factors influencing the uptake of NPs, which include type, morphology, and size of the NPs; species, growth stage, and condition of plants; and phyllospheric, as well as rhizospheric factors like root exudates and microorganisms, which play a major role in changing morphological properties of NPs and their dissolution and transformation [76].

NPs reach the plant cell membrane after getting internalized through leaves, roots, or cell wall pores. It can be further internalized from the cell membrane either by apoplastic or symplastic pathways. By the apoplastic pathway, NPs can pass through the intercellular spaces between the cell wall and cell membrane of a cell to other cells from the epidermal to the cortex region, but not through the endodermis because of the presence of Casparian strips. Some NPs can penetrate the cell membrane and reach the cytoplasm either by endocytosis, certain membrane-bound transporter proteins, or by inducing new pores utilizing ion carrier molecules and thus enter the symplastic pathway and pass through plasmodesmatal connections between adjacent cells till vascular bundles from where they will be transported to the entire length of plants through xylem and phloem [78,79]. Thus, entered NPs can interact with intracellular organelles and elements and can cause stress and accumulation of reactive oxygen species (ROS), which may interrupt both primary and secondary metabolic pathways of plants and thus can lead to elicitation of SMs [5].

5. NANOMATERIALS AS ELICITORS OF PLANT SECONDARY METABOLITES

Nanomaterials are classified into three main sub-classes: (1) metallic NPs (MNPs), (2) metal oxide NPs (MONPs), and (3) carbon-related nanomaterials for elicitation of plant SMs [5]. Several nanomaterials have been studied for their effect on various plant species and cultures in stimulating the secondary metabolism and thus the production of important SMs, as listed in Table 2.

Table 2. Few examples of SMs elicitation using NPs as elicitors in PTC. [Click here to view]

6. MECHANISM OF NANOPARTICLE-MEDIATED ELICITATION OF SECONDARY METABOLITES

Elicitation mechanisms can involve thousands of mediators from numerous signaling channels, and their interactions make them extremely complicated. The origin, specificity, exposure type, timing, and concentration of the elicitors, as well as plant-dependent factors like the stage of development, cellular cycle, type of tissue, nutritional conditions, elicitor uptake by media/soil/aerial, physicochemical environment, and many other factors, affect how these events change [5]. Therefore, it is very difficult to suggest an all-encompassing model for the mechanism by which nanomaterials elicit the synthesis of plant SMs. The scheme of the general mechanism of the elicitation mechanism in plant secondary metabolism is depicted in the Figure 3.

Figure 3. Mechanism of NP-mediated elicitation of SMs. NPs: Nanoparticles, RLK: Receptor-like kinase; GPCR: G protein-coupled receptor, PLC: Phospholipase-C, PIP: Phosphatidylinositol 4,5-bisphosphate, IP3: Inositol 1,4,5-triphosphate, DAG: Diacylglycerol, CalPs: Calcium-binding proteins, JA: Jasmonic acid, SA: Salicylic acid, MAPK: Mitogen-activated protein kinases, TFs: Transcription factors, ROS: Reactive oxygen species. [Click here to view]

A chain of events gets started as soon as the NPs reaches inside the cell. There are numerous receptors on the cell surface and membranes, and multiple elicitor binding sites have been discovered [74]. The initial event is the receptor–elicitor contact, where the NPs that are being applied as the elicitor act as the primary messenger molecule. The interaction of NPs with the transmembrane receptor-like kinases (RLKs) results in a signal transduction cascade. The binding of NPs with RLKs causes Ca²⁺ influx from intracellular stores [5]. Release of Ca²⁺ from apoplastic space also occurs, which adds to the Ca²⁺ level in the cytosol [128]. Elicitor contact with specific receptors will also lead to plasma membrane depolarization, which will further lead to the activation of certain ion channels in the plasma membrane to result in the efflux of Na⁺, K^+, and Cl^- and H⁺ influx. This will further increase the influx of Ca⁺ and deactivation of ATPase, leading to cytoplasmic acidification and extracellular alkalinization [74,129]. The interaction of NPs with G protein-coupled receptors will activate the G-protein and further initiate the activation of phospholipase-C (PLC) on the membrane [130]. PLC then hydrolyzes phosphatidylinositol 4,5-bisphosphate and yields two secondary messenger molecules inositol triphosphate (IP₃) and diacylglycerol. IP₃ then diffuses into the cytosol and leads to the release of Ca²⁺ from intracellular calcium reservoirs through Ca²⁺-gated ion channels [131].

