**3.1 Using elicitors for terpene production**

It has been shown that certain stress conditions can increase or inhibit terpene production and change the emission pattern and/or amount. Stress conditions generally affect both constitutive and induced terpene emission rates [15]. Treatment of *in vitro* cultures with biotic and abiotic elicitors has been seen as essential to increase the production of desired products [37]. This technique has become the subject of an increasing number of studies today. The elicitor triggers signal transduction and generates secondary signals, stimulating regulatory proteins (transcription factors) that coordinate the expression of biosynthetic genes [38]. Elicitors can be classified as biotic and abiotic elicitors depending on their source and effect on plants (**Figure 2**).

Elicitor type, dose, and application time are the main factors in terpene production [39]. Apart from NaCl, an abiotic elicitor source, there is a lot of literature on methyl jasmonate (MeJA) and salicylic acid (SA). MeJA is a plant growth regulator belonging to the jasmonate family. Extensive research has been conducted on phytohormones, as they can influence many physiological and metabolic processes in plants. Given the great diversity of these compounds with specific biological functions, they are also referred to as biostimulants [24]. Biostimulants are essential as a signaling molecule that mediates intra- and inter-plant communication and modulates plant defense responses, including antioxidant systems. Farag et al. [37] studied the effects of six

**Figure 2.**

*Classification of elicitors used in secondary metabolite production.*

different biotic and abiotic elicitors, including MeJA, SA, ZnCl2, glutathione, and β-glucan (BG; fungal stimulant) wounding, on terpene accumulation in soft coral *Sarcophyton ehrenbergi*. Based on the elicitation process, it has been discovered that the mere inclusion of 0.1 mM SA and 1.0 mM ZnCl2 resulted in a remarkable increase in the levels of sarcophytolide I by 132 and 17 times, respectively, in just 48 hours.

Again, in *Mentha x piperita* plants, where SA or MeJA was applied exogenously, a significant increase in menthol, pulegone, linalool, limonene, and menthone concentrations was achieved with the application of 2.0 mM MeJA [40]. Depending on the dose and time, significant increases in the accumulation of TIAs (eburenin, quebrachamine, fluorocarpamine, pleiocarpamine, tubotaiwine, tetrahydroalstonine, and ajmalicine) occurred in hairy root cultures of *Rhazya stricta*, elicited by the application of different concentrations of MeJA [38]. The roots of *Salvia miltiorrhiza*, a plant rich in tanshinones, an essential active diterpene, are used medicinally for cardiovascular diseases and inflammation. In a study on *Salvia miltiorrhiza*, with 0.2% (v/v) bacterial inoculum as a biotic elicitor, the total tanshinone content of the roots increased more than 12-fold [41]. Li et al. [42] reported that selected genes in the tanshinone biosynthetic pathway of Ag (+), MeJA, and yeast extract were significantly upregulated in *S. castanea* f tomentosa Stib hairy root cultures. Yeast extracts increased the expression level of isopentenyl diphosphate isomerase 13.9 fold at 12 hours. It was determined that yeast extracts increased the expression level of isopentenyl diphosphate isomerase 13.9-fold in 12 h. In contrast, the contents of tanshinone IIA were increased by 1.8-fold and 1.99-fold compared to the control with Ag (+) and MeJA elicitation, respectively.

The addition of the biotic elicitor Anabaena sp. (265 cells/mL) to cell suspension cultures of *Azadirachta indica* triggered the synthesis of the triterpene azadirachtin (0.32 g/μL) [43]. UV-B caused a significant increase in lochnericine concentrations in hairy root cultures of C. roseus. It has also been shown that increasing the exposure time to UV-B up to 20 minutes causes significant increases in lochnericine, serpentine, and ajmalicine and a decrease in horhammericine [44].

## **3.2 Effect of salt as an elicitor**

NaCl is one of the most important abiotic stress factors that cause different changes in plants' morphological, physiological, and biochemical responses, limit their growth and development, and consequently negatively affect total crop production [45, 46]. In the early stages of salinity-induced stress, the ability of roots to absorb water is highly affected and reduced [47]. Higher NaCl concentrations pose a significant threat to the plant by inhibiting physiological processes through osmotic stress, nutrient imbalance, ionic toxicity, and oxidative stress [48]. Oxidative stress is a process in which reactive oxygen species occur as a result of excessive accumulation of sodium (Na+ ) and chloride (Cl<sup>−</sup> ) in plant tissues [49]. After exposure to excess NaCl, plants first sense the potential source of stress and then activate a multifaceted response that includes a signaling network and the synthesis of several compounds that help reduce the effects of high salinity and maintain cellular homeostasis. At this point, secondary metabolites play critical roles in plant adaptation to NaCl stress [4]. Depending on the salinity levels that plants are exposed to *in vitro*, the type and amount of these metabolites they synthesize to survive may vary. Thus, plants can often produce species-specific secondary metabolites in shoots, roots, leaves, etc., at different stages of plant development [10]. Some studies have reported that terpenes exhibit antioxidant activities and thus their function in overcoming oxidative

### *Influence of Salinity on* In Vitro *Production of Terpene: A Review DOI: http://dx.doi.org/10.5772/intechopen.111813*

stress [4]. During NaCl stress, terpenes can reduce the consequences of oxidative stress either by reacting directly with intercellular oxidants or altering the signaling of reactive oxygen species. Various terpenes can minimize NaCl stress in different plant species by providing membrane stabilization and direct antioxidant effects. In addition to their antioxidant effects, isoprenes and monoterpenes can react rapidly with ozone and reduce the toxicity caused by NaCl stress. Amphipathic isoprene can prevent membrane and protein degradation by improving hydrophobic interactions between membrane proteins and lipids [20].

