**7. Parameters of elicitors**

Elicitation has been widely used to increase the production or to induce de novo synthesis of secondary metabolites in in vitro plant cell cultures [141]. This opened up a new area of research that could have important economic benefits for pharmaceutical industry. Several parameters such as elicitor concentration and selectivity, duration of elicitor exposure, age of culture, cell line, growth regulation, nutrient composition, and quality of cell wall materials are also important factors influencing the successful production of secondary metabolite [142]. Some of these parameters were highlighted on elicitation of some medicinal plants for the production of secondary metabolites.

#### **7.1. Elicitor concentration**

Elicitor concentration plays a very important role in elicitation process. High dosage of elicitor has been reported to induce hypersensitive response leading to cell death, whereas an optimum level was required for induction [143–145]. At 0.1% (w/v) sodium chloride, ginseng saponin content and productivity were increased to approximately 1.15 and 1.13 times control values, respectively [146]. In the cell culture of *S. miltiorrhiza*, the effects of different concen‐ trations of SA were affected the accumulation of salvianolic acid B and of caffeic acid. The increased accumulation of salvianolic acid B and of caffeic acid was observed in the applica‐ tions of 3.125–25.0 mg/L of SA at 8 and 96 h when compared to the 32.0–50.0 mg/L of SA. After 96 h treatments with 3.125–25.0 mg/L of SA, the concentration of the phenolic acids decreased drastically compared to the amount 8 h after the treatments but still accumulated the higher concentrations of compound than that of the control [147]. The various concentrations (50, 100, 150, 200, and 250 μM) of MeJA and SA were used in the cell suspension cultures of *Gymnema sylvestre*. The MeJA at 150 μM and SA at 200 μM enhanced the accumulation of gymnemic acid content [148]. In the hairy root culture of *W. somnifera*, the MeJA (15 μM) and SA (150 μM) enhanced the production of withanolide A, withanone, and withaferin A content [95]. In the cell suspension culture of *V. vinifera*, the cobalt at all three used concentrations (5.0, 25, and 50 μM), Ag, and Cd at low concentration (5.0 μM) were most effective to stimulate the phenolic acid production, increasing the 3–O–glucosyl–resveratrol up to 1.6–fold of the control level (250.5 versus 152.4 μmol/g), 4 h after the treatments [67]. In the *A. paniculata* cell culture, MeJA at 5 μM showed 5.25 higher accumulation of andrographolide content compared with control [97]. The root cultures of *Taverniera cuneifolia* treated with increasing concentrations of MeJA (1.0, 2.5, 5, 10, 100, and 1000 μM) [135]. The glycyrrhizic acid content increased gradually with increase in MeJA (1–100 μM) concentration. Approximately 2.5–fold increase in glycyrrhizic acid production was noticed in MeJA (100 μM) treated roots, as compare to the unchallenged root culture. However, further increase in MeJA (1000 μM) concentration resulted in decrease in glycyrrhizic acid production [135].

#### **7.2. Duration of elicitor exposure**

The cell suspension culture of *G. sylvestre* was treated with MeJA and SA for 24 h, 48 h, and 72 h. With the MeJA treatment, the maximum gymnemic acid production was recorded 72 h after treatment with 150 μM (135.41 ± 0.43 mg/g DCW). The gymnemic acid content was 15.4–fold higher than the control cultures that were free of the elicitor. When the MeJA concentration exceeded 150 μM, there was a drastic fall (36.3%) in the gymnemic acid accumulation [148]. A high concentration of 200 μM SA was required to induce substantial quantities of gymnemic aid (43.27 ± 0.80 mg/g DCW) in the suspensions that reached a maximum after 48 h treatment. The SA–induced response toward gymnemic acid accumulation resulted in a 4.9–fold increase in comparison to the control cultures [148]. The different biotic elicitors (*A. rhizogenes*, *Bacillus subtilis*, *Escherichia coli, Aspergillus niger*, and *Saccharomyces cerevisiae*) required different duration of time (24, 48, 48, 72, and 72 h, respectively) to elicit the gymnimic acid in the cell suspension culture of *G. sylvestre* [149]. The MeJA and SA for 4 h exposure time enhanced the production of withanolide A, withanone and withaferin A content in the hairy root culture of *W. somnifera* [95]. The yields of atropine content in the *Datura metel* hairy roots were increased by nanosilver as an elicitor after 12, 24, and 48 h, but atropine accumulation in *D. metel* hairy roots was reduced by AgNO3, *Bacillus cereus*, and *Staphylococcus* after 12, 24, and 48 h [150]. In the cell culture of *Andrographis paniculata,* the MeJA induced the highest accumulation of andrographolide at 24 h compared with 48 and 72 h of treatments [97]. In the cell system of *V. vinifera*, a rapid accumulation of trans–resveratrol was recorded with MeJA treatment, starting from 2 h and reaching its maximum value at 96 h and the highest levels of viniferins recorded in cell cultures elicited with chitin (chitosan) for 144 h [91]. The MeJA produced the highest amount of bacoside A, 1.5–fold higher than the control shoots in the *B. monnieri* shoot culture after 48 h [96]. MeJA elicitation can cause an initial rapid increase in amount of various secondary metabolites from 24 to 72 h compared to controls after which a subsequent decrease can be found [151].