The released Ca²⁺ also regulates the PLC, which will further increase the Ca²⁺ concentration within the cytoplasm and also the production of other secondary messengers like JA, SA, ethylene, etc., which can directly or indirectly modulate secondary metabolism [132]. It is also known that Ca²⁺ activates several oxidases, like cell membrane NADPH oxidase, apoplastic POD, and several other oxidases in cellular organelles like chloroplasts, mitochondria, and peroxisomes. The activation of these oxidases will lead to oxidative burst by which enormous amounts of ROS are generated within the cell [129]. It includes hydrogen peroxide, superoxide anions, hydroxyl radicals, and singlet oxygen. NPs interfere with the electron transport chain in chloroplasts and mitochondria, which also leads to the generation of ROS and reactive nitrogen species and nitric oxide [128,129]. These are known to cause cellular damage by protein modification, lipid peroxidation, and DNA damage. These are also known to activate certain enzymatic and non-enzymatic antioxidant systems in plants like SOD, ascorbate peroxidase, catalase, and glutathione-S-transferase, which help to convert ROS and detoxify them. Based on the balance between generation and scavenging of ROS within the cell, they either cause oxidative damage or act as signaling molecules . ROS also causes damage to the plasma membrane and activation of PLC, which will further lead to a Ca²⁺ spike [133]. ROS is also known to activate another cascade called the mitogen-activated protein kinase (MAPK) cascade, and also the production of jasmonates, ethylene, etc. NPs' perception by membrane receptors may also activate the MAPK cascade. All these will lead to an increase in Ca²⁺ concentration approximately within 5 minutes of NPs administration, which marks a very early response [129,130].

The increased Ca²⁺ level within the cytoplasm can be sensed by certain calcium-binding proteins or calcium sensors like calmodulin, calmodulin-like proteins, phospholipase-D, annexins, calreticulin, calnexin, and pistil-expressed calcium binding proteins or by calcium-dependent protein kinases. These Ca²⁺ signaling will further transfer the information downstream to initiate a cascade of phosphorylations, which includes activation of MAPK cascades, which will lead to regulation of expression of genes involved in secondary metabolism mainly by altering the phosphorylation status of certain transcription factors (TFs) of target genes [128].

The complex signaling pathways that occur on exposure to the NPs, like MAPK cascades, Ca²⁺, ROS, JA, SA, and ethylene signaling, and the cross-talks between these, together activate several TFs that will bind to the target genes and regulate the defense mechanism, thereby enhancing SM production [134]. The major TFs involved in secondary metabolism are: i) MYB TF, which contains the MYB DNA-binding domain. It is involved in the biosynthetic pathways of anthocyanin, proanthocyanidins, glucosinolate, phenylpropanoids, flavonoids, and hydroxycinnamic acid amide (HCCAs). ii) bHLH TFs, which are found to act in combination with MYB TF in the regulation of anthocyanin production. bHLH TF-like MYC2 plays a major role in terpenoid indole alkaloid (TIA), nicotine, anthocyanin, alkaloids, glucosinolate, diterpenoid, phytoalexin, and saponin biosynthesis. iii) AP2/ERF family TFs have a DNA-binding AP2 domain, such as in the ORCA protein, which has a major role in controlling multiple genes involved in TIA biosynthesis. It is also found to be involved in artemisinin, artemisinic acid, nicotine, vinblastine, bisindole alkaloids, steroidal glycoalkaloids, saponin, and lignin biosynthesis. iv) WRKY family TFs are found to have a role in biosynthesis of gossypol, artemisinin, volatile terpene, camalexin, phytoalexin, HCCA, resveratrol, benzylisoquinoline alkaloid, taxol, TIA, etc. v) NAC TFs are found to be involved in camalexin and putrescine biosynthesis. vi) bZIP TFs have major roles in lignin, phenolic acids, flavonoids, terpenoids, alkaloids, anthocyanin, tanshinone, and artemisinin biosynthetic pathways [6,135,136].

