*3.1.2. Osmotic stress*

**Elicitor Plant species Nature of culture Compounds References**

*Vitis vinifera* Cell suspension Resveratrol [67] *Salvia castanea* Hairy root Tanshinone [173] *Datura metel* Hairy root Atropine [150]

*Datura stramonium* Root Sesquiterpenoid [79]

*Bacopa monnieri* Shoot Bacoside [170] *Datura stramonium* Root Sesquiterpenoid [79]

vincristine

tanshinone IIA

tanshinone IIA

[55]

[78]

[78]

Sodium salicylate *Salvia officinalis* Shoot Carnosol [180]

Sodium chloride *Catharanthus roseus* Embryogenic tissues Vinblastine and

252 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

Sorbitol *Perovskia abrotanoides* Adventitious roots Cryptotanshinone and

Silver (Ag) *Perovskia abrotanoides* Adventitious roots Cryptotanshinone and

Cadmium (Cd) *Vitis vinifera* Cell suspension Resveratrol [67]

Cobalt (Co) *Vitis vinifera* Cell suspension Resveratrol [67] Copper (Cu) *Ammi majus* Shoot Xanthotoxin [181]

**Table 1.** Effect of Different Abiotic Elicitors on the Production of Various Secondary Metabolites in Plants

Physical elicitors include light, osmotic stress, salinity, drought, and thermal stress.

The light is a physical factor that can affect the metabolite production. Light can stimulate such secondary metabolites include gingerol and zingiberene production in *Zingiber officinale* callus culture [22]. The effect of light irradiation on anthocyanin production in cell suspension cultures of *Perilla frutescens* was reported [23]. The effect of light and hormones on the digitoxin accumulation in *Digitalis purpurea* L. was reported by Hagimori et al. [24]. Moreover, in hairy root cultures of *Artemisia annua*, the effect of light irradiation influenced the artemisinin biosynthesis [25]. The effect of white light on taxol and baccatin III accumulation in cell cultures of *Taxus cuspidate* was reported by Fett–Neto et al. [26]. Ultraviolet (UV) radiation stimulates secondary metabolite production. Increasing UV–B exposure in field–grown plants not only increased the total essential oil and phenolic content but also decreased the amount of the possibly toxic beta–asarone [27]. These findings are to be expected as phenolics are known UV protectants [28]. *Catharanthus roseus* plants, exposed to UV–B light, show significant increases in the production of vinblastine and vincristine, which have proven effective in the treatment of leukemia and lymphoma [29]. UV–C irradiation promotes the phenylpropanoid pathway

**3.1. Physical elicitors**

*3.1.1. Light*

Osmotic stress (water stress) is an abiotic physical elicitor [35] and is one of the important environmental stresses that can alter the physiological and biochemical properties of plants and increase the concentration of secondary metabolites in plant tissues [36]. Proline acts as an osmolyte, as protective agent for cytoplasmic enzymes, as a reservoir of nitrogen and carbon sources for post–stress growth, or even as a stabilizer of the machinery for protein synthesis, regulation of cytosolic acidity and scavenging of free radicals [37]. However, the various roles of proline have been proposed, but the main role could be the osmotic adjustment in osmoti‐ cally stressed plant tissues and the protection of plasma membrane integrity [38]. Polyethylene glycol (PEG) is an osmotic agent (nonpenetrating osmoticum) that has been used for induction of water stress in many plants [39]. The proline and PEG enhanced the production of steviol glycosides content in both callus as well as suspension culture of *Stevia rebaudiana* [40]. PEG elicited the pharmacologically active compounds, such as hypericin and pseudohypericin, in *Hypericum adenotrichum* [41]. Sucrose is a typical osmotic stress agent used for the induction of water stress in plants that also serves as a vital carbon and energy source [42]. It has been shown that water and osmotic imbalance can strongly influence the synthesis of hypericin and hyperforin in *Hypericum perforatum* plants [43]. In addition, it has been reported that both hypericin and pseudohypericin concentrations decreased, while hyperforin concentration increased significantly in the plants grown under water stress conditions [36].

### *3.1.3. Salinity*

Salinity reduces plant growth and development and alters a wide array of physiological and metabolic processes [44,45]. Plants have developed complex mechanisms for adaptation to the osmotic, ionic, and oxidative stresses that are induced by the salt stress. Exposure to salinity is known to induce or stimulate the production of secondary plant products, such as phenols, terpenes, and alkaloids [46–48]. *C. roseus* grown under salt stress showed increased levels of the alkaloid vincristine [49]. In *Grevillea*, a significant increase in anthocyanin concentration was reported under salinity exposure in both the salt–tolerant *Grevillea ilicifolia* and the salt– sensitive *Grevillea arenaria* [50]. In contrast to this, salt stress decreased the anthocyanin level in the salt–sensitive species [51]. In *Datura innoxia*, salt treatment increased the total alkaloid content in young leaves, and the results indicated that at the organ level, tropane alkaloid accumulation was related to plant growth [52]. Glycine betaine was increased under salinity in numerous species including *Triticum aestivum* [53] and *Trifolium repens* [54]. Salinity also increased the diamine and polyamine content in *Oryza sativa* [53]. An improved synthesis of vinblastine and vincristine was observed in *C. roseus* embryogenic tissue culture by using NaCl as an elicitor [55].

