**6. Biotic elicitors**

In the production of secondary metabolites from plants, the use of biotic elicitors had an important role (Table 2).


cultures [84–86]. JA elicitation are reported to induce the production of rosmarinic acid in *Mentha piperita* [84], anthocyanin in *V. vinifera* [87], and plumbagin in hairy roots of *Plumbago indica* [88]. JA and MeJA have been used as elicitors for stilbene biosynthesis in *V. vinifera* foliar cuttings [89], *V. vinifera* cell cultures [90,91], and *Vitis rotundifolia* hairy root cultures [92]. The addition of MeJA to *V. vinifera* cell cultures also promoted anthocyanin accumulation [93]. MeJA with transgenic technology highly enhanced the production of tanshinones in *Salvia miltiorrhiza* hairy roots [94]. In the hairy root culture of *Withania somnifera*, MeJA elicited the production of withanolide A, withanone, and withaferin A [95]. The MeJA enhanced the production of bacoside A, a valuable triterpenoid saponin having nootropic therapeutic activity in in vitro shoot cultures of *Bacopa monnieri* [96]. In *Andrographis paniculata* cell culture, the MeJA induced the production of andrographolide content [97]. In *Glycyrrhiza glabra*, methyl jasmonate induced the production of the saponin soyasaponin [98]. It enhanced the production of paclitaxel in *Tacus canadensis* and *T. cuspidate* [99], and in *Rubus idaeus*, it stimulated the

Salicylic acid, well known for the systemic acquired resistance it induces in the plant response to many pathogens, can also elicit the production of secondary metabolites in plants [101,102]. SA with transgenic technology highly enhanced the production of tanshinones in *S. miltior‐ rhiza* hairy roots [94]. The higher production of withanolide A, withanone, and withaferin A was reported in the elicited–hairy roots of *W. somnifera* [95]. SA induced the stilbene production in the cell suspension of *V. vinifera* [34]. It stimulated the production of alkaloids such as vincristine and vinblastine in periwinkle [103], the tropane alkaloid scopolamine in hairy root cultures of *Brugmansia candida* [77], and pilocarpine in jaborandi leaves [104]. Anthraquinone production was greatly increased in *Rubia cordifolia* after a SA treatment [105]. SA also affects terpenoid secondary metabolism in plants. It induced accumulation of the triterpenoids ginsenosides in ginseng and glycyrrhizin in licorice [106,107]. Recent evidence demonstrated

that suitable concentrations of SA can also promote monoterpene production [108].

Gibberellin (GA), a phytohormone, is also well known as an effective elicitor for the production

In the production of secondary metabolites from plants, the use of biotic elicitors had an

production of the raspberry ketone benzalacetone [100].

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

**5.2. Salicylic acid**

**5.3. Gibberellic acid**

**6. Biotic elicitors**

important role (Table 2).

of secondary metabolites [109].

**Table 2.** Effect of Different Biotic Elicitors on the Production of Various Secondary Metabolites in Plants

#### **6.1. Polysaccharide**

The biotic elicitors have been utilized to increase secondary metabolite production in medicinal plants. In a *Panax ginseng* cell suspension, the cell wall–derived elicitor oligogalacturonic acid significantly increased the ginseng saponin content [110]. The treatment of cultured cells of *Lithospermum erythrorhizon* with the polysaccharide agropectin induced the production of the naphthoquinone shikonin [111]. The chitosan treatment of cultures of *Plumbago rosea* increased the plumbagin content [112]. The application of chitin or chitosan induced the production of coumarins and fluoroquinolone alkaloids in shoot cultures of *Ruta graveolens* [113]. Chitosan enhanced the production of trans–resveratrol and viniferins in the cell system of *V. vinifera* [91]. Chitin induced the phenylpropanoid and naphtodianthrone production in cell suspension cultures of *H. perforatum* [114].

#### **6.2. Yeast origin**

For decades, scientists are using yeast extract as one of the biotic elicitors. Yeast extracts stimulated ethylene biosynthesis in tomato [115] and bacterial resistance in bean (*Phaseolus vulgaris*) [116]. Yeast extract elicited the production of tanshinone in the root culture of *Perovskia abrotanoides* [78].