7. CONCLUSION AND FUTURE PROSPECT

Since there is a very high demand for SMs in various fields and because of the improved knowledge about commercially important plant-based SMs, currently, the use of PTC techniques has increased for their large-scale production. PTC is an advantageous technique in order to synthesize biocompounds from plants that are even on the verge of extinction for their conservation. It also provides a good yield irrespective of any environmental or geographical conditions if sterile and appropriate conditions are properly maintained.

In order to enhance the production potential of important SMs in terms of both quantity and quality in culture systems, the elicitation technique has been proven to be highly advantageous. Use of nanomaterials (MNPs, MONPs, and other carbon-related nanomaterials) due to their specificity and enhanced properties is reported to be a potential approach. Various research studies proved the improved production of diverse plant SMs of commercial importance on elicitation using different types of nanomaterials in various culture conditions. Concentration, size, exposure time, time of addition of nanomaterials, as well as the plant species, type, and age of culture, are the main factors to be given importance during the elicitation process for the maximum result.

Further research by addressing the limitations of present methods, as well as by utilizing the emerging technologies, will definitely help to produce important SMs more sustainably while giving enhanced yield. For this, there is an importance to move forward by acknowledging the current limitations. Some studies have reported certain concentrations of NPs to be toxic to the plants. Therefore, the usage of smaller concentrations of NPs seems to be more efficient in generating higher amounts of SMs without causing phytotoxicity to the plants. Further studies incorporating metabolomics, transcriptomics, and genomics will help provide a wide understanding of the cellular and molecular-level events that take place on elicitor administration in plants. Thus, specific steps in metabolic pathways, specific genes and proteins that experience alterations and are responsible for SM generation can be identified, and thus the elicitation mechanism can be improved accordingly. Moreover, there are still many sources unveiled for green synthesis of safer, less toxic NPs as well as for SM production. More research improving the existing techniques and approaches more sustainably, without compromising efficiency and productivity, has to be introduced to bring out a major impact, generating better economic productivity and environmental sustainability.

8. ACKNOWLEDGMENT

The authors wish to express their profound appreciation to the Rashtriya Uchchatar Shiksha Abhiyan (RUSA 2.0) program of the Government of India for its funding of infrastructure and research activities under Research Innovation and Quality Improvement (Component-10), which is being implemented through the University of Mysore.

9. AUTHOR’S CONTRIBUTION

All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agreed to be accountable for all aspects of the work. All the authors are eligible to be an author as per the International Committee of Medical Journal Editors (ICMJE) requirements/guidelines.

10. ETHICS APPROVAL

This study does not involve experiments on animals or human subjects.

11. DATA AVAILABILITY

All the data is available with the authors and shall be provided upon request.

12. PUBLISHER’S NOTE

All claims expressed in this article are solely those of the authors and do not necessarily represent those of the publisher, the editors and the reviewers. This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.

13. USE OF ARTIFICIAL INTELLIGENCE (AI)-ASSISTED TECHNOLOGY

The authors declare that they have not used artificial intelligence (AI)-tools for writing and editing of the manuscript, and no images were manipulated using AI.

14. CONFLICT OF INTEREST

The authors report no financial or any other conflicts of interest in this work.

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