#### *3.1.4. Drought stress*

One of the most important abiotic stress is drought, which affect plant growth and their developmental process [56]. The available water in the soil is reduced to such critical levels, and atmospheric conditions add to the continuous loss of water; the situation is called drought stress. The high temperature in the environment and solar radiations add up the water deficit in the soil, which leads to drought stress. Drought stress tolerance is observed in all types of plants, but its extent varies from species to species [56]. Drought stress, which can also greatly reduce plant growth, can increase secondary metabolite content. Mild water stress significantly increased the content of the anti–inflammatory saikosaponins in *Bupleurum chinense* [57]. Moderate water stress increased the content of salvianolic acid in roots of *Salvia miltiorrhiza*, although the content of other bioactives, including tanshinone, was lowered [58]. Moderate drought stress also increased the production of rosmarinic, ursolic, and oleanolic acid in *Prunella vulgaris* [59]. A weak water deficit greatly increased the glycyrrhizic acid content in roots of *Glycyrrhiza uralensis* [60]. In *Hypericum brasiliense*, the amounts of various phenols and betulinic acid were drastically increased under drought stress [61].

#### *3.1.5. Thermal stress*

Although thermal stress can greatly reduce plant growth and induce senescence, elevated temperatures (heat stress) or low temperatures (cold stress) have also been shown to increase secondary metabolite production. Temperature strongly influences metabolic activity and plant ontology, and high temperatures can induce premature leaf senescence [62]. Elevated temperatures increase leaf senescence and root secondary metabolite concentrations in the herb *Panax quinquefolius* [63]. A 5°C increase in temperature significantly increased the ginsenoside content in roots of *P. quinquefolius* [63]. A temperature variation has multiple effects on the metabolic regulation, permeability, and rate of intracellular reactions in plant cell cultures [62]. Temperature range of 17–25°C is normally used for the induction of callus tissues and growth of cultured cells [16]. The temperature and light quality influences on the production of ginsenoside in hairy root culture of *Panax ginseng* [64]. The *Melastoma malaba‐ thricum* cell cultures incubated at a lower temperature range (20 ± 2°C) grew better and had higher anthocyanin production than those grown at 26 ± 2°C and 29 ± 2°C [65]. Fifteen days at 35°C significantly increased the hypericin and hyperforin content in shoots of *Hypericum perforatum* [66].

#### **4. Chemical elicitors**

Heavy metals have become one of the main abiotic stress agents for living organisms because of their increasing use in the developing fields of industry and agrotechnics and high bioaccumulation and toxicity [67]. Although a lot of information is available concern‐ ing the effects of heavy metals on plant growth and physiology, much less is known regarding their effects on the production of secondary metabolites. Heavy metal–induced changes in metabolic activity of plants can affect the production of photosynthetic pig‐ ments, sugars, proteins, and nonprotein thiols. These effects can result from the inhibition of enzymes involved in the production of these natural products, likely through impaired substrate utilization [68]. Metals may alter the production of bioactive compounds by changing aspects of secondary metabolism [2]. Metals including Ni, Ag, Fe, and Co have been shown to elicit the production of secondary metabolites in a variety of plants [69].

An increased oil content up to 35% in *Brassica juncea* was seen due to the effective accumulation of metals (Cr, Fe, Zn, and Mn) [70]. The highest accumulations of secon‐ dary metabolites such as shikonin [71] and also the production of digitalin [72] were observed by treating Cu2+ and Cd2+. The production of betalains in *Beta vulgaris* also stimulated by Cu2+ [73]. Co2+ and Cu2+ have a stimulatory effect on the production of secondary metabolites in *Beta vulgaris* [73]. The betalaines production was enhanced by exposing the hairy root culture to metal ions [74]. The stimulatory effects of Cu2+ on the accumulation of betacyanins in callus cultures of *Amaranthus caudatus* were reported by Obrenovic [75]. The addition of Zn2+ (900 μM) improved the yield of lepidine in cultures of *Lepidium sativum* [76]. However, Cu proved more effective than Zn in enhancing the yield product [76]. In hairy root cultures of *Brugmansia candida*, silver nitrate (AgNO3) or cadmium chloride (CdCl2) elicited the overproduction of two tropane alkaloids, scopolamine, and hyoscyamine [20]. The production of taxol in cell culture of *Taxus* sp. was enhanced by the rare–earth metal (lanthanum) [77]. AgNO3 stimulated the production of tanshinone in the root culture of *Perovskia abrotanoides* [78]. The treatment of root cultures of *Datura stramoni‐ um* with cadmium salts at external concentrations of approximately l mM has been found to induce the rapid accumulation of high levels of sesquiterpenoid–defensive compounds, notably lubimin and 3–hydroxylubimin, but not alkaloid [79].