#### **7.3. Age of culture**

research that could have important economic benefits for pharmaceutical industry. Several parameters such as elicitor concentration and selectivity, duration of elicitor exposure, age of culture, cell line, growth regulation, nutrient composition, and quality of cell wall materials are also important factors influencing the successful production of secondary metabolite [142]. Some of these parameters were highlighted on elicitation of some medicinal plants for the

Elicitor concentration plays a very important role in elicitation process. High dosage of elicitor has been reported to induce hypersensitive response leading to cell death, whereas an optimum level was required for induction [143–145]. At 0.1% (w/v) sodium chloride, ginseng saponin content and productivity were increased to approximately 1.15 and 1.13 times control values, respectively [146]. In the cell culture of *S. miltiorrhiza*, the effects of different concen‐ trations of SA were affected the accumulation of salvianolic acid B and of caffeic acid. The increased accumulation of salvianolic acid B and of caffeic acid was observed in the applica‐ tions of 3.125–25.0 mg/L of SA at 8 and 96 h when compared to the 32.0–50.0 mg/L of SA. After 96 h treatments with 3.125–25.0 mg/L of SA, the concentration of the phenolic acids decreased drastically compared to the amount 8 h after the treatments but still accumulated the higher concentrations of compound than that of the control [147]. The various concentrations (50, 100, 150, 200, and 250 μM) of MeJA and SA were used in the cell suspension cultures of *Gymnema sylvestre*. The MeJA at 150 μM and SA at 200 μM enhanced the accumulation of gymnemic acid content [148]. In the hairy root culture of *W. somnifera*, the MeJA (15 μM) and SA (150 μM) enhanced the production of withanolide A, withanone, and withaferin A content [95]. In the cell suspension culture of *V. vinifera*, the cobalt at all three used concentrations (5.0, 25, and 50 μM), Ag, and Cd at low concentration (5.0 μM) were most effective to stimulate the phenolic acid production, increasing the 3–O–glucosyl–resveratrol up to 1.6–fold of the control level (250.5 versus 152.4 μmol/g), 4 h after the treatments [67]. In the *A. paniculata* cell culture, MeJA at 5 μM showed 5.25 higher accumulation of andrographolide content compared with control [97]. The root cultures of *Taverniera cuneifolia* treated with increasing concentrations of MeJA (1.0, 2.5, 5, 10, 100, and 1000 μM) [135]. The glycyrrhizic acid content increased gradually with increase in MeJA (1–100 μM) concentration. Approximately 2.5–fold increase in glycyrrhizic acid production was noticed in MeJA (100 μM) treated roots, as compare to the unchallenged root culture. However, further increase in MeJA (1000 μM) concentration resulted in decrease

The cell suspension culture of *G. sylvestre* was treated with MeJA and SA for 24 h, 48 h, and 72 h. With the MeJA treatment, the maximum gymnemic acid production was recorded 72 h after treatment with 150 μM (135.41 ± 0.43 mg/g DCW). The gymnemic acid content was 15.4–fold higher than the control cultures that were free of the elicitor. When the MeJA concentration exceeded 150 μM, there was a drastic fall (36.3%) in the gymnemic acid accumulation [148]. A high concentration of 200 μM SA was required to induce substantial quantities of gymnemic

production of secondary metabolites.

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

in glycyrrhizic acid production [135].