#### **6.3. Fungal origin**

Biotic elicitors produced by pathogens have mainly been used to induce the plant defense response. In the past, biological mixtures were prepared from pathogens without identification of the active compounds. The use of pathogenic and nonpathogenic fungal preparations as elicitors has become one of the most effective strategies to induce phenylpropanoid/flavonoid biosynthetic pathways in plant cells [117,118]. Necrotrophic pathogens such as *Botrytis* sp. usually kill the host cells often through secretion of toxins before deriving nutrients from them [119]. On the other hand, biotrophic pathogens *Fusarium* sp. or *Phoma* sp. try to avoid killing the host cells, and derive their nutritional benefits from extensive contact with them and by altering the host metabolism and secretion systems [120,121]. An early defense reaction of the plant cell attacked by fungal pathogen includes the rapid and transient production of reactive oxygen species (ROS). Plant cells are usually protected against the detrimental effects of ROS by a complex of nonenzymatic and enzymatic antioxidant systems [122]. It has been demon‐ strated that the phenylalanine ammonia lyase (PAL) enzyme that catalyses the entry of L– phenylalanine into the phenylpropanoid pathway has reputedly a crucial role in the synthesis of antioxidant/defense–related compounds [117]. The mycelia extracts from the above mentioned fungi induced partitioning of the phenylpropanoid pathway and a rapid stimula‐ tion of the monolignol pathway in *Linum usitatissimum* cultured cells [123]. Cultures of *Phytophthora* elicited microbial resistance in soybean [124] and potato [125]. Extracts from microbial–enriched composts stimulated systemic resistance to *Phytophthora* in pepper (*Capsicum annuum*) [126]. As the plant defense response and the production of secondary metabolites are closely related, it is not surprising that a number of elicitors have also been shown to increase the production of secondary metabolites in medicinal plant cell culture. Similar to plant defense, initial work on secondary metabolite elicitation was performed using

biological mixtures. Fungal cell wall fragments increased the production of the indole alkaloids ajmalicine, serpentine, and catharanthine by up to five times in cell suspensions of *C. roseus* [69,127] and the 12–oxo–phytodienoic acid, raucaffrincine, in *Rauwolfia canescens* [128]. Fungal mycelia increased the diosgenin content in *Dioscorea deltoidea* cells by 72% [129]. In *Papaver somniferum*, fungal spores increased the content of codeine, morphine, and sanguinarine by over eightfold [130,131]. A mixture of fungal polysaccharides increased the amount of the antimicrobial alkaloid acridone epoxide up to 100–fold in cultures of *R. graveolens* [132]. *Taxus chinensis* cells treated with an endophytic fungus found in the bark of the *T. chinensis* tree produced three times as much taxol as nonelicited cells [133].

The content of thiarubrine A was enhanced 3–fold in *Ambrosia artemisiifolia* hairy root cultures through the utilization of autoclaved cell wall filtrates from the fungus *Protomyces gravidus*, a pathogen of *Ambrosia artemisiifolia* [134]. The fungal challenged root cultures of *Taverniera cuneifolia*, increased the glycyrrhizic acid content [135]. Moreover, maximum increase in glycyrrhizic acid was noticed in *Mucor hiemalis* treated cultures. In case of *Fusarium monili‐ forme* and *Aspergillus niger*, threefold increase in glycyrrhizic acid was observed as compared to control unchallenged root culture. However, marginal increase in glycyrrhizic acid content was noticed, in *Penicillium fellutanum* and *Aspergillus tenuis* challenged cultures [135]. Similarly, biotic elicitors from *Claviceps purpurea* were included in *Azadirachta indica* hairy root cultures, leading to a 5–fold increase in the production of azadirachtin [136]. The transformed cell suspension cultures of *W. somnifera* when treated with the dual elicitation of copper sulfate (100 μM) and the cell extract of *Verticilium dahaliae* (5% v/v) showed highest production of withaferin A content when compared with the individual elicitors [137].

#### **6.4. Bacterial origin**

**6.1. Polysaccharide**

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

cultures of *H. perforatum* [114].