Although significant progress has been made on the accumulation mechanisms of terpene compounds in medical plants *via* NaCl elicitation [50], studies are still ongoing on terpene compounds whose chemistry and role are still unknown. To the present day, especially in recent years, research on accumulating these compounds has been presented in **Table 1**. Terpenes and terpenoids are the main components of volatile oils of medical and aromatic plants; it has a variety of chemical compositions ranging from monoterpenes, sesquiterpenes, triterpenes, alcohols, ethers, aldehydes, esters, and ketones [60, 61]. There are 49 different terpenes in the leaves and resins of the male and female mastic tree (*Pistacia lentiscus*) [62]. Tilkat et al. [16] investigated the effects of different NaCl concentrations applied to juvenile shoots of *P. lentiscus in vitro* on the accumulation of anticancer triterpenes, such as ursonic, moronic, oleanolic, masticadienolic, oleanolic, and ursolic acids. Accordingly, while 50 mM NaCl elicitation increased the amount of ursonic acid 2.16 times, 25 mM NaCl elicitation increased it 3.71 times. In addition, masticadienolic acid, which is not found in the control group, was induced *in vitro* leaves *via* elicitated 100 mM NaCl; and ursolic acid was induced *in vitro* leaves and stems by 25, 50, and 100 mM NaCl. It is known that C. roseus, which contains 200 different TIAs, is also a source of vinblastine and vincristine, which have high economic and medical importance. However, since it has been observed that these compounds accumulate in insufficient amounts in the natural growing environment of this plant, it has been tried to increase the production of these compounds with various strategies [63]. Studies have shown that TIA accumulation can be significantly altered through stress mechanisms such as NaCl stress [53, 64] or studies such as overexpression of these genes [65, 66]. In a survey conducted by Fatima et al. [53] using different levels of NaCl concentrations (0, 25, 50, 75, 100, and 125 mM) as an elicitor in various embryogenic tissues of C. roseus grown *in vitro*, the content of vinblastine and vincristine was increased. It was noted that the maximum accumulation of vinblastine and vincristine was obtained by elicitation with 25 mM NaCl. Thymol is a natural volatile monoterpene phenol and the main active ingredient of essential oil obtained from *Thymus vulgaris, Ocimum gratissimum, Carum copticum,* and *Nigella sativa*. Black seed (*N. sativa* L.) is a medicinal plant used to treat many diseases since ancient times. It has been reported that 250 mM NaCl elicitation in *N. sativa* calli leads to high thymol accumulation [19]. Diterpene steviol glycosides obtained from the leaves of the *Stevia rebaudiana* plant are calorie-free natural sweeteners. NaCl and Na2CO3 salts were investigated on steviol glycoside production in callus and suspension cultures. In calli, steviol glycosides increased from 0.27% (control) to 1.43% and 1.57%, respectively, with 0.10% NaCl and 0.025% Na2CO3. However, in suspension cultures, the same concentrations of NaCl and Na2CO3 increased the steviol glycoside content from 1.36 (control) at day 10 to 2.61% and 5.14%, respectively [52]. In addition to the studies on increasing the terpene compounds with a single type of elicitation *in vitro*, more than one type of elicitation has been tried in many plants, and successful results have been obtained. In this context, it has been reported that elicitors, such as NaCl, chitosan, MeJA, SA,


#### **Table 1.**

*Effect of salt stress on the accumulation of some terpene compounds in plants.*

and jasmonic acid (JA), are widely used in combination or separately for terpene production [25, 67–69]. Razavizadeh et al. (2020) investigated the potential effects of terpene accumulation by examining the combined effect of chitosan and NaCl on *C. copticum* shoot and callus cultures. They reported that combined elicitation led to an increased accumulation of thymol and p-cymene in both shoots and calli [25]. In another study on the production of vincristine and vinblastine from TIAs in C. roseus calluses of both osmotic and NaCl stresses, 75 mM NaCl elicitation significantly increased the content of both bioactive compounds [51]. In another combined elicitation study, the effect of NaCl and phenylalanine on rosmarinic acid production *in vitro* cultures of *Mentha longifolia* was investigated. Five different phenylalanine concentrations (0, 0.5, 5, 10, and 15 mg/l) and four different NaCl concentrations (0, 2000, 4000, and 6000 mg/l) were tested. Shoot tips were more efficient in rosmarinic acid production than callus cultures. In addition, low-concentration NaCl elicitation led to a high accumulation of rosmarinic acid [70].

As discussed above, salt, an abiotic stress factor, actually increases the synthesis of terpenes in plants and is an essential factor for plants to combat the negativities they encounter under natural conditions (**Figure 3**). In addition, it is possible to increase

**Figure 3.** *Terpenes production under NaCl stress [71–73].*

the production of terpenes with economic value by plants through salt elicitation carried out *in vitro*. From this point of view, it can be seen that salt, which negatively affects agricultural production, is an essential factor that triggers different metabolic pathways in plants and contributes to the production of many other secondary metabolites, including terpenes, that are beneficial for humanity. Terpenes, in particular, and all secondary metabolites in general, which plants synthesize to make their own life easier and more efficient under salinity, form active compounds used in agricultural and industrial fields and necessary pharmaceutical raw materials for human beings.