**7.2. Duration of elicitor exposure**

**7.1. Elicitor concentration**

Age of culture plays is an important parameter in the production of bioactive com‐ pounds by elicitation. The treatment with MeJA and SA in the hairy root culture of *W. somnifera* showed highest accumulation of withanolide A, withanone, and withaferin A content after 40 days of culture [95]. In a study, 20–day–old cell cultures of *C. roseus* showed higher yields of ajmalicine on elicitation. The optimum level of ajmalicine (166 μg/g DW) was observed in 20–day–old cells elicited with extracts of *Trichoderma viride* followed by 90 and 88 μg/g DW ajmalicine in cells elicited with *A. niger* and *F. moniliforme*, respectively [127,152]. A similar type of observation was noticed from various workers Rijhwani and Shanks [153] and Ganapathi and Kargi [142]. The selenium addition at inoculum time did not significantly affect ginseng saponin accumulation. However, the addition of 0.5 mM selenium as an elicitor, after 21 days of culture, ginseng saponin content and productivity increased to about 1.31 and 1.33 times control levels, respectively [146]. The MeJA, at a concentration of 10 μM and 100 μM when introduced to cell suspension of *C. roseus* on day 6 of cell growth increased ajmalicine and serpentine production, respectively, re–elicita‐ tion showed a negative effect on both growth and alkaloid synthesis [154].

#### **7.4. Nutrient composition**

The composition of the medium or selection of medium also played a vital role in elicitation process. In the callus culture of *Erythroxylum coca*, the amounts of cocaine, cinnamoylcocaine, chlorogenic acid (CGA), and 4–coumaroyl quinate (CQA) were significantly affected by the culture medium [155]. Cocaine production was nearly an order of magnitude greater on Anderson rhododendron medium (ARM) [156], Gamborg B5 (GB5) [157], and Murashige– Tucker medium (MMT) [158], but the amounts produced on MMT and GB5 were not signifi‐ cantly different from each other. Cinnamoylcocaine was affected in the same way. The major factor controlling tropane alkaloid (TA) accumulation was medium composition, with cocaine levels on ARM being nearly an order of magnitude greater than on the other media. Many nutrients, including cobalt, copper, molybdenum, calcium, magnesium, iron, boron, iodine, manganese, zinc, and myo–inositol, and the growth regulators and the ammonium:nitrate ratio are at equivalent levels in ARM as in one of the other media, and can therefore be excluded as factors promoting TA accumulation. However, a number of factors differed between ARM and the other media, and might be responsible for the elevated TA content. Total ion concen‐ tration is lower in ARM, and could be an important factor given the importance of salt content in controlling secondary metabolism [155]. Nitrate concentration was also lower in ARM, and there are numerous reports in the literature of an inverse relationship between nitrate availa‐ bility and accumulation of secondary metabolites in many plant species, including *Arabidopsis thaliana* [159], *Hordeum vulgare* [160], and *Nicotiana tabacum* [161]. Similarly, the reduction of nitrate concentration in the culture medium of *Atropa belladonna* hairy roots increases alkaloid content [162]. In regards to CGA, the media were all significantly different from each other with the lowest production on ARM and highest on MMT. Less CQA was produced on ARM than on either of the other two media, which did not differ from each other [155]. Apart from these characteristics, the efficiency of elicitation also depends on elicitor specificity, cell line or clones of microbial elicitor used the presence of growth regulators, nutrient composition of the medium, and the environmental conditions.

### **8. Conclusion**

The development of plant tissue cultures for the production of secondary metabolites has been underway for more than three decades. Although there are well–established plant tissue culture techniques, their application to large scale production is still limited to a few processes. Various stimulation and process strategies have been exercised to improve secondary metab‐ olite production in plant tissue cultures. Elicitation has been widely applied for enhancement of secondary metabolite production in plant cell and organ cultures. The effects of various abiotic and biotic elicitors on secondary metabolite production in plant tissue cultures are dependent on the specific secondary metabolites. The exploration of the production of useful secondary metabolites through regulation of biosynthetic pathway of the various plant cell and tissue cultures of medicinal plants has been carried out by a group of plant scientists in several countries during the last decade. Although, elicitation enhances secondary metabolism in plant cells in vitro, but the exact mechanism is not exactly understood. There is a tremendous scope for the large–scale production of secondary metabolites in the plant tissue culture system by using the elicitors as an agent.