**6.2. Yeast origin**

*abrotanoides* [78].

**6.3. Fungal origin**

The biotic elicitors have been utilized to increase secondary metabolite production in medicinal plants. In a *Panax ginseng* cell suspension, the cell wall–derived elicitor oligogalacturonic acid significantly increased the ginseng saponin content [110]. The treatment of cultured cells of *Lithospermum erythrorhizon* with the polysaccharide agropectin induced the production of the naphthoquinone shikonin [111]. The chitosan treatment of cultures of *Plumbago rosea* increased the plumbagin content [112]. The application of chitin or chitosan induced the production of coumarins and fluoroquinolone alkaloids in shoot cultures of *Ruta graveolens* [113]. Chitosan enhanced the production of trans–resveratrol and viniferins in the cell system of *V. vinifera* [91]. Chitin induced the phenylpropanoid and naphtodianthrone production in cell suspension

For decades, scientists are using yeast extract as one of the biotic elicitors. Yeast extracts stimulated ethylene biosynthesis in tomato [115] and bacterial resistance in bean (*Phaseolus vulgaris*) [116]. Yeast extract elicited the production of tanshinone in the root culture of *Perovskia*

Biotic elicitors produced by pathogens have mainly been used to induce the plant defense response. In the past, biological mixtures were prepared from pathogens without identification of the active compounds. The use of pathogenic and nonpathogenic fungal preparations as elicitors has become one of the most effective strategies to induce phenylpropanoid/flavonoid biosynthetic pathways in plant cells [117,118]. Necrotrophic pathogens such as *Botrytis* sp. usually kill the host cells often through secretion of toxins before deriving nutrients from them [119]. On the other hand, biotrophic pathogens *Fusarium* sp. or *Phoma* sp. try to avoid killing the host cells, and derive their nutritional benefits from extensive contact with them and by altering the host metabolism and secretion systems [120,121]. An early defense reaction of the plant cell attacked by fungal pathogen includes the rapid and transient production of reactive oxygen species (ROS). Plant cells are usually protected against the detrimental effects of ROS by a complex of nonenzymatic and enzymatic antioxidant systems [122]. It has been demon‐ strated that the phenylalanine ammonia lyase (PAL) enzyme that catalyses the entry of L– phenylalanine into the phenylpropanoid pathway has reputedly a crucial role in the synthesis of antioxidant/defense–related compounds [117]. The mycelia extracts from the above mentioned fungi induced partitioning of the phenylpropanoid pathway and a rapid stimula‐ tion of the monolignol pathway in *Linum usitatissimum* cultured cells [123]. Cultures of *Phytophthora* elicited microbial resistance in soybean [124] and potato [125]. Extracts from microbial–enriched composts stimulated systemic resistance to *Phytophthora* in pepper (*Capsicum annuum*) [126]. As the plant defense response and the production of secondary metabolites are closely related, it is not surprising that a number of elicitors have also been shown to increase the production of secondary metabolites in medicinal plant cell culture. Similar to plant defense, initial work on secondary metabolite elicitation was performed using

The bacterial elicitors stimulated the biosynthesis of scopolamine in adventitious hairy root cultures of *Scopolia parviflora* via the inhibition of H6H (hyoscyamine 6β–hydoxylase) expres‐ sion [138]. In bacterial elicitation, the maximum glycyrrhizic acid increase was observed in *Rhizobium leguminosarum* challenged culture as compared to unchallenged control roots of *Taverniera cuneifolia* [135]. Furthermore, in *Bacillus aminovorans*, *Agrobacterium rhizogenes,* and *Bacillus cereus* challenged cultures, significant increase in glycyrrhizic acid content was observed. However, root culture challenged by *Agrobacterium tumefaciens* did not show any significant increase in glycyrrhizic acid content [135]. The gradual increase in hypericin and pseudohypericin was observed in seedlings of *H. perforatum* after challenging with Rhizobac‐ terium [139]. Coronatine, phytotoxin produced by the *Pseudomonas syringae* species signifi‐ cantly induced taxane synthesis in taxane media cell cultures [140], also induced the viniferins production in the cell culture of *V. vinifera* [91]*.*
